Structure and function of the SH3 domain from Bruton - DiVA Portal

Structure and function of the SH3 domain from Bruton´s tyrosine kinase

Henrik Hansson

Department of Biotechnology Royal Institute of Technology

Stockholm 2001

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ISBN 91-628-5080-6

Tryck: Universitetsservice US AB

Stockholm 2001

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Contents

Contents ..................................................................................................................................... iii Abstract ...................................................................................................................................... iv Main References.........................................................................................................................v Acknowledgements ..................................................................................................................vi Abbreviations ...........................................................................................................................vii 1 Overview.............................................................................................................................1 2 Introduction .......................................................................................................................2

2.1 Signal transduction....................................................................................................2 2.2 Non-receptor tyrosine kinases ................................................................................2 2.3 SH3 domains..............................................................................................................6 2.4 X-linked agammaglobulinemia and Bruton´s tyrosine kinase............................9

3 Objective...........................................................................................................................15 4 Methodology ....................................................................................................................16

4.1 NMR spectroscopy .................................................................................................16 4.2 Structure determination of proteins using NMR methods ..............................19 4.3 Preparation of samples for protein NMR...........................................................21 4.4 Studies of molecular interactions..........................................................................25

5 Results and discussion ....................................................................................................28 5.1 Structure of the SH3 domain from Bruton´s tyrosine kinase..........................28 5.2 Structure of the SH3 domain in the PRR-SH3 fragment. ................................30 5.3 Dimerization of Btk PRR-SH3.............................................................................31 5.4 Dimerization of Bruton´s tyrosine kinase, biological relevance ......................34

6 Conclusions ......................................................................................................................37 7 References.........................................................................................................................39

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Henrik Hansson (2001): Structure and function of the SH3 domain from Bruton´s tyrosine kinase. Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.

Abstract

Mutations in the gene coding for Bruton´s tyrosine kinase (Btk) lead to a lymphocyte differentiation block, which results in an extreme deficiency of B cells and plasma cells in the blood. These mutations are one cause of the hereditary immunodeficiency X-linked agammaglobulinemia. Evolutionarily, Btk belongs to the Tec family of non-receptor protein tyrosine kinases. Members of this family share a common domain composition with an N-terminal Pleckstrin homology domain, the Tec homology (TH) region, an Src homology (SH) 3 domain, an SH2 domain and a C-terminal catalytic (kinase) domain. The TH region, N-terminal to the SH3 domain in Btk, contains two proline-rich sequences that conform to the consensus sequence of SH3 ligands and which can recognize and bind to the Btk SH3 domain.

The solution structure of the SH3 domain from Btk has been determined using NMR methods. Studies of a protein fragment containing the proline-rich sequences of the Btk TH region and the Btk SH3 domain revealed that the Btk SH3 domain interacts with the TH region in an inter-molecular manner and that experimental data are consistent with dimerization. Intermolecular NOEs between two monomers of a dimer indicate a normal polyproline type II helix conformation of the proline-rich sequences in the interaction with the SH3 domain.

Activation of Btk by Src-family kinases leads to an auto-phosphorylation of tyrosine 223 within the binding site on the SH3 domain. It is possible that intermolecular interactions between SH3 and the proline-rich region within a Btk dimer play a key role for the regulation of Btk activity.

Keywords: nuclear magnetic resonance, Src homology 3, Bruton´s tyrosine kinase, X-linked agammaglobulinemia, gel permeation chromatography, signal transduction.

©Henrik Hansson, 2001

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Main References

This thesis is based on the following papers, referred to in the text by their Roman numerals.

I. Hansson, H., Mattsson, P.T., Allard, P., Haapaniemi, P., Vihinen, M., Smith, C.I.E. and Härd, T. (1998). Solution Structure of the SH3 Domain from Bruton´s tyrosine kinase. Biochemistry 37, 2912-2924.

II. Hansson, H., Okoh, M.P., Smith, C.I.E., Vihinen, M. and Härd, T. (2001)

Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton´s tyrosine kinase. FEBS Letters 489, 67-70.

III. Hansson, H., Smith, C.I.E. and Härd, T. (2001) Both proline-rich

sequences in the TH region of Bruton´s tyrosine kinase stabilize intermolecular interactions with the SH3 domain. FEBS Letters 501, 11-15.

IV. Hansson, H. and Härd, T. 1H, 13C and 15N backbone resonance

assignments of an N-terminally extended SH3 domain of Bruton's tyrosine kinase in its self-associated state. Manuscript.

V. Hansson, H., Kjellberg, A. and Härd, T. Structural studies of a dimeric

SH3 domain. Manuscript.

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Acknowledgements

Det finns några personer som jag särskilt skulle vilja tacka och som har bidragit, i stort eller i smått och medvetet eller omedvetet, till att denna avhandling blev verklighet och slutförd. Först och främst vill jag förstås tacka Torleif, min handledare, för att jag fått möjlighet att doktorera för dig i din grupp. Alexandra, din insats bidrog mycket till att denna avhandling kunde skrivas nu. Tack också till Ted, Pekka, Mauno och alla andra medförfattare, ett särskilt tack vill jag rikta till Ted. Helena, för all hjälp, allt stöd och tidig korrekturläsning. Peter, samma sak: ställer upp när man behöver hjälp. Magnus, tack för hjälpen med finishen av denna lilla utgåva, formatet börjar bli standard nu... Tack också för sällskap på is och i fjäll. Susanne och Esmeralda: nuvarande rumskamrater som får stå ut med mig. De har ju varit några stycken genom åren... Esme: tack också för din genomläsning. Ett tack också till alla övriga nuvarande NMR-gruppsmedlemmar: Christofer, Elisabet, Anders, Vildan, Niklas, Martin, Inger, Per-Åke och Jakob. Och före detta: Lotta, Peter Agback, Anders Ö., Anja och Thomas (NMR dream team förblir nog bara en dröm...). Och så förstås Mange. För mycket (ospecificerat). CSB-lunchgängen, som tillsammans med delar av ovanstående höll snacket igång: Erik, Lennart, Johan och Pekka som Tango-sällskap och Gudrun, Mikael, Philip och Peter H. som CSB-bordssällskap. Tack också till Ana, Jan, Hans, Caroline, Matthew och alla andra som jag lärt känna under tiden på Novum. Tack också till: Deborah, Teo och Fredrik, Mamma, pappa, Johan, Ann och Johanna, Britta, Björn, Pelle och Anna. Tove, min älskade fru, för stöd genom åren och för all hjälp med denna avhandling.

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Abbreviations

ATP Adenosine triphosphate Btk Bruton’s tyrosine kinase CD Circular dichroism DNA Deoxyribonucleic acid GPC Gel-permeation chromatography GSH Glutathione GST Glutathione-S-transferase HSQC Heteronuclear single quantum coherence spectroscopy Itk Interleukin 2 inducible T-cell kinase NMR Nuclear magnetic resonance NOE Nuclear Overhauser enhancement NOESY NOE spectroscopy PCR Polymerase chain reaction PDGF Platelet-derived growth factor PH Pleckstrin homology PI3K Phosphatidylinositide 3-kinase PPII Polyproline type II helix PRR proline-rich region PTK Protein tyrosine kinase rf Radio frequency Rho-GAP Rho GTPase activating protein rmsd Root mean square deviation SAB SH3-binding protein that preferentially associates with Btk SH3 Src homology 3 TH Tec homology TOCSY Total correlation spectroscopy TROSY Transverse relaxation-optimized spectroscopy WASP Wiscott-Aldrich syndrome protein XLA X-linked agammaglobulinemia

Overview 1

1 Overview All living cells need to communicate with their environment. Regardless of whether a cell is one of the thousands of cooperating spleen cells in a human tissue, or one of the thousands of competing bacterial cells on a human index finger, they have in common a need to “know” about the environment. Also, to be able to react to any change in this environment, chemical or physical, they need to transfer inputs (i.e. chemical or physical stimuli) from specific receptors on the cell surface to the interior, which in the case of eukaryotic cells is most often to the nucleus. The mediators of these signals are often proteins that interact with other proteins. Disturbances in this signal transduction are commonly the cause of diseases and can lead to both uncontrolled cell growth (as in cancer) and cell apoptosis or growth arrest (as in XLA; see below). An understanding of the basis for these diseases, and of signal transduction in general, requires detailed research on different levels, i.e. tissue, cellular and molecular levels.

For an understanding on the molecular level, a structural analysis of interacting proteins based on NMR spectroscopy or X-ray crystallography and a functional analysis based on structural knowledge are of great importance. The great advantage of NMR-spectroscopy, compared to X-ray crystallography, is that there are methods that enable the study of molecular interactions directly in solution as well as the investigation of the dynamics and flexibility of proteins. Many signal transduction proteins are built up by small protein modules, and their interaction with ligands can often be studied in isolation. NMR methods are particularly useful for the study of these small protein modules and their interactions with ligands, which can be both other proteins and small molecules such as drugs.

This thesis deals with the structure and the functional role of one of these signaling protein modules, an SH3 domain in an enzyme called Bruton´s tyrosine kinase, which is involved in the development of antibody-producing B-lymphocytes in humans. If this enzyme is not working properly, it causes a hereditary immunodeficiency known as X-linked agammaglobulinemia (XLA). As a step towards better understanding of the SH3 domain and its role, we have determined the structure of this protein module using NMR-methods. We have also studied its self-association behavior, involving the SH3 domain and another part of the enzyme, the TH region.

2 Structure and function of the SH3 domain from Bruton´s tyrosine kinase

2 Introduction

2.1 Signal transduction

Signal transduction is used as a general term for the flow of information that occurs between the nucleus of an eukaryotic cell and other cell compartments or the cell surroundings. Many proteins and other molecules are involved in this information flow. The use of the term transduction can be understood with the insulin-receptor as an example. The insulin receptor spans the cell membrane and when an insulin molecule physically interacts with the extracellular parts of the receptor, it switches on enzymatic activity in the intracellular part of the receptor. This makes the insulin receptor a transducer of signals, by translating one type of chemical signal on the surface to another chemical signal within the cell.

Bruton´s tyrosine kinase is a so-called non-receptor protein tyrosine kinase and is involved in the signal transduction in B-lymphocytes. Its role is important for correct differentiation and development of plasma cells, which are needed to produce gammaglobulins as an immunological response to e.g. a bacterial infection.

2.2 Non-receptor tyrosine kinases

Approximately 20% of the human genes encode proteins involved in signal transduction. These include many types of proteins: transmembrane receptors, G-protein subunits, adaptor proteins, kinases and phosphatases. Among these are the protein tyrosine kinases (PTK), a large and diverse group of enzymes that are unique to metazoans and not found in unicellular eukaryotes such as yeast [1, 2]. This is in contrast to serine/threonine kinases, such as cyclin-dependent kinases and MAP (mitogen-activated protein) kinases, which are conserved throughout eukaryotes and regulate processes both in unicellular and multicellular eukaryotes. More than 90 genes in the human genome code for PTKs, and these can be divided into two subgroups: receptor and non-receptor PTKs. The non-receptor PTK subgroup consists of 32 genes that can be divided into 10 families (Figure 2.1A). Many of these genes and their gene-products play significant roles in different human disease states, such as cancer. Src, the gene coding for Src kinase, was for instance the first discovered cellular counterpart (c-Src) to a retroviral transforming oncogene (v-Src) [3]. The

Introduction 3

phosphorylation catalyzed by these kinases, together with the dephosphorylation catalyzed by phosphatases, can be regarded as molecular on/off-switches.

Some of the non-receptor PTKs are themselves regulated by tyrosine phosphorylation, catalyzed either by receptor or other non-receptor PTKs. For instance, the Src family kinase c-Src becomes inactive when a tyrosine within the C-terminal of the molecule is phosphorylated [4]. Tec-family kinases, including Bruton´s tyrosine kinase, are also regulated by tyrosine phosphorylations (see below).

Crystal structures of inactive Hck and Src kinases have revealed part of this switch mechanism affecting the regulation of the Src-family kinases [5, 6]. The Src family kinases have three distinct domains (Figure 2.1A), which were first discovered based

SH3 SH2 SH3Grb-2 adaptorSH3 SH2SH3SH3Nck cytoplasmic adaptor

PI3-kinase, p85αααα-subunit SH2RhoGAPSH3 SH2Vav C1 SH2 SH3RhoGEFCH PH

B.

SH3 SH2 kinaseCsk

Src SH3 SH2 kinase

SH3 SH2 kinase DBD actinAbl

Jak kinase-like kinaseFERM

Ack SH3 cdc42kinase

Fak kinase FABDFERM

Frk SH3 SH2 kinase

Syk SH2 kinaseSH2

SH2 kinaseCIP4Fes

A.

