University of Groningen

Structure and function of substrate-binding proteins of ABC-transportersBerntsson, Ronnie Per-Arne

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Chapter 3

Contribution of a hydrophobicpocket to peptide selection andpositioning in lactococcal OppA

Ronnie P-A Berntsson, Nienke Kuipers, Silvia Ekkerman, FabriziaFusetti, Andy-Mark WH Thunnissen, Bert Poolman and Dirk JanSlotboom

3.1 Abstract

OLIGOPEPTIDE-BINDING PROTEIN A (OppA) of Lactococcus lactis has anexceptionally broad substrate specificity, binding peptides from 4 toat least 35 residues in length with very little sequence preference. A

voluminous binding cavity and specific interaction (hydrogen bonds) restricted toa stretch of 5 backbone positions allows this promiscuity. We recently proposedthat OppA can bind its ligands in different registers, and that a single hydrophobicpocket determines the positioning of the peptide. This hydrophobic pocket alsoseemed responsible for the limited sequence preference, and may contribute to thefree energy of binding. To test these hypotheses, Phe493, located at the bottom

50 Chapter 3

of the pocket was replaced by Ala, Arg, Cys, Glu, Gln and Lys. Ligand bindingto wild-type and mutant OppA was studied with fluorescence titration, isothermaltitration calorimetry and differential scanning calorimetry, revealing that the pocketindeed plays a significant role in peptide binding and selectivity. OppA was alsoco-crystallized together with a newly designed peptide (SLSQSLSQS) in the closed-liganded conformation. The structure determined at 1.5 A resolution revealed thatthe nonapeptide was bound in a different register when compared to a previouslydetermined structure structure with bradykinin (RPPGFSPFR).

3.2 Introduction

Lactococcus lactis is a multiple amino acid auxotroph that requires for growth eitheramino acids, peptides or proteins as source of nitrogen (Reiter and Oram, 1962). Inmilk, L. lactis uses a cell wall-anchored protease, PrtP, to digest milk proteins (α-, β- and κ-caseins), and the degradation products are subsequently taken up andused as nutrients (Kunji et al., 1998). The oligopeptide permease, Opp, plays amajor role in this uptake in L. lactis (Kunji et al., 1995). Opp is an ABC-transporter,composed of five subunits; two nucleotide-binding proteins (OppD, OppF), twotransmembrane proteins (OppB, OppC), and a substrate-binding protein (OppA),which is anchored to the cell membrane via lipid modification of the N-terminalcysteine. OppA binds peptides and delivers them to the translocator by forminga transient complex with the OppBCDF complex. The quintuple complex is onlyformed when OppA is in its ligand-bound conformation (Doeven et al., 2008).

OppA from L. lactis binds peptides of very different length, from 4 up to 35amino residues long, with little sequence selectivity. Recently, we have determinedthe structural basis for peptide binding in OppA* (a derivative of OppA lackingthe signal sequence and lipid modification). A very voluminous binding cavitycan accommodate peptides of large size. Interactions between the protein andthe bound peptide are restricted to hydrogen bonds with the peptide backboneover a stretch of only five residues. On the basis of the structures of OppA* inits open-liganded and closed-liganded conformation, we proposed a new modelfor peptide binding (Berntsson et al., 2009). The main observations were: (i)the closed-liganded complex, obtained with a mixture of endogenous peptidesof variable length, showed a decrease in ligand occupancy towards the peptidetermini; (ii) the closed-liganded structure revealed that the termini of the bound

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 51

peptide (bradykinin) did not form salt-bridges with the protein, contrary to thesituation in homologous proteins; (iii) the density for the bound ligand lookedsimilar in four structures of open-liganded conformations obtained with differentsynthetic peptides; and (iv) in the structure of the closed-liganded conformationobtained with a mixture of peptides, only one side-chain could unambiguouslybe identified as a isoleucine; situated in the only well-defined side-chain pocketof OppA*. The model thus suggested that peptides can bind in different register,with the restriction that a hydrophobic side-chain is preferentially located in thishydrophobic side-chain pocket.

To further demonstrate that peptides can bind in different register, we crystallizedOppA* in the presence of a nonapeptide with a hydrophobic residue at position 6rather than 5 as in bradykinin. To substantiate the importance of the hydrophobicside-chain pocket, we replaced phenylalanine-493, a residue located at the bottomof this pocket, by alanine, arginine, cysteine, glutamine, glutamic acid and lysine.The mutants were studied with isothermal titration calorimetry (ITC), fluorescencetitrations and differential scanning calorimetry (DSC).

With the first structure of a substrate binding protein determined in 1974, thatof L-arabinose binding protein (Quiocho et al., 1974), and numerous more since,a thorough understanding of the structure and function relationship of SBDs isavailable (for a review, see Chapter 1). When it comes to the subclass of peptidebinding proteins, several proteins have been thoroughly characterized via differentpeptide binding and transport assays to find out their ligand specificities as well ascrystallized, an example being OppA from S. typhimurium (Tame et al., 1994, 1995;S. Sleigh et al., 1997; S. H. Sleigh et al., 1999). The peptide binding proteins bindtheir ligands with hydrogen bonds to the peptide backbone, keeping the peptidetermini fixed with salt-bridges, and place the side-chains in large well- hydratedpockets. This serves as the basis for the well-documented promiscuity

3.3 Results

3.3.1 Structure of OppA* with bound SLSQSLSQS

To investigate whether the hydrophobic pocket in OppA determines the registerin which a peptide binds, four nonapeptides were designed, each with one out oftwo leucines at a different position: SLSLSQSQS, SLSQLSSQS, SLSQSLSQS, and

52 Chapter 3

SLSQSSLQS. The dissociation constant of OppA* for SLSQSLSQS was 4.2 ± 0.3µM as determined by intrinsic protein fluorescence titrations (data not shown),which compares to a value of 0.07 ± 0.02 µM for bradykinin (RPPGFSPFR). Thesepeptides were used in co-crystallization trials with OppA* (Berntsson et al., 2009).Co-crystallization was successful with one of the peptides (SLSQSLSQS) producingcrystals (space group P1) that diffracted to 1.5 A resolution. The structure wassolved by molecular replacement, using the previously solved structure of OppA*with bradykinin bound (PDB code: 3DRG) as a model. The structure of OppA*complexed with SLSQSLSQS was in the closed conformation and very similarto OppA* with bradykinin bound (r.m.s.d of 0.3 A). During refinement, residualelectron density appeared in the ligand-binding cavity, at a position similar tothat observed for bradykinin in the previously determined structure. The electrondensity could be modeled to SLSQSLSQS with the density of the entire peptidebackbone and all side-chains defined, although at the N-terminal end of the peptide(position 1-2) the electron density became weaker.

Superimposition of bradykinin (from 3DRG) and SLSQSLSQS revealed that the twopeptides were bound by OppA* in a different register. The backbone of residues 2-9 of SLSQSLSQS superimposed on the backbone residues 1-8 of bradykinin withan r.m.s.d. of 0.44 A. In fact, the side-chain of leucine at position six of SLSQSLSQSwas now located in the well-defined, hydrophobic side-chain pocket of OppA*; thispocket contained the side chain of phenylalanine at position 5 of bradykinin in thepreviously determined structure. The entire peptide had thus shifted register byone residue when compared to bradykinin (Fig. 3.1).

3.3.2 Engineering of the hydrophobic pocket in OppA*

To investigate the contribution of the hydrophobic pocket to peptide binding andto the stabilization of the closed state, a series of substitution mutants was madeby replacing Phe493 for more polar and/or charged or less bulky residues. WhenSLSQSLSQS is bound the side-chain of leucine at position 6 of the peptide fitssnugly in the hydrophobic pocket, just like the isoleucine side-chain in the structureof OppA* in complex with endogenous ligands (PDB code: 3DRF). With bradykinin(RPPGFSPFR) bound (PDB code: 3DRG), the Phe493 of OppA* adopted a differentconformation and had rotated away from the ligand to make room for the largerside chain of phenylalanine (5th residue of the peptide). In addition, the pocketharbors two water molecules in the structure with endogenous ligand and only

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 53

Figure 3.1. Peptide binding to OppA* in different register. A) Bradykinin (RPPGFSPFR) and B) SL(SLSQSLSQS). Ligand is visualized in gray with electron density (2Fo-Fc map) displayed as a gray meshat 1 σ. OppA* residues forming the only defined side-chain pocket visualized in orange.

54 Chapter 3

one in the structure with bound bradykinin. Due to the conformational flexibilityof the side-chain of Phe493 it was expected not be crucial for protein stability, andtherefore it was chosen for mutagenesis to change the hydrophobic character of thepocket. Mutants were made in which F493 was changed to an alanine, glutamine,glutamic acid, lysine, arginine and cysteine.