Btk/Tec PH SH3 SH2 kinaseTH

SH3 SH2 SH3SH3 SH2 SH3Grb-2 adaptorSH3 SH2SH3SH3 SH3 SH2SH3SH3Nck cytoplasmic adaptor

PI3-kinase, p85αααα-subunit SH2RhoGAPSH3 SH2 SH2RhoGAPSH3 SH2Vav C1 SH2 SH3RhoGEFCH PH C1 SH2 SH3RhoGEFCH PH

B.

SH3 SH2 kinaseSH3 SH2 kinaseCsk

Src SH3 SH2 kinaseSH3 SH2 kinase

SH3 SH2 kinase DBD actinSH3 SH2 kinase DBD actinAbl

Jak kinase-like kinaseFERM kinase-like kinaseFERM

Ack SH3 cdc42kinase SH3 cdc42kinase

Fak kinase FABDFERM kinase FABDFERM

Frk SH3 SH2 kinaseSH3 SH2 kinase

Syk SH2 kinaseSH2 SH2 kinaseSH2

SH2 kinaseCIP4 SH2 kinaseCIP4Fes

A.

Btk/Tec PH SH3 SH2 kinaseTHPH SH3 SH2 kinaseTH

Figure 2.1. SH3 domain-containing proteins. A. Families of human non-receptor tyrosinekinases (family-names according to the conentional three letter abbreviation), SH3 - Srchomology 3 domain, SH2 - Src homology 2 domain, DBD – DNA-binding domain, actin –actin binding domain, cdc42 - cdc42-binding motif, FERM – Integrin-binding domain, CIP4-FES/CIP4 homology domain, FABD – Focal adhesion-binding domain, PH, Pleckstrinhomology domain, TH – Tec homology region. B. Other non-kinase proteins containing SH3domains, CH - C Homology domain, RhoGEF – RhoGTPase-exchanging factor, C1 - C1-domain, RhoGAP – RhoGTPase-activating protein homologous domain


on sequence homology between Src and other kinases [7-9]: Src homology (SH) 2, SH3 and the kinase domain (formerly SH1). Both the SH2 and the SH3 domains are involved in intramolecular contacts (Figure 2.2) to keep the kinase active site in an “open” inactive conformation. The SH2 domain binds to a region in the C-terminal of the protein. This region contains the inactivating tyrosine phosphorylation mentioned

SH2

SH3

kinase domain

C-termSH2

SH3

kinase domain

C-term

Figure 2.2. Intramolecular binding of SH3 and SH2 domains in Src family kinaseillustrated by the X-ray structure of inactive c-Src kinase (Xu et al. [5]). The SH2 domainbinds to a phosphorylated tyrosine close to the C-terminal and the SH3 domain binds to thePPII forming linker between the SH2 domain and the kinase domain. These interactions keepthe kinase active site in an open and inactive conformation. This figure and all other proteinillustrations figures in this thesis were created using the program MOLMOL (Koradi et al.[16]).

Introduction 5

above [10]. The SH3 domain binds to a region formed by the linker between the SH2 and kinase domains. Although it was previously reported that the SH3 domain was involved in keeping the kinase inactive [11, 12], the intramolecular ligand within the protein was not known before the three-dimensional structures were determined. The active, “closed”, conformation of the kinase is known from the crystal structure of an active kinase domain from the Lck kinase [13] and structure comparisons reveal the rearrangements within the kinase catalytic site and its vicinity that might switch on tyrosine kinase activity. This is discussed in detail in Sicheri and Kuriyan [14] and in Williams et al. [15].

The displacement of the SH3 and SH2 domains upon activation of the kinase can also be considered as a part of the activation event. Based on molecular simulations and expression of different mutant proteins in a model expression system, Young et al. suggested that the linker connecting the SH3 and SH2 domains works as an inducible “snap lock” [17]. The term “snap lock” was introduced by Laity et al. to describe the folding upon binding of small conserved linkers located between DNA-binding tandem Cys2-His2 zinc-fingers [18]. Calculations on Hck kinase showed dynamic correlations between the two domains when the regulating C-terminal tyrosine, Y527, is phosphorylated. Removal of the phosphate group from the tyrosine i.e. activating the enzyme, also removed the correlation. Replacement of residues within the linker to make it less rigid also resulted in loss of correlation. In an in vivo test system, the same replacements were introduced via mutations, which resulted in a constitutively active kinase. A “snap lock” function of the connector might not only contribute to the affinity of the intramolecular binding but may also contribute to the kinetics in activation/inactivation. It might also provide necessary flexibility in the active state for the SH3 and SH2 domains to be able to interact with other proteins. Much of what is known about non-receptor PTKs comes from studies on Src family tyrosine kinases. Although there is a high degree of homology between this family and other PTK-families, both in terms of primary and tertiary structure of the different domains, there are noticeable differences regarding their biological functions.


2.3 SH3 domains

The SH3 domains are a group of protein modules, found in a single copy in many protein tyrosine kinases. They are also frequently found, either as a single copy or in multiple copies, in other proteins involved in signal transduction (Figure 2.1B). The domain consists of approximately 60 residues and the structural motif can be described as a five-stranded β-barrel-like tertiary structure (Figure 2.3). In some, but not all, SH3 domain structures, there is also a 310 helix between the fourth and fifth β-strand.

Since the first structures were reported [19, 20], SH3 domains have been given a central role as model systems in the understanding of the fundamental biophysical chemistry (such as the thermodynamic, dynamic, and structural aspects) of both protein folding and protein-protein or protein-ligand interactions [21-27].

SH3 domains have so far been found only in eukaryotic organisms, although structurally homologous proteins have been identified in lower organisms. Some examples are the DNA-binding Sso7d protein from Sulfolobus solfatricus, the DNA-

ββββ1 ββββ2 ββββ3 ββββ4ββββ5

333310101010

Figure 2.3. Illustration of the secondary elements of a typical SH3 domain (Xu et al. [5]).

Introduction 7

binding domain of HIV-1 integrase and photosystem I protein PsaE from Synechococcus [28-30]. It is interesting that some SH3-like proteins bind DNA, although no report has yet been published on DNA-binding SH3 domains.

This protein module seems to have evolved for protein-protein interaction and the general motif for its binding are proline-rich sequences that can adopt the typical polyproline type II (PPII)-helix (see Stapley & Creamer for a survey on PPII-helices [31]) The affinity of the SH3 domains to these motifs are usually quite low compared to for instance, antibody-antigen interactions. The relatively low affinity, with dissociation constants at the µM level, makes sense since it would not slow down the transmission of signals. A high affinity interaction would probably cause the involved molecules to be bound to each other too long for an effective transmission of a signal. The peptide-interacting surface on the SH3 domain is built up mainly of aromatic sidechains that are conserved among different proteins.

The consensus sequence for all SH3-binding motifs is PxxP, in which x denotes any amino acid residue, and the motifs are usually divided into class I and II, according to the orientation of the peptide backbone when interacting with an SH3 domain. A conserved aspartate on the edge of the binding pocket on the SH3 domain

Figure 2.4. Schematic drawing to illustrate the notation used for the binding site positions(according to Yu et al. [34]). Residues of the Btk SH3 binding site are indicated atapproximate locations relative to the PPII helix.


interacts with a basic residue on the PPII helix, and thereby determines the orientation of the peptide backbone [32, 33]. The consensus sequence for a class I motif, i.e. N- to C-terminal orientation, is R/KpxPpxP, and for a class II motif it is xPpxPpR/K, where uppercase reflects the critical residues [32]. Five of these seven peptide residues actually interact with the SH3 domain and their relative positions along the peptide backbone are P-3, P-1, P0, P+2 and P+3 (Figure 2.4), using a notation introduced by Yu et al. [34]. The P-3 position is the basic residue in both motifs, but in class I motifs the critical prolines are in the P0 and P3 positions, whereas in class II motifs they are in P-1 and P+2 positions.

In an extensive study, Nguyen and coworkers demonstrated the roles of the prolines at these positions. They showed that, in the case of the Sem5 SH3 domain interaction with class II motifs, the residues in positions P-1 and P+2 need to be N-substituted while in P0 it needs to be a normal Cα-substituted residue [35]. Proline is the only natural occurring amino acid that is N-substituted. The authors substituted all the residues at the different positions, one at the time, to either alanine or sarcosine, which is an N-substituted equivalent to alanine. Affinity decreased more than 50-fold when the residue in either the P-1 or the P+2 positions was an alanine, but not if it was an N-substituted sarcosine. For the residue in position P0 the situation was reversed, i.e. the residue in P0 needs to be Cα-substituted and cannot be N-substituted. Nguyen et al. also determined the structures of several SH3-ligand complexes. Altogether they claim that the SH3 PPII-helix interaction cannot be explained by a traditional lock-and-key mechanism, i.e. an interaction that does not involve conformational changes. Given the rigidity of a polyproline peptide backbone, lock-and-key would be an expected mechanism for the interaction. Only a portion of each proline sidechain was involved in direct contact with the SH3 surface. The conclusion of the authors was that the rigidity of the peptide backbone still is important in PPII-SH3 interaction but not for the traditional lock-and-key mechanism. Instead the rigidity causes less surface area to be buried upon binding, which would decrease the affinity. Still needed for the specificity of the interaction are the specific contacts that only the proline sidechains are able to make in P-1 and P+2 positions and that only Cα-substituted residues can make in P0 positions.

Introduction 9

2.4 X-linked agammaglobulinemia and Bruton´s tyrosine kinase

2.4.12.4.12.4.12.4.1 XXXX----linked agammaglobulinemia (XLA)linked agammaglobulinemia (XLA)linked agammaglobulinemia (XLA)linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is a hereditary disease characterized by an increased susceptibility to bacterial infections. In 1952 there was a report by Bruton [36] on an 8-year-old boy who had suffered from recurrent bacterial infections since the age of 4½. Bruton found that the levels of serum immunoglobulins were undetectable and that the boy suffered from a general inability to synthesize antibodies. This was the first human immune disorder in which an underlying defect - the absence of gammaglobulins - was clearly identified and reported as a distinct disease. The study of XLA has since then been important in the understanding of the basics of immunology in general and especially of the humoral immunity. It has been shown that mutations in the gene coding for a protein called Bruton´s tyrosine kinase (Btk) can cause the immunodeficiency [37] and more than 380 unique XLA-causing mutations are known and registered in the mutation database BTKbase [38]. Btk is expressed throughout the development of B cells and in XLA the development is blocked at the time of transition from pro-B to pre-B cell resulting in B cell apoptosis [39] (Figure 2.5). The exact role of Btk in this development is not clear. For a review on XLA, both causes and history, see Sideras & Smith [40].

Figure 2.5. Schematic illustration of the B-cell development. Btk is expressed and activethroughout the development, but is actively down-regulated in plasma cells. XLA causes a blockin the development at the transition from pro-B cell to Pre-B cell.

Stemcell

Lymphoid progenitor

Pro-Bcell

Pre-Bcell

B lympho-cyte

Plasma cell

Differentiation block in XLA


2.4.22.4.22.4.22.4.2 Bruton´s tyrosine kinase (Btk)Bruton´s tyrosine kinase (Btk)Bruton´s tyrosine kinase (Btk)Bruton´s tyrosine kinase (Btk)

The PH domain and the Btk motif Btk belongs to the Tec-family of non-receptor PTKs, showing sequence homology and sharing domain composition (Figure 2.6) with human and murine kinases Bmx, Itk, Tec and Txk, and with skate PTK and Drosophila Btk29a [41]. Similar to other families of non-receptor PTKs, the Tec family PTKs contain both SH3 and SH2 domains next to the kinase domain. On the other hand, at the N-terminus a Pleckstrin homology (PH) domain followed by the Tec homology (TH) region is unique for the Tec family. The TH region contains two different motifs but is probably not a structured domain in itself. The N-terminal part of the TH region, immediately next to the PH domain, harbors a 25-residue conserved sequence called the Btk motif and at the C-terminal part, preceding the SH3 domain, there is a proline-rich region (PRR) with SH3 binding motifs (Figure 2.7) [42]. The Btk motif contains a zinc-binding site, with missense mutations reported in XLA-patients [43]. The structure of the PH domain extended with the Btk motif has been determined by X-ray crystallography [44, 45].

PH domain followed by a Btk motif is not only found in Tec family kinases, but also in some Ras GTPase activating proteins [46]. The findings that G-protein βγ-subunits and the α-subunits Gαq and Gα12, can stimulate Btk and interact with the PH

SH3 SH2 kinasePHTH

SH3 SH2 kinasePHTH

Figure 2.6. Schematic presentation of the different domains and regions of a Tec family kinaseand individual structures of the different domains present in Bruton´s tyrosine kinase. Thestructure of the SH2 domain from Btk is not known, whereas the structure of the kinase domainis not available in the pdb database. This illustation shows the structures of Btk PH and SH3domains and c-Src SH2 and kinase domains [5, 44-45, 68 and I ].

Introduction 11

domain and Btk motif, link regulation of the kinase to G-protein coupled receptors [47-49]. The PH domain is also involved in membrane association. Constitutively active Btk is the consequence of a point mutation within the PH domain, E41K, and the result is increased membrane localization [50, 51]. Targeting of Btk to the membrane by fusion of Btk to membrane proteins gives a similar behavior as the point mutation, indicating that the membrane association itself might be a step in kinase activation. In the wild-type protein, the association to membrane occurs through recognition of phosphoinositides, which couples the Btk signaling to the phosphatidylinositide 3-kinase (PI3K) signaling pathway [45, 52]. PI3K can indirectly regulate Btk via a specific interaction with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), a product of PI3K that is normally not present in membranes but are produced upon stimulation. Divalent cations are needed for the interaction between PtdIns(3,4,5)P3 and Btk PH. Raised Ca2+-levels is one way to activate PI3K. The interaction between PtdIns(3,4,5)P3 and Btk PH is highly dependent on an arginine residue, R28, which is mutated in some XLA patients and also in the murine X-linked immunodeficiency Xid. Mutation of R28 abolishes binding of inositolphosphates to the PH domain [45].

The proline-rich region and the SH3 domain The C-terminal part of the TH region contains the proline-rich region, PRR (Figure 2.7). This region, which in Btk contains two SH3-binding motifs [42], differs slightly within the Tec family. As in Btk, the Tec kinase contains what seem to be two SH3 binding motifs while the Bmx, Itk and Txk kinases have only one such motif [41]. The most N-terminal of the two motifs in Btk, PRRN, is identical in sequence to the single PRR in Itk. In vitro studies have shown that this sequence, KKPLPPTPE, can be recognized by the SH3 domains of Src family kinases Fyn, Lyn and Hck [53, 54]. This is of interest since Src-family kinases are known to be upstream regulators of Btk activity [55, 56]. In these in vitro binding-studies neither the GST-fused PRRN peptide nor the PRRC peptide bound to the SH3 domain of Btk itself. In contrast, Patel et al. [57] used fluorescence titration spectroscopy to determine the dissociation constants for binding of different peptides to Btk SH3, among these a peptide corresponding to PRRN, which was shown to have a KD of 54.8 µM. For Itk, an NMR structure showed that the single PRR sequence within the TH region binds intramolecularly to the SH3


domain [58] in a suggested self-regulating manner. In the same study an intramolecular interaction between TH region and the SH2 domain of Itk was inferred, but a more detailed study of this potential interaction has not yet been reported. This self-regulation should be analogous to what has been reported for the Src-family kinases, i.e. an intramolecular interaction to suppress the kinase activity, although here with a totally different mechanism. For Btk we have reported an intermolecular interaction involving the SH3 domain and both PRR sequences of the TH region, leading to a possible dimerization (II and III). A dimerization behavior has also been reported in the protein crystals of the Btk PH domain, where unusually large contacts between two molecules in a crystal unit cell were seen [45]. The crystal contacts between the two protein molecules in the unit cell were stabilized by several intermolecular hydrogen bonds between residues within the PH domains.

We have determined the structure of Btk SH3 using NMR methods (I). The SH3 domain of Btk is a classical SH3 domain consisting of 56 residues. An XLA-causing point mutation at a 5’ splice site results in exclusion of one coding exon (no. 8) and a loss of 21 residues, 14 of which form the C-terminus of the SH3 domain [59]. This mutant SH3 domain contains only approximately 44 residues and lacks several residues important for folding and peptide recognition. Patel et al.[57] have measured the peptide affinities for this mutant SH3 domain as well, and although it could bind peptides with measurable affinities, these were lower than for the full-length SH3 domain. No other XLA-causing mutations are known from the SH3 part of Btk.

Although Btk SH3 might be involved in dimerization (see above) there exist in addition known external protein ligands for this domain. Active Btk phosphorylates, for instance, the protein product of the oncogene c-cbl, p120cbl, and the recognition is mediated via the SH3 domain [60]. Tzeng and coworkers [61] have determined the

PRRN PRRC

GSSHRKTKKPLP PTPEEDQILK KPLPPEPAAA PVSTSELKKVVALYDY…

SH3

178 190 200 210

PRRN PRRC

GSSHRKTKKPLP PTPEEDQILK KPLPPEPAAA PVSTSELKKVVALYDY…

SH3SH3

178 190 200 210

Figure 2.7. Peptide sequence of the proline-rich region in Btk TH region. Two such SH3-binding proline-rich motifs (underlined residues) are present in Btk and these are denoted PRRN

and PRRC, respectively.

Introduction 13

solution structure of Btk SH3 in complex with a peptide derived from p120cbl. Also WASP (Wiskott-Aldrich Syndrome Protein) and SAB, (SH3-domain binding protein that preferentially associates with Btk) bind the SH3 domain of Bruton´s tyrosine kinase. [62-64]. Forced overexpression of SAB-proteins in B-cells reduces the B-cell receptor induced phosphorylation of Btk, which indicates a negative regulation of the SAB-Btk interaction.

For the Btk-related Itk SH3 domain it has been reported that it can interact intermolecularly with the Itk SH2 domain [65]. The binding surfaces of the SH2 domain on the SH3 domain, and vice versa, were mapped using NMR. The binding site for SH2 on the SH3 domain was suggested to be the ordinary peptide binding site.

The SH2 and kinase domains of Btk One recently reported interaction of Bruton´s tyrosine kinase is the SH2 domain binding to the adaptor protein BLNK (B-cell linker protein). This leads to the downstream activation of phospholipase Cγ2 [66]. Interestingly, this suggests a possible activation pathway for Btk. Baba et al. [67] have found that if the adaptor protein BLNK is associated with Btk, the protein Syk (Spleen tyrosine kinase) can then phosphorylate and activate Btk. This gives BLNK a role as a direct connector.

Another known pathway is activation by Src-family kinases [55, 56]. Both pathways are mediated by the B-cell receptor and lead to phosphorylation of a critical tyrosine within the Btk kinase domain, Y551, which activates Btk. A recent crystal structure of the inactive (unphosphorylated) Btk kinase domain [68] has shown that although this tyrosine is located in the activation loop, the loop conformation is different from other known kinase domains in that the aromatic sidechain of Y551 is not located in the active site [5, 69]. The activation “trigger” is suggested to be mediated by an arginine, R544, which is hydrogen bonded to a glutamate, E445. Mutations of both these residues have been found in XLA-patients and E445 is believed to orient ATP in the active enzyme. Phosphorylation of Y551 would break the hydrogen bond between R544 and E445, releasing the glutamate to interact with ATP and a lysine, K430, in the active site. This would close the active site and thereby activate the enzyme [68]. The active kinase has both external and internal ligands [70, 71]. Phosphorylation and activation by Src family kinases have also been shown to lead to autophosphorylation of a tyrosine residue in the SH3 domain, Y223 [70]. This tyrosine is located at one edge of the binding pocket on the SH3 domain (I) and


phosphorylation would possibly alter the binding properties of the SH3 domain. This has also been shown [72] in in vitro studies of GST-fused Btk SH3 domain. While unphosphorylated Btk SH3 could bind to both p120cbl and WASP, phosphorylated Btk SH3 could only bind p120cbl.

Among the downstream effects of active Btk is activation of the transcription factor NF-κB [73]. Another transcription factor, STAT5, has been shown to be a direct substrate for Bruton’s tyrosine kinase [71]. Mahajan et al. have studied binding of STAT5 to Btk both in cell-systems and using recombinant protein in surface plasmon resonance experiments. The reported affinity is quite high (KD of 44 nM) and the binding seems to be dependent only on the kinase domain itself. These observations are interesting because they reveal pathways between the cell-surface B-cell receptor and a nuclear response, especially since active Btk can be translocated to the nucleus [74]. Btk utilizes known nuclear export signals for this translocation, exportin 1/CRM-1. A mutant lacking the whole SH3 domain was predominantly localized to the nucleus but its autophosphorylation activity was low. This indicates a role for the Btk SH3 domain in the shuttling from the nucleus. Overall, these observations link Btk interaction with transcription factors to Btk activity within the nucleus, which may be critical in gene regulation during B-cell differentiation.

Objective 15

3 Objective Bruton´s tyrosine kinase has a crucial role in the differentiation and development of B-lymphocytes to become antibody-producing plasma cells. Overall it is of great interest to learn more about the specific regulation of this enzyme and its role in signaling of differentiating and developing B-cells. This thesis focuses on a part of the Bruton´s tyrosine kinase: the TH region and the SH3 domain. The objective was to use biophysical and biochemical methods to study the structure and the functional role of the SH3 domain and its intermolecular interaction with the proline-rich motifs within the TH-region.


4 Methodology

4.1 NMR spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a technique that reveals information about the environment of magnetically active atomic nuclei. In an appropriately strong static magnetic field, NMR-active nuclei can absorb electromagnetic radiation in the radio-frequency (rf) region. The exact frequency of the radiation being absorbed is affected by chemical bonds, dynamic processes, electronegativity of neighboring atoms and molecular conformation, such as presence of aromatic rings.

The following brief description of NMR spectroscopy is based on Evans “Biomolecular NMR spectroscopy” [75] and Cavanagh et al. “Protein NMR Spectroscopy: Principles and Practice” [76].

The magnetic field strength of an NMR magnet, B0, is measured in Tesla (T) and is directly proportional to the frequency of NMR. The resonance frequency (ω0, the Larmor frequency) of an isolated proton (1H-nucleus) is 600 MHz at a magnetic field strength of 14.1 Tesla, which is about 105 times stronger than the magnetic field at the surface of the Earth (ω0= -γB0 in rad/s and γ is the gyromagnetic ratio, a nucleus-dependent constant). NMR-active nuclei frequently used in protein NMR spectroscopy are 1H, 13C, 15N, 31P and 2H, all of which have spin quantum number ½ except 2H that has spin 1.

Chemical shift The NMR frequency is not only dependent on γ and B0 directly but is also dependent on the electronic environment. All nuclei are surrounded by electrons, motions of which give rise to a non-negligible secondary magnetic field. These local fields shield the nuclei from the influence of the B0-field and result in a local effective field for each nucleus. Hence, each nucleus (denoted i) in a molecule will experience a slightly different magnetic field than the static field of the NMR magnet:

and consequently each nucleus will have a slightly different resonance frequency (ωi) compared to the Larmor frequency:

(1) ( )ii BB σ−= 10

Methodology 17

The shielding factor, σi, is dimensionless and the difference in resonance frequency is referred to as the chemical shift. Chemical shifts are measured in units of Hz or in parts per million (ppm) of the static magnetic field. The latter unit, ppm, is more practical since it makes the chemical shifts independent of B0. In practice, the chemical shifts are measured in ppm relative to a reference resonance signal from a standard.

The chemical shifts are very sensitive to any changes in the molecular environment that might follow due to, for instance, conformational changes and ligand interaction. This phenomenon is used in studies of molecular interaction, utilizing perturbations of chemical shifts as a probe to localize changes in a structure (see section 4.4).

Scalar coupling If two spin-½ nuclei are adjacent with one or few separating chemical bonds, the electrons of the chemical bonds act to couple the spins. This scalar coupling, or spin-spin coupling, modifies the energy levels of the system and therefore the NMR spectrum is modified. In protein NMR this is used in many ways. For instance the presence of scalar coupling can be used to identify which nucleus that has a certain resonance. This is usually done with NMR experiments that are designed to select for magnetization polarization (coherence) transfer between scalar coupled nuclei e.g. COSY (correlation spectroscopy) and TOCSY (total correlation spectroscopy) experiments [76].

One way to estimate the magnitude of a scalar coupling is to measure the splitting of a resonance affected by the coupling. The magnitudes of scalar couplings are dependent on local bond angles and measurements of certain scalar couplings in the backbone of a protein, e.g. the coupling between HN and Hα, can be used to derive dihedral angle constraints for a structure determination [77, 78].