3.3.3 Peptide binding studies

Peptide binding to wild type and mutant OppA* was measured by fluorescencetitrations (using changes in intrinsic protein fluorescence upon peptide binding),isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC).Fluorescence titrations were performed with bradykinin (RPPGFSPFR) and a caseinderived peptide SLSQSKVLP. The dissociation constant of wild type OppA* forbradykinin was 0.07 ± 0.02 µM. Four mutants (F493Q, F493E, F493A and F493C)had lower affinities for bradykinin (KD values were 3 to 53 fold higher, Table 3.1).For the other two mutants (F493K and F493R), the change in fluorescence (∆Fmax)upon bradykinin binding was too small to accurately fit the data. Fluorescencetitrations were also used to determine binding of the SLSQSKVLP peptide, but onlywild-type OppA* produced sufficiently large fluorescence changes to accuratelydetermine the binding (KD = 2.9 ± 0.9 µM).

Using ITC, the affinities of wild-type OppA* and all 6 mutants for bradykinin weredetermined (Fig. 3.2 and Table 3.1). Within experimental error, the determinedKD values were the same as those determined by fluorescence titrations, but nowit was possible to also obtain values for F493K and F493R (KD of 13 ± 1.3 µMand 44 ± 3.3 µM, respectively). Next, ITC experiments were performed with theSLSQSKVLP peptide. The binding affinities of wild-type OppA* and the F493Q andF493E mutants for SLSQSKVLP were 3.5 ± 1.4 µM, 18 ± 1.7 µM and 27 ± 2.7 µM,respectively. For the other mutants the change in heat upon peptide binding wastoo small to estimate the KD. It is possible that charge interaction between F493Eand the lysine at position 6 in SLSQSKVLP increases the affinity of this mutantfor the peptide. In summary, all mutations at position 493 of OppA* resulted ina decreased affinity for the peptides; the introduction of a positive charge had thebiggest impact, with both the F493K and F493R mutants having a two-order ofmagnitude decreased affinity.

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 55

Figure 3.2. ITC measurements of bradykinin binding to wild-type OppA* and the mutants F493C,F493E, F493Q, F493K and F493R. The upper graphs show the heat released by the protein upon bindingof bradykinin. The number of binding sites (N) was 1 for all measured mutants (as expected) exceptfor F493C, which had N of 0.5 and this number was consistently observed in three independent sets ofmeasurements. Although we do not have direct evidence, the reduced number of binding sites could becaused by oxidation or irreversible modification of the cysteine in half of the molecules. The integratedpeaks were fitted to the one-site binding model (black line, lower graphs).

The enthalpic and entropic contributions to ∆G determined by ITC varied drasti-cally for the different mutants and could differ for mutants displaying the sameaffinity for the ligand. For example, the F493Q and F493A mutants had KD valuesfor bradykinin of around 1 µM, yielding ∆G values of -32.8 and -32.2 kJ/mol,respectively. The F493Q mutant had an almost negligible entropic contribution,while the F493A mutant had a large, unfavorable contribution of ∆S, similar to

56 Chapter 3

Table 3.1. Peptide binding data, obtained from the ITC measurements and fluorescence titrations.∆Fmax corresponds to the maximal fluorescence change, measured as intrinsic protein fluorescencechange, upon ligand binding. The error in the KD is the standard deviation based on a minimum oftwo independent measurements (for fluorescence titrations) or the error of the fit (ITC).

Peptide OppA KDFluorescence(µM)

∆Fmax(%)

KD ITC(µM)

∆H(kJ/mol)

T∆S(kJ/mol)

∆G(kJ/mol)

Bradykinin WT 0.07 ± 0.02 9.6 0.12 ± 0.02 -64.39 -24.94 -39.46F493C 0.21 ± 0.07 8.2 1.0 ± 0.1 -72.09 -37.90 -34.19F493Q 1.4 ± 0.8 11.8 1.3 ± 0.1 -33.19 -0.41 -32.78F493A 1.0 ± 0.2 7.6 2.2 ± 0.2 -60.42 -28.18 -32.24F493E 3.8 ± 1.2 2.5 2.3 ± 0.1 -70.37 -38.15 -32.22F493K - - 13 ± 1.3 -22.20 5.69 -27.89F493R - - 44 ± 3.3 -37.97 -13.09 -24.87

SLSQSKVLP WT 2.9 ± 0.9 10.9 3.5 ± 1.4 -23.1 8.1 -31.1F493Q - - 18 ± 1.7 -36.0 -8.9 -27.2F493E - - 27 ± 2.7 -24.9 -1.2 -23.8

wild-type OppA*. Similarly, while the F493K and F493R mutants both have alow affinity for bradykinin, with KD values of 13 and 44 µM, respectively, theentropic contribution to the binding energy was unfavorable for the F493R mutantbut favorable for the F493K mutant. The free energy of binding of the nonapetideSLSQSKVLP to wild-type OppA* had an enthalpic contribution of -23.1 kJ/mol andalso a favorable entropic contribution (T∆S was 8.1 kJ/mol). In contrast the F493Qand F493E mutants showed unfavorable entropic contributions to the free energyof SLSQSKVLP binding, although the magnitudes differed significantly. (Table 3.1).

The stability of OppA* wild-type and mutants was analyzed by differential scan-ning calorimetry (DSC). In the absence of ligand, wild-type OppA* and F493Chad the highest melting temperature (Tm) ( 44 ◦C) when compared to the othermutants (38-41 ◦C). Upon addition of bradykinin, wild-type OppA* displayed thehighest degree of stabilization, with a Tm of 52.2 ◦C. Unfolding of OppA* was notreversible, and the Tm was dependent on the rate of temperature increase in themeasurement. Therefore, the enthalpy and heat capacity of unfolding could not bedetermined, but melting temperatures, of wild-type and mutant OppA*, measuredat the same rate of temperature increase can be compared to each other. All mutantswere stabilized by the addition of bradykinin, albeit to a different degree and lessthan the wild-type protein (Table 3.2). There is a weak correlation (logarithmicdependence) between the Tm shift and the KD of the mutants for bradykinin. Thismeans that the hydrophobic pocket does not only play a role in ligand selectionand positioning, but it also contributes significantly to the stability of the closed-liganded state.

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 57

Table 3.2. Comparison of KD for bradykinin and melting temperatures in the absence and presence(50x the KD for WT, F493C, F493Q, F493A, F493E, 20x the KD for F493K, F493R) of bradykinin. The KDvalues were taken from Table 3.1.

OppA KD (µM) Tm without ligand (◦C) Tm with bradykinin (◦C) Tm shift (◦C)WT 0.07 ± 0.02 43.9 52.2 8.3F493C 0.21 ± 0.07 44.3 48.3 4.0F493Q 1.4 ± 0.8 38.3 43.3 5.0F493A 1.0 ± 0.2 39.9 43.3 3.4F493E 3.8 ± 1.2 38.2 40.5 2.3F493K 13 ± 1.3 40.2 40.6 0.4F493R 44 ± 3.3 40.9 42.0 1.1

3.3.4 Identification of endogenous ligands in OppA* mutants

When OppA* is expressed in the cytosol of L. lactis endogenous peptides bind tothe protein. The binding of at least a subset of these peptides is so strong thatthey co-purify with the protein, unless guanidinium-chloride treatment is used topartially (and reversibly) unfold the protein. Endogenous ligands co-purified withwild-type OppA* have previously been identified by mass spectrometry (Berntssonet al., 2009). We now determined the peptides that co-purified with the F493K andF493R mutants as well as with wild-type OppA*. In total 316 unique peptides wereidentified, corresponding to 89 unique cytosolic proteins; the peptides ranged insize from 5 to 21 residues (data not shown). When compared to the amino acid com-position of the L. lactis proteome, the endogenous ligands were slightly enriched inproline content and a large fraction of the peptides contained an isoleucine. Thesame trends have been observed for wild-type OppA* (Berntsson et al., 2009), andconfirm that proline-rich peptides with at least one isoleucine residue are preferredligands. Although wild-type OppA*, OppA*(F493K) and OppA*(F493R) differhighly in their binding affinity for bradykinin and SLSQSKVLP, these differenceare not reflected in the composition of the endogenous ligands that were co-purified(Fig. 3.3).