One situation where two spin-½ nuclei are adjacent is when a proton is attached to a 15N or 13C, a situation frequently utilized in protein NMR spectroscopy, where enrichment of 13C and 15N in proteins is used. Heteronuclear correlation spectroscopy is a way to obtain correlations between two such NMR-active but different nuclei. Most multidimensional heteronuclear NMR experiments correlate a proton resonance

(2) ( )ii B σγω −−= 10


with the resonance of either 13C or 15N nuclei by transfer of coherences between the two nuclei. One conventional method for obtaining the correlations by a coherence transfer is the heteronuclear single quantum coherence (HSQC) experiment [79], which is a 2D-experiment by itself but can be combined with other experiments to give 3D-experiments (e.g. TOCSY-HSQC and NOESY-HSQC) [80, 81].

NOE Magnetization polarization can at certain conditions be transferred through space by dipole-dipole interactions between nuclei, giving rise to the nuclear Overhauser enhancement (NOE). The efficiency of the NOE-transfer for a protein in solution is proportional to the inverse sixth power of the distance between the interacting nuclei. This distance information is local and can be utilized to derive distance constraints for structure determination. The correlation of neighboring resonances/nuclei is performed in NOESY (NOE spectroscopy) experiments [76, 82]. Both 13C15N-labeling of a protein and 3D-techniques to select for 13C- or 15N-attached protons are commonly used to obtain the distance constraints or to retrieve selective distance information for larger proteins. One advantage of the labeling is that in complexes of 13C15N-labeled protein and unlabeled ligand, one can select for only intermolecular NOEs in e.g. 13C-edited-13C15N-filtered NOESY experiments. This method was used in (II) and in (V) to obtain NOEs between two monomers in a dimer following the principles described in [83].

Relaxation The NMR relaxation, i.e. the return of perturbed magnetization of isolated spins to thermal equilibrium in NMR, is of two types: longitudinal and transverse relaxation (with the rates R1 and R2, respectively). Longitudinal relaxation, also called spin-lattice relaxation, accounts for the return of magnetization to energy levels that follow the Boltzmann distribution, by definition along the z-axis i.e. along the B0-field. Longitudinal relaxation in NMR is a relatively slow process, compared to for instance relaxation in light spectroscopy, where the half lives of excited states are much shorter.

The transverse relaxation, or spin-spin relaxation, is the decay of magnetization in the xy-plane. This relaxation does not involve any energy exchange with the lattice and it is also more rapid than the longitudinal relaxation for slowly tumbling molecules.

Methodology 19

Measurements of the relaxation rates of the 15N nuclear spin are frequently used to examine the dynamics of the protein backbone on several timescales. The applications to proteins were first reported by Kay et al. [84] and the methods are described in [84, 85]. The relaxation rates are sensitive to rapid ps-ns molecular motions and with different NMR experiments, values of R1 and R2 can be estimated for each 15N-nucleus. The R2 relaxation rates are also sensitive to motions in the ms-µs time-scale and can be used to probe such motions in a protein. The ratio between the two relaxation rates can give an approximation of the rotational correlation time, τc, which describes the overall tumbling of a protein. This has been used to estimate a rotational correlation time for the PRR-SH3 fragment in (II). Another way to probe fast motions of protein backbone is the so-called steady-state {1H}-15N NOE [76, 84, 85]. The steady-state {1H}-15N NOE is a function of the longitudinal relaxation rate of the 15N-nucleus and the cross-relaxation resulting from dipolar interaction between the 1H-15N pair of nuclei.

The three relaxation parameters, the (15N)R1, (15N)R2 and the {1H}-15N NOE depend on the values of the spectral density function at certain frequencies. The spectral density depends, in turn, on the motions of the corresponding 1H-15N vector. A quantitative evaluation of the spectral density function can be done using the so-called Model-free formalism, which was first developed by Lipari and Szabo [86, 87]. From this evaluation, values for the rotational correlation time of the molecule, the internal motions and S2, an order parameter describing the rigidity of the backbone, can be derived.

Interesting motions, from a protein functional point of view, are residue sidechain movements on the µs-ms timescale and allosteric transitions and folding/unfolding on the µs-s time-scale. NMR spectroscopy has been used to relate protein flexibility to molecular recognition and biological function, for instance in case of the transcription factor TFIIIA binding to DNA [18] and the interaction between the estrogen receptor DNA-binding domain and DNA [88].

4.2 Structure determination of proteins using NMR methods

Until 1984 structural information at atomic resolution could only be determined with X-ray diffraction techniques on protein crystals. The introduction of NMR spectroscopy as a technique for protein structure determination has made it possible


to obtain three-dimensional structures of proteins also in solution [89]. The NMR method is mainly based on local inter-proton distances and peptide backbone dihedral angle information. The local distances can be obtained from NOEs while information on peptide backbone conformation can be obtained from scalar coupling constants. To obtain structural information, a majority of the resonances of hydrogen-nuclei (protons) present in a protein must be identified and assigned in an NMR spectrum. In protein NMR, the protons each amino acid residue can be regarded as an isolated spin-system, where a spin-system denotes a group of protons that are scalar coupled. This is possible because the scalar coupling between protons over the peptide bond, which connects the amino acid residues, is very weak, while the scalar coupling between the protons within a residue is strong. The three basic steps of the assignment procedure in spectra of unlabeled and 15N-labeled proteins are first to identify the spin-systems in spectra using correlated scalar coupled resonances, e.g. TOCSY-type spectra. The second is to sequentially assign the different spin-systems to the residues along the peptide backbone, which is done using NOESY-type spectra usually by identifying NOEs between HN on one residue and Hα on the preceding residue. And the third step is to assign all sidechain resonances, which can be done in TOCSY-type spectra once the residue type is known. The detailed principles of this assignment procedure is further described by Wüthrich [89]. The larger the size of the protein, the more proton resonances there will be, and consequently the more complicated the spectra, with an increased risk of spectral overlap of the resonances. This makes the assignment difficult for large proteins when using unlabeled and/or only 15N-labeling. With the introduction of isotope-labeling of proteins with the NMR-active nuclei 13C and 15N, new techniques for the sequential assignment of amino acid residues have been established, which use triple-resonance experiments [81]. The methods for obtaining and assigning spectra of 13C15N-labeled proteins have been described, for instance, by Cavanagh et al.[76], Sattler et al.[90] and Güntert [91]. The first step for chemical shift assignments using this approach is to identify backbone HN-NH pairs in a 15N-HSQC spectrum. The second step uses pairs of triple-resonance experiments that will correlate the chemical shifts of the amides with those of Cα, Cβ and/or C´ of two neighboring residues along the peptide backbone. The chemical shifts of Cα and Cβ are used to identify the residue type and then to sequentially assign the HN-NH pairs. These methods have increased the size of proteins for which the structure can be determined using NMR spectroscopy.

Methodology 21

With increasing size, the relaxation behavior of the NMR signal becomes more and more unfavorable. The methods to overcome this problem have not been utilized for the work presented in this thesis but are still worth mentioning. 2H has a different relaxation behavior than 1H so that 2H-labeling of proteins makes it possible to study larger proteins than otherwise would be possible using NMR spectroscopy. The 2H-labeling can be combined with the use of the recently developed TROSY-methods (T2-relaxation optimized spectroscopy) [92, 93] to efficiently assign large proteins. A drawback with uniform 2H-labeling is that it eliminates the possibility to obtain the distance information that is needed for a structure determination. This is because with uniform 2H-labeling only exchangeable protons, i.e. mostly backbone amides, will give rise to NOEs and distance restraints. In practice, this is overcome by the use of fractional 2H-labeling, usually around 80%, or by specific 1H-labeling of methyl-groups in a uniformly 2H-labeled protein. This can give enough distance information for a structure determination [94]. Other types of information that may be used as restraints in a structure determination are residual dipolar couplings and angles derived from dipole-dipole cross-correlations [95, 96].

Methods for the protein structure calculation from NMR data have recently been reviewed by Peter Güntert [91].

4.3 Preparation of samples for protein NMR

4.3.14.3.14.3.14.3.1 In generalIn generalIn generalIn general

Until quite recently, the natural source was the only source from which protein could be efficiently isolated for the purposes of e.g. biophysical studies. Even today, many interesting protein targets must be isolated from their natural sources, although the technology for recombinant protein expression has become the dominant way of producing proteins for isolation and further use in all biosciences. NMR spectroscopy, as a method to study the structure of proteins, has evolved along with the development of recombinant expression techniques. Usually taken for granted, these rapidly developing techniques to produce protein actually form one basis for the now successful use of NMR spectroscopy in biosciences. The reason for this is that it is now possible to incorporate into proteins naturally rare isotopes, e.g. 13C or 15N, which are useful in NMR spectroscopy (see section 4.1 and 4.2). Uniform labeling of a


protein is done by expressing the recombinant protein in bacteria grown with a 13C-enriched carbon source and a 15N-enriched nitrogen source.

Despite fast development of the hardware of NMR spectroscopy, i.e. stronger magnetic fields, more sensitive probes, etc, the time-requirements for obtaining data-sets that can be used for structure determination is quite long. This means that an NMR sample needs to be stable at a certain temperature, usually >20ºC, for at least a week but generally longer than that. The requirements in purity of a protein sample for NMR purposes are therefore very high. Any trace of protease activity can easily ruin a protein sample. It is frequently necessary to take this possibility into account when choosing a fusion-protein/protease-cleavage strategy.

The need of pure and stable protein samples is actually not larger in NMR spectroscopy than in other biophysical sciences. A difference might be the amount of material needed for a reasonable protein NMR sample, which is about 1 mM in protein concentration, i.e. 10 mg/ml for a 10 kDa protein, and well comparable with what is required for crystallography studies. This reveals one of the special demands that structural biology puts on the protein to be studied: solubility. This is also a part of the long-term stability demands. Self-aggregation and precipitation can easily and certainly make life difficult for a protein NMR-spectroscopist. One way to optimize stability of a protein sample is screening for suitable buffers and additives.

4.3.24.3.24.3.24.3.2 SiteSiteSiteSite----specific mutagenesisspecific mutagenesisspecific mutagenesisspecific mutagenesis

The possibility for substitute amino acid residues within a protein by site-specific mutagenesis provides a very useful tool. There are several reasons for this; it allows one to mimic disease-causing mutations in recombinant proteins and to study the behavior of these proteins in an isolated form. Another reason is that with certain substitutions, the biology of the protein of interest can be altered. For instance, a lucky point mutation can turn an insoluble protein more soluble, which can make it easier to study [97]. Or, as in our case (III), where the purpose was to alter the binding properties of a protein-protein interaction.

There are several methods to introduce such substitutions on the genetic level by introducing mutations in the DNA-sequence of the gene that is used for recombinant protein expression. Many companies provide special kits for this purpose. For the work presented in this thesis, we have used a method provided by Stratagene, called QuikChange™. This is a fast, non-PCR method for site-specific mutagenesis based on

Methodology 23

the fact that most E.coli strains incorporate a methyl-group on DNA-bases, which is a way for the bacteria to distinguish between own and alien (e.g. viral) DNA. One of the advantages with this method is that the mutagenesis is done directly on the gene-containing plasmid, which only needs to be prepared in a bacterial strain that introduces the mentioned methylation. The desired base-substitution, which will cause a certain amino-acid residue substitution in the final protein, is incorporated into two complementary synthesized DNA-oligonucleotides. In a thermocycling step, copies of the gene-containing plasmid are created with a DNA-polymerase that uses the oligonucleotides as primers, thereby introducing the base-substitution. The polymerase cannot catalyze methylation of the bases. The next step is to eliminate the parental and methylated plasmid DNA, which is done with a special restriction enzyme that recognizes only methylated DNA. What remains after the digestion, is nicked (unligated) plasmid DNA, which contains the desired base-substitution and can be transformed into common cloning strains of E. coli, e.g. XL1 Blue, where the plasmid will be ligated by E. coli enzymes.

In our work, presented in Main reference III, we have replaced proline residues with alanine residues. The purpose of the mutations in our study was to abolish the participation of certain crucial prolines within the proline-rich region in binding to the SH3 domain, in order to gain further information about the relative roles of the two PRR-sequences in PRR-SH3 dimerization. Both proline and alanine are hydrophobic residues, but alanine is smaller than proline. A bulkier substitute for proline would be valine, which has the same number of carbon atoms. We chose to use alanine as a substitute for prolines for three reasons. First, the small size of alanine minimizes the risk of unwanted changes in protein conformation; and second, it demands only one base of a triplet-codon to be substituted, which increases the probability of success in the mutagenesis; and third, it is conventional for this type of study (e.g. in [34, 35, 57]) and thereby made our study compatible with similar studies in the field.