3.4 Discussion

Previously we hypothesized that OppA* from L.lactis can bind peptide ligands indifferent register so that a hydrophobic side chain within the peptide is located inthe single hydrophobic pocket of the binding cavity (Berntsson et al., 2009). Here,we designed four nonapeptides based on a natural casein-derived peptide, with aleucine at positions 4, 5, 6 or 7 in addition to a fixed leucine at position 2. OppA*

58 Chapter 30%

3.75%

7.50%

11.25%

15.00%

A C E D G F I H K M L N Q P S R T W V Y

0%

3.75%

7.50%

11.25%

15.00%

A C E D G F I H K M L N Q P S R T W V Y

% of total OppA WT % of total OppA F493K% of total OppA F493R

Figure 3.3. Amino acid composition of the endogenous ligands extracted from wild-type OppA*,F493K and F493R mutants. In total, 129, 96 and 162 peptides were identified for wild-type OppA*,F493K and F493R mutants, respectively. There was very little overlap (<5%) between the identifiedpeptides from the tree different samples.

was successfully crystallized with the peptide that contained leucine at position 6(SLSQSLSQS). Although the electron density at the N-terminal end of the peptidewas weak, we could model the peptide in a unique register on the basis of theother side-chain densities and the well-defined C-terminus of the peptide. Whencompared to OppA* in complex with bradykinin, SLSQSLSQS had shifted registerby one residue, resulting in the leucine at position 6 residing in the hydrophobicpocket. The crystal structure presented in this work indicates that indeed OppA*can bind peptides of the same length in different register.

The analysis of the substitution mutants of F493 support the importance of thehydrophobic side-chain pocket in peptide binding and selectivity. Introductionof a positive charge at this position disturbs the binding of bradykinin the most,lowering the affinity by two orders of magnitude (Table 3.1). ITC measurementsprovided more detailed thermodynamic information of the mutants and alloweddissection of the enthalpic and entropic contributions to the ∆G of ligand binding.The disparity between the enthalpic and entropic contributions between mutants,e.g. F493Q and F493A that had similar ∆G for binding, is consistent with thepossibility of binding in different register, in this case due to the different properties

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 59

of the mutants rather than the peptide. It seems unlikely that the binding ofpeptide would have so large differences in its entropic and enthalpic contributionswhen binding in the same register. ITC experiments have also been performed todetermine the binding of 20 tripeptides KXK (with X being any naturally occurringamino acid) in OppA from S. typhimurium (S. H. Sleigh et al., 1999). These dataindicated that OppA from S. typhimurium binds these ligands with affinities inin the (sub)micromolar range. The ∆G of binding varied between -30 and -43 kJ/mol, representing a 160-fold difference in binding affinities between thedifferent tripeptides. The entropic and enthalpic contributions to ∆G varied largely,with a large favorable contribution of T∆S (37 - 71 kJ/mol) and a medium tolarge unfavorable contribution of ∆H (8 - 40 kJ/mol). Compared to OppA*, theenthalpic and entropic contributions are reversed in the S. typhimurium protein. Butsimilar to OppA* from L. lactis, peptide binding occurs with entropic and enthalpiccontributions that vary widely within a relatively small range of ∆G values.

Two substrate-binding proteins that have been studied by DSC are the maltose-binding protein (MBP) and the histidine-binding protein (HisJ) (Ganesh et al., 1997;Novokhatny and Ingham, 1997; Kreimer et al., 2000). Both proteins have a meltingtemperature (Tm) between 55 and 65 ◦C (depending on pH), and the unfoldingcan be well described by a two-state process with two independent transitions,i.e. each of the major two domains unfold independently of each other. Additionof substrate increases the Tm of the proteins by 8-15 ◦C (depending on pH). Inaddition, it has previously been shown for MBP that, upon mechanical stress, theprotein first goes from a closed-liganded to an open conformation, before it startsto unfold (Bertz and Rief, 2009). It is plausible that this mechanism, i.e. proteinopening before unfolding, is generally valid as the mode of binding seems highlyconserved in substrate-binding proteins (see Chapter 1). This would likely alsohold true upon unfolding by heat in a DSC experiment. Since the unfolding ofOppA* was irreversible, as well as dependent on the rate of temperature increase,values of the enthalpy difference and heat capacity change upon unfolding couldnot be determined. However, melting temperatures can be comfortably comparedbetween wild-type OppA* and the various mutants. Wild-type OppA* exhibiteda Tm of 43.9 ◦C, significantly lower than measured for MBP and HisJ. Moreover,the degree of stabilization by ligand (bradykinin) increased with the affinity ofthe protein for the peptide. It must mean that the hydrophobic pocket playsa significant role not only in peptide binding, but also in stabilizing the closedconformation of the protein

60 Chapter 3

With this knowledge in mind, new light is shed on previous results. Peptidebinding to OppA*, using combinatorial peptide libraries, has previously beenstudied in great detail. Those libraries contained nonameric peptides, randomizedat all positions except one. For every defined position there were 19 variations(cysteine was not used), resulting in a total of 171 libraries (Detmers et al., 2000).Only small differences in KD were observed between different amino acids at thesame position in the nonapeptide, with substitutions at a given position givingrise to only 2-fold differences in affinity. This was interpreted as ligand bindingto OppA* being largely indifferent to the peptide sequences. This now seemspartly true, as one also need to take into account that peptides can bind in differentregister. Thus, the peptides in a randomized library, with a defined residue at agiven position, may not all bind in the same register and differences in side-chainpreference may partly average out.