4.3.34.3.34.3.34.3.3 Protein purificationProtein purificationProtein purificationProtein purification

An efficient, and usually fast, way of purifying proteins is the use of an affinity tag as a fusion to the protein of interest. There are several different variants of affinity tags, although the most commonly used are probably poly-histidine (His)-tags and Glutathione-S-transferase(GST)-fusions. In the work presented in this thesis we have used GST as an affinity tag, which means that the gene coding for the protein GST is


attached in-frame to the gene coding for the target protein so that both are translated as one fusion protein. The GST binds glutathione (GSH) with high affinity, which enabled the cleared lysate containing overexpressed GST-fused SH3-fragments to be affinity-purified using GSH-Sepharose. The advantages in putting an affinity step in the beginning of a purification scheme are a reduction in volume, and an early separation of the target protein from other bacterial host proteins (which may cause degradation of the target protein). The drawback of having an affinity tag that needs to be cleaved off is the introduction of a protease that besides cleaving where it is supposed to also might cleave within the target protein. We used thrombin and an introduced thrombin cleavage site between the GST and SH3/PRR-SH3. The best way we found to get stable NMR-sample, besides heating the sample (see below), was to use EDTA both in the purification (after protease cleavage) and in the NMR tube. Many proteases need divalent cations to be active and the presence of EDTA in the NMR sample could potentially lower the activity of any remaining impurities.

Once established, a purification scheme usually does not need to be modified. However, the report that the SH3 domain of Btk thermally unfolds only at temperatures greater than +80ºC [98] made us change the scheme by removing an ion exchange step and adding a heat-stabilizing step, which also turned out to be a very efficient way to remove remaining GST.

As the last step of our purification scheme, the polishing step, we used classical size exclusion chromatography. This step can be used to change the buffer for a suitable NMR buffer, but can also be performed in a volatile buffer, e.g. ammonium acetate. The latter is often an advantage since after size exclusion chromatography, the protein is pure but in a large volume and must be concentrated to be used for NMR studies. Using a volatile buffer, a lyophilization step can be used to concentrate the sample without the risk of protease activity during concentration. When dissolving the lyophilized protein after such a step, the pH of the sample needs to be checked and possibly adjusted.

Methodology 25

4.4 Studies of molecular interactions

4.4.14.4.14.4.14.4.1 The use of NMR to study molecular interactionsThe use of NMR to study molecular interactionsThe use of NMR to study molecular interactionsThe use of NMR to study molecular interactions

NMR is particularly useful in the study of interactions between molecules. If a protein structure is known, interactions of proteins with e.g. small ligands, DNA, and other proteins, can be probed by observing perturbation of the protein chemical shifts. This is usually done in an ordinary 15N-HSQC spectrum and the chemical shift perturbations can be mapped onto the surface of the protein, yielding a potential binding surface [20, 99, 100].

The same basic principle, probing chemical shift perturbations, is used in “SAR (structure-activity relationships) by NMR” but here it is used as a screening tool for high-affinity protein ligands [101]. Weakly binding molecules found in a first round can be tethered to obtain protein-ligands with higher affinity. In this case, the chemical shift perturbations can be used to identify the possible ligands, both in terms of finding a ligand and detecting where it interacts with the target. The latter can be important to avoid tethering of two ligands that bind to the same surface.

The interaction of small molecules with proteins can also be studied directly. Dissociation constants in the mM-range can be determined for, e.g. chloride-protein equilibria and ADP/ATP-protein interaction using the NMR-active nuclei of 35Cl and 31P, respectively [102, 103].

Recently, a novel method using cross saturation for determining interaction surfaces between large proteins has been proposed [104]. Protons on an unlabelled protein molecule are saturated non-selectively and this saturation is then transferred to another protein molecule by cross relaxation. If that other molecule is deuterated, then this cross saturation is limited to the interface. The detection is then done via TROSY-based experiments and the deuterated protein must also be 13C15N-labeled.

4.4.24.4.24.4.24.4.2 Analytical Gel Permeation Chromatography (GPC)Analytical Gel Permeation Chromatography (GPC)Analytical Gel Permeation Chromatography (GPC)Analytical Gel Permeation Chromatography (GPC)

We used analytical gel permeation chromatography to study the dimerization of PRR-SH3. This was done by a series of analyses of PRR-SH3 on a Superdex75HR column keeping all parameters constant except for the protein concentration, which was varied between 50 µM and 1.1 mM. By modeling the non-linear retention behavior of the GPC profile, the dissociation constant Kd, could be estimated.


GPC is a method of separation of molecules based on size. This separation can be established simply because a small particle will experience a larger volume than a large particle, thus causing the larger particle to travel faster through the column than the small particle. A monomeric and a dimeric PRR-SH3 will experience different volumes from which they are excluded. During propagation along the gel bed, dimeric PRR-SH3 will be able to travel faster than monomeric PRR-SH3, because a large molecule will be more excluded from the gel matrix than a small molecule. In another dynamic situation, with slower equilibrium kinetics, this would be seen as two peaks in the profile, one for the dimer and one for the monomer. In our case, the dimer-monomer equilibrium is much faster than the propagation, which causes the non-linear behavior of the elution profile usually referred to as tailing. During the GPC, the volume in which the sample exists will increase, causing a dilution of the protein sample. If same sample volume is applied in all analyses, the volume increase itself will be kept constant. The retention behavior can be characterized by an average partition coefficient σav:

in which Ve is the elution volume corresponding to the maximum of the elution profile, VM is the void volume of the column mobile phase and VS is the volume available for a totally included solute. In the case of a self-association, Ve is directly dependent on the fractions of dimer and monomer and therefore also dependent on the dissociation constant, Kd, of the dimerization. The fraction of monomer, α, is given by:

in which c0 is the total protein concentration. There is, to the best of our knowledge, no derived closed-form expression for the relation between σav and concentration for a species in a rapid monomer-dimer equilibrium. However, the non-linear retention behavior can be modeled using the Craig method [105] by letting the injected volume propagate through a grid of theoretical plates and then calculate the equilibrium

−

+= 118

40

0 d

d

Kc

cKα (4)

−=S

Meav V

VVσ (3)

Methodology 27

fraction of monomer and dimer and their retention at each of the plates. The retention at each plate is characterized by a concentration-dependent effective partition-coefficient, σeff, which for a monomer-dimer equilibrium can be written

in which σ1 and σ2 represent the partition coefficients for monomer and dimer, respectively. Kd can then be estimated, together with the unknown parameters σ1 and σ2, by fitting the experimentally determined σav as a function of loaded protein concentration. Given initial guesses of Kd, σ1 and σ2, the elution profiles (σav) are simulated for each given data point (loaded concentration) and the results are compared to experimental σav values. The input parameters are adjusted and optimized according to the minimization algorithm, in our case the Nelder-Mead simplex algorithm.

( )212 σσασσ −+=eff (5)


5 Results and discussion

5.1 Structure of the SH3 domain from Bruton´s tyrosine kinase

Following our report of the solution structure of the isolated Btk SH3 domain (I), the structure of Btk SH3 domain in complex with a peptide has also been reported (pdb-id: 1qly) [61]. The peptide used in that study was a fragment derived from the protein product of the oncogene c-cbl (p120cbl), a putative ligand for the Btk SH3 domain [57, 60]. The difference between the two Btk SH3 structures, isolated and peptide-bound, in terms of an average rmsd value is 0.95 Å, when calculated for the heavy backbone atoms of 55 residues in a comparison between the mean structures of each ensemble (pdb-id: 1aww and 1qly). Overall, the backbone structures of peptide-bound and isolated Btk SH3 domains are similar (Figure 5.1). There seem to be small structural differences, mainly in the so-called RT-loop, in the n-Src loop (neuronal Src), and in a helix-like turn between the 4th and 5th β-strand.

The peptide-binding surface we suggested (I) was based on comparisons with other SH3 domains. Our proposed surface agrees well with what is reported in the peptide-complex of Btk SH3. The binding surface contains four aromatic residues. One of these aromatic residues, Y268, is attached to the helix-like turn and this might explain the difference in structure between the peptide complex and the free Btk SH3 domain. Y268, as well as the other three aromatic sidechains building up the binding surface, were reported to have NOEs to the p120cbl-peptide [61]. However, since the sidechain positions for these aromatic residues are not well determined, a detailed comparison of the surface between isolated and peptide-bound Btk SH3 could not be performed. The part of the binding surface that is located between the RT- and n-Src loops is sometimes referred to as the “specificity pocket” [106]. The RT- and n-Src loops vary in sequence among SH3 domains and this sequence variation has been suggested to be part of the specificity for SH3 domain to recognize different peptide sequences. In the free SH3 domain these regions seem to be slightly less ordered. The RT-loop, named after an arginine and a threonine residue in Src, corresponds to M228 and N229 in Btk. These two residues and also S247 within the n-Src loop have lower amplitudes of the generalized order parameter, S2, compared to most of the other residues of Btk SH3 (I). This is an indication of a higher degree of flexibility within

Results and discussion 29

these two loops in the peptide-free state. In peptide titration studies and also in the PRR-SH3 domain, we observed that some residues within these two loops showed chemical shift perturbations (II & V). These chemical shift perturbations might originate from a direct involvement in the peptide recognition or they might arise indirectly from a decrease in flexibility of the peptide backbone, if neighboring residues are involved in the recognition.

RTloop

helix-likeloop

n-Srcloop

RTloop

helix-likeloop

n-Srcloop

Figure 5.1. Superposition of free Btk SH3 in black (pdb-id:1awx) and Btk SH3 structurefrom peptide complex in gray (1qly). From 1qly-ensemble, model no. 1 was used for the figure.1awx is an averaged-minimized structure of the 1aww-ensemble (I). The superposition was donewith MolMol [16] using the backbone heavy atoms from residue K218 to A272.


5.2 Structure of the SH3 domain in the PRR-SH3 fragment.

Previously, the structure of the SH3 domain from the Btk-related kinase Itk was determined using NMR (pdb-id: 1awj) [58]. In this report the protein fragment had an N-terminal extension containing the single SH3-binding proline-rich sequence present in the Itk TH region. This PRR-sequence bound intramolecularly to the SH3 domain forming the traditional PPII helix seen in many peptide-complexes. The difference between the structures of the Btk SH3 domain and this Itk SH3 domain was unexpectedly large, relative to the differences in the comparisons with Fyn and c-Src SH3 domains (I). In contrast to the situation in Itk SH3, where the N-terminal extension binds intramolecularly, we could detect a self-association involving the two PRR-sequences present in Btk (II & III; further discussed below), which indicated a dimerization rather than intramolecular binding. The β-structures of the SH3 domain itself seemed to be kept fairly constant in the dimer compared to the free SH3 domain. Although the structure of the dimeric SH3 domain is not yet available, data from 1H-2H-exchange studies supports this assumption (V). Secondary structure elements of proteins, including antiparallel β-sheets, are stabilized by hydrogen bonds between carbonyl oxygens and amide protons. In β-sheets, these hydrogen bonds are between the strands. Exchanging protons (1H) for deuterons (2H) is one way of getting experimental evidence for hydrogen bonds. This method relies on the fact that hydrogen atoms involved in bonding exchange more slowly than other exchangeable hydrogen atoms. One way of finding potential donors (amides) of hydrogen bonds is to run multiple 2D 15N-HSQC experiments some time after dissolving in 2H2O a lyophilized 15N-enriched protein sample that has been produced in 1H2O. If slowly exchanging amides can be detected, these are involved as donors in hydrogen bonds. Doing this for PRR-SH3 revealed slowly exchanging protons of all amides that were found to be donors in hydrogen bonding in the structure of Btk SH3 (I & V). The conclusion was that dimerization did not seem to cause any large structural changes of the β-sheets of the SH3 domain.

Another reason to believe that the overall structure of the SH3 domain itself in the dimer is similar to the free SH3 domain is that there are usually only small differences between free SH3 and peptide-bound SH3 domains. A superposition of backbone heavy atoms for 56 residues of the NMR structures of unliganded and peptide-bound c-Src SH3 domains (pdb-id: 1srl and 1rlq) [20, 32] give a rmsd value of 1.16 Å. Corresponding superposition of crystal structures of unliganded and peptide


bound Abl SH3 from Abelson´s protein tyrosine kinase (pdb-id: 1abq and 1abo) [107] gives a rmsd value of 0.95 Å. The same feature has also been reported in a structural and thermodynamic characterization of the interaction of a peptide from p85α-subunit with Fyn SH3 domain [21]. The peptide in this study was shown to be unstructured by itself in solution, as judged from CD-measurements, and while the structure of the Fyn SH3 domain was unchanged, the peptide folded to a PPII helix upon binding. Unfavorable entropy of binding was compensated by a favorable enthalpy of binding, which possibly is derived from burying hydrophobic surfaces.

Taking into account that the structure of Btk SH3 is relatively unchanged by the peptide interaction with p120cbl (see above) and that the hydrogen-bond network of the β-strands seems to be preserved in PRR-SH3, the overall structure of the SH3 domain is likely to be preserved also in PRR-SH3.

5.3 Dimerization of Btk PRR-SH3

Initially we wanted to investigate if the SH3-binding motifs of Btk could bind intramolecularly to the SH3 domain in the same manner as had been reported for the related Itk kinase [58]. We found however that our PRR-SH3 construct self-associated. The chemical shifts of the PRR-SH3 fragment were clearly concentration dependent, indicating the self-association We were able to determine the dissociation constant of this presumed dimerization to 60 µM using analytical gel permeation chromatography (GPC). We could also show that the NMR line widths decreased by the introduction of a short peptide corresponding to the PRRN-sequence (see Figure 2.7 for notation and sequence) indicating the dissociation of a complex (II). Specific NOEs between 13C15N-enriched PRR-SH3 and unlabeled PRR-SH3 in 13C-edited, 13C15N-filtered NOESY experiments further supported self-association involving the PRR-motifs of the TH-region. Furthermore, the introduction of the PRRN-peptide to the free SH3 domain gave the same chemical shift perturbations as was observed in a chemical shift comparison between the PRR-SH3 fragment and the free SH3 domain, which indicate that dimerization occurs via binding of the PRR to the SH3 domain. In this study we could not separate the relative roles of the two different SH3 binding motifs.

To investigate their relative roles, we selectively replaced the prolines with alanines in either one or both PRRs to inhibit the SH3-binding. The results indicated that both


PRRs, denoted PRRN and PRRC, can be involved in the dimerization, and that the interaction is somewhat more dependent on PRRN than on PRRC. Furthermore, substitution of only two prolines in each PRR was not sufficient for a total elimination of the dimerization (Table 5.1). Studies on SH3 domain interactions with peptides (e.g. [51]) have shown that class I sequences interact in such a way that the residues in position P0 and P+3 (Figure 5.2) must be prolines. One would expect class I interaction of the PRRs in Btk because both PRRN and PRRC sequences agree with the consensus sequence of class I peptides, and furthermore because class I orientation has also been shown for the intramolecular interaction of Itk SH3, in which PRR has exactly the same sequence as PRRN in Btk [58]. The corresponding residues in P0 position are P189 (PRRN) or P203 (PRRC) and in P+3 position P192 (PRRN) or P206 (PRRC), respectively. This would place K186 or K200 in the P-3 position, which is needed for a salt bridge to D232 in the SH3 domain. Both PRRs in Btk contain four prolines, hence forming two PxxP motifs. Substitution of the two prolines that are supposed to be in P0 and P+3 positions actually preserve one PxxP motif. When these two prolines were replaced with alanines in both PRRs, there was still a tendency towards dimerization, as measured by analytical GPC. One explanation to this could be that the remaining prolines of the interacting PRR, instead of being in P-2 and P+1 positions have shifted to occupy the P0 and P+3 positions, and that

Y225

Y268

W251

D232

Y223A B

Figure 5.2. A. Schematic representation of the positions of the PRR peptide backbone in thePRR - Btk SH3 interaction discussed in the text. The orientation of the backbone is indicatedwith an arrow. Sidechains on the SH3 domain binding surface are indicated with the Btknumbering. B. PRR binding site on the SH3 domain with binding surface residues shown.


possibly K185 or K199 could occupy a P-3 position. However, this would give only one residue, instead of two, between the P-3 and P0 positions and the sequence of the quadruple substituted PRR-SH3 (PRRN2C2) does not agree with the consensus sequence of class I peptides (Table 5.1).

Another explanation to the behavior of PRRN2C2 could be that the structure at the peptide-SH3 interface is essentially the same as in PRR-SH3 and not affected much by the substitutions, such that the complex can be preserved despite alanines in positions P0 and P+3. However, there are counter-arguments also for this explanation. First, the fact that class I peptides have conserved prolines at these positions [32-34] argues against it. Secondly, Nguyen and coworkers [32-34] have shown that an essential feature of the conserved proline residues at the P-2 and P+1 positions in class II orientation is the N-substitution, and since prolines are the only naturally occurring N-substituted amino acid, prolines are crucial in these positions (see also section 2.2).

Name in (III) Sequence of PRRN Sequence of PRRC

PRR-SH3 K P L P P T P K P L P P E P

PRRN2 K P L A P T A K P L P P E P

PRRN4 K A L A A T A V K P L P P E P

PRRC2 K P L P P T P K P L A P E A

PRRC4 K P L P P T P K A L A A E A

PRRN2C2 K P L A P T A K P L A P E A

PRRN4C4 K A L A A T A V K A L A A E A

Positions -3-2-1 0+1+2+3 -3-2-1 0+1+2+3

Class I consensus

K p x P p x P K p x P p x P

PRRN2C2 shifted in positions

K _ K P L A P T K _ K P L A P E

Table 5.1. The different PRR-SH3 constructs (used in Main reference III) and the relativepositions (according to Yu et al. [34]) of the residues if a binding in a class I orientation ispresent. Replaced/mutated residues are underscored. For PRRN2C2 a PxxP is still present, if theresidues are shifted in position.


The same principle would then be expected to apply also for class I peptides, although at the P0 and P+3 positions.

Each of the PRR-sequences appears to be able to individually contribute to dimerization, at least when the SH3-binding of the other PRR-sequence is destroyed. Whether they both can contribute to dimerization independently of each other also in PRR-SH3 is still an open question. There seems to be evidence for this in the appearance of resonances of the N-terminal extension containing the two PRR-sequences (IV). For two residues between the PRRs, there exist two different states with different chemical shifts assigned for each. One possible explanation could be that one of these states represents dimerization through PRRN and the other dimerization through PRRC. Which state corresponds to dimerization through PRRN and which through PRRC has not been determined.

In addition, the NOEs between the monomers indicate that both PRRN and PRRC participate. NOEs from the binding site on the SH3 domain to two residues in the TH-region have been assigned (V). One is to a threonine, most likely within the PRRN. If PRRN interacts in the assumed class I way, T190 would be in the P+2 position (Table 5.1). The P+2 position would put the sidechain in a location in the binding pocket so that NOEs could arise to the sidechain of both Y268 and Y223 of which NOEs to Y223 actually can be safely assigned (V). The other residue in the TH-region with assigned NOEs to the SH3 domain is I197. The location of I197 is C-terminal to PRRN (Figure 2.7), and the NOEs are between one of the methyl groups on this residue and both sidechains of Y263 and W251. If PRRN binds, these NOEs would not be possible because the I197 and Y263/W251 would be on different sides of the molecule. Consequently, these NOEs provide indirect evidence for an interaction involving PRRC.

5.4 Dimerization of Bruton´s tyrosine kinase, biological relevance

Various roles have been suggested for the Btk SH3 domain in the regulation of Bruton’s tyrosine kinase activity. We speculated previously (I) that the Btk SH3 domain, presumably together with the SH2 domain, interacts intramolecularly in a manner similar to what has been reported for Src family PTKs. This suggestion can be questioned, since Tec family PTKs lack the C-terminal phosphorylation site, which is conserved in Src family PTKs and which interacts with the SH2 domain. A


fundamental difference between these two PTK families seems to be that Src family PTKs are phosphorylated in the inactive state while Tec family PTKs are not. The recent crystal structure of the kinase domain from Btk in an inactive state indicates that this kinase domain can be held inactive, without any interaction from other domains [68]. This has also been shown in in vitro studies of the kinase activity[108].

One suggestion for the in vivo role of the Btk TH region and SH3 domain is that they interact to regulate and suppress the kinase activity (the same role that has been suggested for Itk TH and SH3) [109]. Other suggested roles for the TH region and SH3 domain are as a binding motif for the regulation by Src family kinases (TH region), as a motif for nucleocytoplasmic shuttling (SH3 domain) and as a binding motif for both Btk regulating factors and for downstream targets of Btk activity (SH3 domain) [54, 62, 64, 72, 74, 110]. Our data suggest a dimerization of Btk involving the TH region and SH3 domain. Although there is no direct evidence for the existence of a Btk dimer, neither in vivo nor in vitro, there is reported dimerization behavior also for the PH-domain of Btk [45]. Thus, there might exist two different dimerization surfaces in the full-length Btk, which then probably would decrease the dissociation constant (Figure 5.3).

Dimerization involving poly-proline motifs and SH3 domains has also been reported both in vitro and in vivo for the p85α-subunit of phosphatidylinositol-3-kinase [111]. In the dimeric state of the p85α-subunit there is also an intermolecular interaction reported between the Rho-GAP domains, in the report referred to as B-cell receptor homology domains. Hence, dimerization via SH3 domains and proline-rich motifs is not necessarily an isolated phenomenon to our PRR-SH3 fragment, but seems to exist in vivo in similar contexts. Neither is the idea of dimerization of a protein tyrosine kinase extraordinary. Many receptor protein tyrosine kinases are active as dimers; among these is the PDGF-receptor, which dimerizes upon activation. One step of this activation is a trans-autophosphorylation of tyrosines [112], i.e. phosphorylation catalyzed by the active site on another protein molecule rather than by the active site on the same molecule (cis-autophosphorylation).

As mentioned previously, Y223 within Btk SH3 is autophosphorylated upon activation of Btk by Src family kinases. Although it is suspected to be a regulatory phosphorylation, because one would expect that a phosphorylation would change the ligand-binding properties for potential intra- or intermolecular ligands, the exact role of the Y223-phosphorylation is not known. For the interaction with WASP, it has


actually been shown that phosphorylation of the SH3 domain gives altered ligand binding properties [72]. Recombinant Btk SH3 can be recognized and phospohorylated in vitro by full-length Btk and our results suggest that one way this recognition could be accomplished is by binding of the SH3 domain to the proline-rich motifs of the full-length kinase.

Y551, located in the activation loop of Btk kinase domain, is the target residue of Src family PTKs and Syk kinase, and phosphoryl-ation of this residue is the activating signal in Tec family kinases [55, 56, 67]. Phosphoryl-ation of Y551 has also been proposed to be performed by Btk itself [108]. A recent study of the effect of the PH/TH-module of Btk kinase activity, show that Y551 is an autophosphorylation site. Interestingly, this tyrosine residue points away from the active site in the crystal structure and can be trans-phosphorylated but not cis-phosphorylated [68]. Thus, a dimerization could have a role in the activation of Bruton´s tyrosine kinase, in the way that a previously active Btk molecule can dimerize with an inactive Btk molecule, thereby spreading the activation signal “horizontally” (Figure 5.3). If the dissociation constant we have determined for PRR-SH3 is in the same range as the dissociation constant for the full-length kinase, this dimerization could be of a transient nature. And if the autophosphorylation of Y223 decreases the affinity of this dimerization, this would further stimulate a separation of a Btk dimer.

SH3

SH2

kinase

PH

TH

?

Y551--P

phos-phoryl-ation

Y551

Y223SH3

SH2

kinase

PH

TH

?

Y551--P

phos-phoryl-ation

Y551

Y223

Figure 5.3 Schematic picture of how dimerizationof Btk might occur. An active Btk-moleculeassociates with an inactive Btk molecule, at whichpoint the active kinase domain can phosphorylateboth Y551 and Y223 on the inactive Btk molecule.

Conclusions 37

6 Conclusions By the use of NMR methods and by combining NMR with other techniques, we have obtained structural and functional information on the molecular level regarding the SH3 domain of Bruton´s tyrosine kinase. Hopefully these results will contribute to an increased understanding of the role of the SH3 domain and the activation mechanism of Btk and related proteins. In addition to the structure determination of the SH3 domain from Btk we focused on intermolecular interactions involving the TH region and the SH3 domain of Btk. Our observations indicated a protein dimerization, of which the characteristics were studied and the interaction surface was localized. The main results and interpretations are summarized below.

Main reference I The solution structure of the SH3 domain from Btk was determined using NMR methods. The average atomic rmsd of the 42 ensemble structures of Btk SH3 was 0.55 Å when calculated for the heavy backbone atoms of structured residues. The structure is a classical SH3 domain. Comparisons with other SH3 domains revealed a peptide-binding surface and also that the SH3 domain from Src family kinase Fyn is the closest structural relative with an rmsd value of 1.39 Å calculated for the heavy backbone atoms of 54 homologous residues (the sequence identity is 47% ). The structure of the SH3 domain from Tec family kinase Itk had in the same comparison an rmsd value of 2.54 Å (46% sequence identity).

Main reference II The self-association behavior of PRR-SH3, an N-terminal extended SH3 domain containing the two SH3-binding sequences of the TH-region of Btk, was studied using NMR spectroscopy and analytical gel permeation chromatography. Both concentration-dependence and peptide-coupled changes of the NMR spectra of PRR-SH3 and SH3 indicated a dimerization involving the SH3-binding sequences of the PRR. The dissociation constant of the monomer-dimer equilibrium was estimated to 60 µM.

Main reference III NMR studies and analytical gel permeation chromatography on variants of PRR-SH3 with crucial prolines in the PRRN and PRRC replaced by alanines showed that the


dimerization occurs due to an intermolecular interaction between the SH3 domain and the PRR-sequences and that both PRRs could act to stabilize dimerization. Replacements of prolines by alanines within PRRN decreased the dimerization more than replacements within PRRC.

Main reference IV More than 96% of the backbone Cα, CO, NH and HN NMR chemical shifts were assigned for non-proline residues in the Lys183 to Ser275 fragment of the PRR-SH3 dimer.

Main reference V The presence of intermolecular NOEs together with the results from an investigation of backbone dynamics by steady-state heteronuclear NOE measurements of PRR-SH3 in its dimerized state provided direct evidence for a normal SH3-PPII recognition between the two molecules of a dimer. The data indicated a multi-conformational and dynamic mode of intermolecular interactions within PRR-SH3.

Activation of Bruton´s tyrosine kinase involves autophosphorylation of tyrosine

residues. The studied intermolecular interaction provide a possible roles for the proline-rich region of TH region and the SH3 domain of Bruton´s tyrosine kinase as dimerization motifs. The implicated in vivo role of a dimerization could be to let autophosphorylation occur trans (between two molecules) rather than cis (within one molecule) and that this might be part of the regulation of both kinase activity and Btk interaction with other Btk molecules or with external ligands.

References 39

7 References 1 Blume-Jensen, P. and Hunter, T. (2001) Oncogenic kinase signalling. Nature

411, 355-365. 2 Robinson, D. R., Wu, Y. M. and Lin, S. F. (2000) The protein tyrosine kinase

family of the human genome. Oncogene 19, 5548-5557. 3 Bishop, J. M. (1985) Viral oncogenes. Cell 42, 23-38. 4 Bjorge, J. D., O'Connor, T. J. and Fujita, D. J. (1996) Activation of human

pp60c-src. Biochem. Cell. Biol. 74, 477-484. 5 Xu, W., Harrison, S. C. and Eck, M. J. (1997) Three-dimensional structure of

the tyrosine kinase of c-Src. Nature 385, 595-602. 6 Sicheri, F., Moarefi, I. and Kuriyan, J. (1997) Crystal structure of the Src

family tyrosine kinase Hck. Nature 385, 602-609. 7 Lehto, V. P., Wasenius, V. M., Salven, P. and Saraste, M. (1988) Transforming

and membrane proteins. Nature 334, 388. 8 Mayer, B. J., Hamaguchi, M. and Hanafusa, H. (1988) A novel viral oncogene

with structural similarity to phospholipase C. Nature 332, 272-275. 9 Stahl, M. L., Ferenz, C. R., Kelleher, K. L., Kriz, R. W. and Knopf, J. L.

(1988) Sequence similarity of phospholipase C with the non-catalytic region of Src. Nature 332, 269-272.

10 Liu, X., Brodeur, S. R., Gish, G., Songyang, Z., Cantley, L. C., Laudano, A. P. and Pawson, T. (1993) Regulation of c-Src tyrosine kinase activity by the Src SH2 domain. Oncogene 8, 1119-1126.

11 Okada, M., Howell, B. W., Broome, M. A. and Cooper, J. A. (1993) Deletion of the SH3 domain of Src interferes with regulation by the phosphorylated carboxyl-terminal tyrosine. J. Biol. Chem. 268, 18070-18075.

12 Moarefi, I., LaFevre-Bernt, M., Sicheri, F., Huse, M., Lee, C.-H., Kuriyan, J. and Miller, W. T. (1997) Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature 385, 650-653.

13 Yamaguchi, H. and Hendrickson, W. A. (1996) Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation. Nature 384, 484-489.

14 Sicheri, F. and Kuriyan, J. (1997) Structures of Src-family tyrosine kinases. Curr. Opin. Struct. Biol. 7, 777-785.


15 Williams, J. C., Wierenga, R. K. and Saraste, M. (1998) Insights into Src kinase functions: structural comparisons. Trends Biochem. Sci. 23, 179-184.

16 Koradi, R., Billeter, M. and Wüthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51-55, 29-32.

17 Young, M. A., Gonfloni, S., Superti-Furga, G., Roux, B. and Kuriyan, J. (2001) Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation. Cell 105, 115-126.

18 Laity, J. H., Dyson, H. J. and Wright, P. E. (2000) DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers. J. Mol. Biol. 295, 719-727.

19 Musacchio, A., Noble, M., Pauptit, R., Wierenga, R. and Saraste, M. (1992) Crystal structure of a Src-homology 3 (SH3) domain. Nature 359, 851-855.

20 Yu, H., Rosen, M. K., Shin, T. B., Seidel-Dugan, C., Brugge, J. S. and Schreiber, S. L. (1992) Solution structure of the SH3 domain of Src and identification of its ligand-binding site. Science 258, 1665-1668.

21 Renzoni, D. A., Pugh, D. J., Siligardi, G., Das, P., Morton, C. J., Rossi, C., Waterfield, M. D., Campbell, I. D. and Ladbury, J. E. (1996) Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase. Biochemistry 35, 15646-15653.

22 Maxwell, K. L. and Davidson, A. R. (1998) Mutagenesis of a Buried Polar Interaction in an SH3 domain: Sequence Conservation Provides the Best Prediction of Stability Effects. Biochemistry 37, 16172-16182.

23 Viguera, A. R., Jiménez, M. A., Rico, M. and Serrano, L. (1996) Conformational Analysis of Peptides Corresponding to beta-Hairpins and a beta-Sheet that Represent the Entire Sequence of the alpha-Spectrin SH3 Domain. J. Mol. Biol. 255, 507-521.

24 Grantcharova, V. P. and Baker, D. (1997) Folding Dynamics of the Src SH3 Domain. Biochemistry 36, 15685-15692.

25 Plaxco, K. W., Guijarro, J. I., Morton, C. J., Pitkeathly, M., Campbell, I. D. and Dobson, C. M. (1997) The Folding Kinetics and Thermodynamics of the Fyn-SH3 Domain. Biochemistry 37, 2529-2537.

References 41

26 Arold, S., O’Brien, R., Franken, P., Strub, M.-P., Hoh, F., Dumas, C. and Ladbury, J. E. (1998) RT Loop Flexibility Enhances the Specificity of Src Family SH3 Domains for HIV-1 Nef. Biochemistry 37, 14683-14691.

27 Pisabarro, M. T., Serrano, L. and Wilmanns, M. (1998) Crystal structure of the abl-SH3 domain complexed with a designed high-affinity peptide ligand: implications for SH3-ligand interactions. J. Mol. Biol. 281, 513-521.

28 Baumann, H., Knapp, S., Lundbäck, T., Ladenstein, R. and Härd, T. (1994) Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus. Nat. Struct. Biol. 1, 808-819.

29 Eijkelenboom, A. P., Lutzke, R. A., Boelens, R., Plasterk, R. H., Kaptein, R. and Hård, K. (1995) The DNA-binding domain of HIV-1 integrase has an SH3-like fold. Nat. Struct. Biol. 2, 807-810.

30 Falzone, C. J., Kao, Y. H., Zhao, J., Bryant, D. A. and Lecomte, J. T. (1994) Three-dimensional solution structure of PsaE from the cyanobacterium Synechococcus sp. strain PCC 7002, a photosystem I protein that shows structural homology with SH3 domains. Biochemistry 33, 6052-6062.

31 Stapley, B. J. and Creamer, T. P. (1999) A survey of left-handed polyproline II helices. Protein Sci. 8, 587-595.

32 Feng, S., Chen, J. K., Yu, H., Simon, J. A. and Schreiber, S. L. (1994) Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science 266, 1241-1247.

33 Lim, W. A., Richards, F. M. and Fox, R. O. (1994) Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains [published erratum appeared in Nature 1995 Mar 2;374(6517):94]. Nature 372, 375-379.

34 Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W. and Schreiber, S. L. (1994) Structural basis for the binding of proline-rich peptides to SH3 domains. Cell 76, 933-945.

35 Nguyen, J. T., Turck, C. W., Cohen, F. E., Zuckermann, R. N. and Lim, W. A. (1998) Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors. Science 282, 2088-2092.

36 Bruton, O. C. (1952) Agammaglobulinemia. Pediatrics 9, 722-727. 37 Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F.,

Hammarström, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C. I. E. and


Bentley, D. R. (1993) The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases [published erratum appeared in Nature 1993 Jul 22;364(6435):362]. Nature 361, 226-233.

38 Vihinen, M. and Smith, C. I. E. (2001) BTKbase: Mutation registry for X-linked agammaglobulinemia. http://www.uta.fi/laitokset/imt/bioinfo/BTKbase/

39 Anderson, J. S., Teutsch, M., Dong, Z. and Wortis, H. H. (1996) An essential role for tyrosine kinase in the regulation of Bruton's B-cell apoptosis. Proc. Natl. Acad. Sci. U.S.A. 93, 10966-10971.

40 Sideras, P. and Smith, C. I. E. (1995) Molecular and cellular aspects of X-linked agammaglobulinemia. Adv. Immunol. 59, 135-223.

41 Smith, C. I. E., Islam, T. C., Mattsson, P. T., Mohamed, A. J., Nore, B. F. and Vihinen, M. (2001) The Tec family of cytoplasmic tyrosine kinases: mammalian Bmx, Btk, Itk, Tec, Txk and homologs in other species. Bioessays 23, 436-446.

42 Vihinen, M., Nilsson, L. and Smith, C. I. E. (1994) Tec homology (TH) adjacent to the PH domain. FEBS Letters 350, 263-265.

43 Vihinen, M., Nore, B. F., Mattsson, P. T., Bäckesjö, C. M., Nars, M., Koutaniemi, S., Watanabe, C., Lester, T., Jones, A., Ochs, H. D. and Smith, C. I. E. (1997) Missense mutations affecting a conserved cysteine pair in the TH domain of Btk. FEBS Letters 413, 205-210.

44 Hyvönen, M. and Saraste, M. (1997) Structure of the PH domain and Btk motif from Bruton's tyrosine kinase: molecular explanations for X-linked agammaglobulinaemia. Embo J. 16, 3396-404.

45 Baraldi, E., Carugo, K. D., Hyvönen, M., Surdo, P. L., Riley, A. M., Potter, B. V., O'Brien, R., Ladbury, J. E. and Saraste, M. (1999) Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate. Structure 7, 449-460.

46 Maekawa, M., Li, S., Iwamatsu, A., Morishita, T., Yokota, K., Imai, Y., Kohsaka, S., Nakamura, S. and Hattori, S. (1994) A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions. Mol. Cell. Biol. 14, 6879-6885.

47 Tsukada, S., Simon, M. I., Witte, O. N. and Katz, A. (1994) Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase. Proc. Natl. Acad. Sci. U.S.A. 91, 11256-11260.

References 43

48 Ma, Y. C. and Huang, X. Y. (1998) Identification of the binding site for Gqalpha on its effector Bruton's tyrosine kinase. Proc. Natl. Acad. Sci. U.S.A. 95, 12197-12201.

49 Jiang, Y., Ma, W., Wan, Y., Kozasa, T., Hattori, S. and Huang, X. Y. (1998) The G protein G alpha12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain. Nature 395, 808-813.

50 Li, T., Tsukada, S., Satterthwaite, A., Havlik, M. H., Park, H., Takatsu, K. and Witte, O. N. (1995) Activation of Bruton's tyrosine kinase (BTK) by a point mutation in its pleckstrin homology (PH) domain. Immunity 2, 451-460.

51 Li, T., Rawlings, D. J., Park, H., Kato, R. M., Witte, O. N. and Satterthwaite, A. B. (1997) Constitutive membrane association potentiates activation of Bruton tyrosine kinase. Oncogene 15, 1375-1383.

52 Saito, K., Scharenberg, A. M. and Kinet, J. P. (2001) Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk. J. Biol. Chem. 276, 16201-16206.

53 Alexandropoulos, K., Cheng, G. and Baltimore, D. (1995) Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc. Natl. Acad. Sci. U.S.A. 92, 3110-3114.

54 Cheng, G., Ye, Z.-S. and Baltimore, D. (1994) Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction. Proc. Natl. Acad. Sci. U.S.A. 91, 8152-8155.

55 Afar, D. E., Park, H., Howell, B. W., Rawlings, D. J., Cooper, J. and Witte, O. N. (1996) Regulation of Btk by Src family tyrosine kinases. Mol. Cell. Biol. 16, 3465-3471.

56 Rawlings, D. J., Scharenberg, A. M., Park, H., Wahl, M. I., Lin, S., Kato, R. M., Fluckiger, A.-C., Witte, O. N. and Kinet, J.-P. (1996) Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science 271, 822-825.

57 Patel, H. V., Tzeng, S. R., Liao, C. Y., Chen, S. H. and Cheng, J. W. (1997) SH3 domain of Bruton's tyrosine kinase can bind to proline-rich peptides of TH domain of the kinase and p120cbl. Proteins 29, 545-552.

58 Andreotti, A. H., Bunnell, S. C., Feng, S., Berg, L. J. and Schreiber, S. L. (1997) Regulatory intramolecular association in a tyrosine kinase of the Tec family. Nature 385, 93-97.


59 Zhu, Q., Zhang, M., Rawlings, D. J., Vihinen, M., Hagemann, T., Saffran, D. C., Kwan, S. P., Nilsson, L., Smith, C. I. E., Witte, O. N. and et al. (1994) Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA). J. Exp. Med. 180, 461-470.

60 Cory, G. O., Lovering, R. C., Hinshelwood, S., MacCarthy-Morrogh, L., Levinsky, R. J. and Kinnon, C. (1995) The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase. J. Exp. Med. 182, 611-615.

61 Tzeng, S. R., Lou, Y. C., Pai, M. T., Jain, M. L. and Cheng, J. W. (2000) Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl. J. Biomol. NMR 16, 303-312.

62 Cory, G. O., MacCarthy-Morrogh, L., Banin, S., Gout, I., Brickell, P. M., Levinsky, R. J., Kinnon, C. and Lovering, R. C. (1996) Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways. J. Immunol. 157, 3791-3795.

63 Matsushita, M., Yamadori, T., Kato, S., Takemoto, Y., Inazawa, J., Baba, Y., Hashimoto, S., Sekine, S., Arai, S., Kunikata, T., Kurimoto, M., Kishimoto, T. and Tsukada, S. (1998) Identification and characterization of a novel SH3-domain binding protein, Sab, which preferentially associates with Bruton's tyrosine kinase (BtK). Biochem. Biophys. Res. Commun. 245, 337-343.

64 Yamadori, T., Baba, Y., Matsushita, M., Hashimoto, S., Kurosaki, M., Kurosaki, T., Kishimoto, T. and Tsukada, S. (1999) Bruton's tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein. Proc. Natl. Acad. Sci. U.S.A. 96, 6341-6346.

65 Brazin, K. N., Fulton, D. B. and Andreotti, A. H. (2000) A specific intermolecular association between the regulatory domains of a Tec family kinase. J. Mol. Biol. 302, 607-623.

66 Hashimoto, S., Iwamatsu, A., Ishiai, M., Okawa, K., Yamadori, T., Matsushita, M., Baba, Y., Kishimoto, T., Kurosaki, T. and Tsukada, S. (1999) Identification of the SH2 domain binding protein of Bruton's tyrosine kinase as BLNK--functional significance of Btk-SH2 domain in B-cell antigen receptor-coupled calcium signaling. Blood 94, 2357-64.

References 45

67 Baba, Y., Hashimoto, S., Matsushita, M., Watanabe, D., Kishimoto, T., Kurosaki, T. and Tsukada, S. (2001) BLNK mediates Syk-dependent Btk activation. Proc. Natl. Acad. Sci. U.S.A. 98, 2582-2586.

68 Mao, C., Zhou, M. and Uckun, F. M. (2001) Crystal structure of Bruton's tyrosine kinase domain suggests a novel pathway for activation and provides insights into the molecular basis of X-linked agammaglobulinaemia (XLA). J. Biol. Chem. 276, 41435-41443.

69 Heyeck, S. D., Wilcox, H. M., Bunnell, S. C. and Berg, L. J. (1997) Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity. J. Biol. Chem. 272, 25401-25408.

70 Park, H., Wahl, M. I., Afar, D. E. H., Turck, C. W., Rawlings, D. J., Tam, C., Scharenberg, A. M., Kinet, J.-P. and Witte, O. N. (1996) Regulation of Btk function by a major autophosphorylation site within the SH3 domain. Immunity 4, 515-525.

71 Mahajan, S., Vassilev, A., Sun, N., Ozer, Z., Mao, C. and Uckun, F. M. (2001) Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells. J. Biol. Chem. 276, 31216-31228.

72 Morrogh, L. M., Hinshelwood, S., Costello, P., Cory, G. O. and Kinnon, C. (1999) The SH3 domain of Bruton's tyrosine kinase displays altered ligand binding properties when auto-phosphorylated in vitro. Eur. J. Immunol. 29, 2269-2279.

73 Petro, J. B., Rahman, S. M., Ballard, D. W. and Khan, W. N. (2000) Bruton's tyrosine kinase is required for activation of IkappaB kinase and nuclear factor kappaB in response to B cell receptor engagement. J. Exp. Med. 191, 1745-1754.

74 Mohamed, A. J., Vargas, L., Nore, B. F., Bäckesjö, C. M., Christensson, B. and Smith, C. I. E. (2000) Nucleocytoplasmic shuttling of Bruton's tyrosine kinase. J. Biol. Chem. 275, 40614-40619.

75 Evans, J. N. S. (1995) Biomolecular NMR Spectroscopy. Oxford University Press, Oxford.

76 Cavanagh, J., Fairbrother, W. J., Palmer, A. G. and Skelton, N. J. (1996) Protein NMR spectroscopy: Principles and practice. Academic Press, London.


77 Vuister, G. W. and Bax, A. (1993) Quantitative J Correlation: A New Approach fo Measuring Homonuclear Three-Bond J(HN-Hα) Coupling Constants in 15N-Enriched Proteins. J. Am. Chem. Soc. 115, 7772-7777.

78 Bax, A., Vuister, G. W., Grzesiek, S., Delaglio, F., Wang, A. C., Tschudin, R. and Zhu, G. (1994) Measurement of homo- and heteronuclear J couplings from quantitative J correlation. Methods Enzymol. 239, 79-105.

79 Kay, L. E., Keifer, P. and Saarinen, T. (1992) Pure Absorption Gradient Enhanced Heteronuclear Single Qantum Correlation Spectroscopy with Improved Sensitivity. J. Am. Chem. Soc. 114, 10663-10665.

80 Marion, D., Driscoll, P. C., Kay, L. E., Wingfield, P. T., Bax, A., Gronenborn, A. M. and Clore, G. M. (1989) Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser- multiple quantum coherence spectroscopy: application to interleukin 1 beta. Biochemistry 28, 6150-6156.

81 Ikura, M., Kay, L. E. and Bax, A. (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29, 4659-4667.

82 Macura, S. and Ernst, R. R. (1980) Elucidation of cross-relaxation in liquids by two-dimensional N.M.R. spectroscopy. Mol. Phys. 41, 95-117.

83 Vuister, G. W., Kim, S.-J., Wu, C. and Bax, A. (1994) 2D and 3D study of phenylalanine residues in proteins by reverse isotopic labeling. J. Am. Chem. Soc. 116, 9206-9210.

84 Kay, L. E., Torchia, D. A. and Bax, A. (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972-8979.

85 Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D. and Kay, L. E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984-6003.

86 Lipari, G. and Szabo, A. (1982) Model-Free Approach to the Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 1. Theory and Range Validity. J. Am. Chem. Soc. 104, 4546-4559.

References 47

87 Lipari, G. and Szabo, A. (1982) Model-Free Approach to the Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 2. Analysis of Experimental Results. J. Am. Chem. Soc. 104, 4559-4570.

88 Wikström, A., Berglund, H., Hambraeus, C., van den Berg, S. and Härd, T. (1999) Conformational dynamics and molecular recognition: backbone dynamics of the estrogen receptor DNA-binding domain. J. Mol. Biol. 289, 963-979.

89 Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York.

90 Sattler, M., Schleucher, J. and Griesinger, C. (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solutions employing pulsed field gradients. Prog. Nucl. Magn. Res. Spec. 34, 93-158.

91 Güntert, P. (1998) Structure calculation of biological macromolecules from NMR data. Q. Rev. Biophys. 31, 145-237.

92 Pervushin, K., Riek, R., Wider, G. and Wüthrich, K. (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl. Acad. Sci. U.S.A. 94, 12366-12371.

93 Salzmann, M., Pervushin, K., Wider, G., Senn, H. and Wüthrich, K. (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl. Acad. Sci. U.S.A. 95, 13585-13590.

94 Rosen, M. K., Gardner, K. H., Willis, R. C., Parris, W. E., Pawson, T. and Kay, L. E. (1996) Selective methyl group protonation of perdeuterated proteins. J. Mol. Biol. 263, 627-636.

95 Tjandra, N. and Bax, A. (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111-1114.

96 Reif, B., Hennig, M. and Griesinger, C. (1997) Direct measurement of angles between bond vectors in high-resolution NMR. Science 276, 1230-1233.

97 Jenkins, T. M., Hickman, A. B., Dyda, F., Ghirlando, R., Davies, D. R. and Craigie, R. (1995) Catalytic domain of human immunodeficiency virus type 1


integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc. Natl. Acad. Sci. U.S.A. 92, 6057-6061.

98 Knapp, S., Mattson, P. T., Christova, P., Berndt, K. D., Karshikoff, A., Vihinen, M., Smith, C. I. E. and Ladenstein, R. (1998) Thermal unfolding of small proteins with SH3 domain folding pattern. Proteins 31, 309-319.

99 Baumann, H., Knapp, S., Karshikoff, A., Ladenstein, R. and Härd, T. (1995) DNA-binding surface of the Sso7d protein from Sulfolobus solfataricus. J. Mol. Biol. 247, 840-846.

100 Foster, M. P., Wuttke, D. S., Clemens, K. R., Jahnke, W., Radhakrishnan, I., Tennant, L., Reymond, M., Chung, J. and Wright, P. E. (1998) Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA. J. Biomol. NMR 12, 51-71.

101 Shuker, S. B., Hajduk, P. J., Meadows, R. P. and Fesik, S. W. (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531-1534.

102 Wolf-Watz, M., Bäckström, S., Grundström, T., Sauer, U. and Härd, T. (2001) Chloride binding by the AML1/Runx1 transcription factor studied by NMR. FEBS Letters 488, 81-84.

103 Miller, R. W., Eady, R. R., Gormal, C., Fairhurst, S. A. and Smith, B. E. (1998) Nucleotide binding by the nitrogenase Fe protein: a 31P NMR study of ADP and ATP interactions with the Fe protein of Klebsiella pneumoniae. Biochem. J. 334, 601-607.

104 Takahashi, H., Nakanishi, T., Kami, K., Arata, Y. and Shimada, I. (2000) A novel NMR method for determining the interfaces of large protein- protein complexes. Nat. Struct. Biol. 7, 220-223.

105 Czok, M. and Guichon, G. (1990) The physical sense of simulation models of liquid chromatography: propagation through a grid or solution of the mass balance equation? Anal. Chem. 62, 189-200.

106 Feng, S., Kasahara, C., Rickles, R. J. and Schreiber, S. L. (1995) Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands. Proc. Natl. Acad. Sci. U.S.A. 92, 12408-12415.

References 49

107 Musacchio, A., Saraste, M. and Wilmanns, M. (1994) High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nat. Struct. Biol. 1, 546-551.

108 Lowry, W. E., Huang, J., Lei, M., Harrison, S. C., Rawlings, D. and Huang, X. Y. (2001) Role of the PH/TH Module in Protein Substrate Recognition by Bruton's Agammaglobulinemia tyrosine kinase. J. Biol. Chem. 276, 45276-45281.

109 Yang, W. C., Collette, Y., Nunes, J. A. and Olive, D. (2000) Tec kinases: a family with multiple roles in immunity. Immunity 12, 373-382.

110 Guinamard, R., Fougereau, M. and Seckinger, P. (1997) The SH3 domain of Bruton's tyrosine kinase interacts with Vav, Sam68 and EWS. Scand. J. Immunol. 45, 587-595.

111 Harpur, A. G., Layton, M. J., Das, P., Bottomley, M. J., Panayotou, G., Driscoll, P. C. and Waterfield, M. D. (1999) Intermolecular Interactions of the p85alpha Regulatory Subunit of Phosphatidylinositol 3-Kinase. J. Biol. Chem. 274, 12323-12332.

112 Heldin, C. H. and Westermark, B. (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev. 79, 1283-1316.

Structure and function of the SH3 domain from Bruton - DiVA Portal

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