3.5 Experimental Procedures

3.5.1 Bacterial strains, plasmids, growth conditions and purifica-tion

The oppA* gene was amplified by PCR, from the template vector pAMP21 (Lanfer-meijer et al., 1999), using cLIC complementary primers, and subsequently ligatedinto the pREcLIC vector (Geertsma and Poolman, 2007); the resulting plasmid(pREcLIC-OppA*) was transformed to E. coli MC1061. The parental pREcLIC-OppA* was subsequently used to generate the following OppA substitution mu-tants: F493C, F493A, F493Q, F493E, F493K and F493R, using the Quikchange PCRprotocol (Stratagene). The mutations were verified via DNA sequencing. TheoppA* wild-type and mutant genes were transferred from pREcLIC to pNZcLIC,via a SfiI digestion and combining of pREcLIC and pERL according to the LIC-VBExprocedure (Geertsma and Poolman, 2007) and the plasmids were transformed to L.lactis NZ9000 (Geertsma and Poolman, 2007). L. lactis NZ9000 expressing wild-typeor mutant variants of OppA* was cultivated semi-anaerobically in 2% (w/v) GistexLS (Strik BV, Eemnes, NL), 65 mM potassium phosphate (KPi) pH 6.5, 1.0% (w/v)glucose and 5 µg/ml chloramphenicol at 30 ◦C.

For isolation of endogenous peptides, OppA*, OppA*(F493K) and OppA*(F493R)were cultivated semi-anaerobically in M17 broth, 1% (w/v) glucose and 5 µg/ml

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 61

chloramphenicol at 30 ◦C to an OD600 of 0.6, where they were induced by theaddition of 0.2% (v/v) culture supernatant of the nisin A producing strain NZ9700(Ruyter et al., 1996). The cells were harvested at 2h after the start of induction,at the start of the stationary growth phase. The endogenous ligands of OppA*,OppA*(F493K) and OppA*(F493R) were purified and isolated as described (Bernts-son et al., 2009).

3.5.2 Crystallization and structure determination

Crystals were grown and cryoprotected as described (Berntsson et al., 2009). Thedrops consisted of 1 µL of protein (10 mg/mL of OppA* in 9 mM Na-MES, pH6.0, 9 mM NaCl and 1 mM SLSQSLSQS peptide) and 1 µL reservoir solution (0.2M NaCl, 0.1 M Na-Hepes, pH 7.0, 20% PEG 6000). Diffracting crystals of OppA*complexed with SLSQSLSQL were obtained after 5 months of incubation at 18 ◦C.Data were collected at ID14-1 at ESRF, Grenoble (Table 3.3). Data processing andreduction were carried out using MOSFLM and programs from the CCP4 package(Leslie, 1992; Collaborative Computational Project Number 4, 1994). The structurewas solved by molecular replacement using Phaser (McCoy et al., 2005), usingthe previously determined structure of OppA* in complex with bradykinin (PDBcode: 3DRG) as a search model. The output from Phaser was further refinedusing Refmac5 and Phenix.refine (Murshudov et al., 1997; Adams et al., 2002),interspersed with manual building rounds in Coot (Emsley and Cowtan, 2004).

3.5.3 Fluorescence measurements

Fluorescence spectra were obtained with a Fluorolog R�-3 (Jobin Yvon) spectrofluo-rometer. A quartz-cuvette containing 800 µL of protein sample (1-2 µM wild-type ormutant OppA* in 20 mM MES pH 6.0, 150 mM NaCl) was incubated for 10 minutesat 25 ◦C under constant stirring before stepwise addition of the peptide substrate(or buffer as control), using a Hamilton syringe pump in 0.5 - 1 µL steps (Harvardapparatus). After each addition the solution was incubated for 5 seconds, to assurefull mixing, before the averaged fluorescence signal was measured over 20 seconds.The protein solution was excited at 280 nm and emission was measured at 315 nm,with slit widths of 1 and 3 nm, respectively. Fluorescence titrations were analyzedas described (Lanfermeijer et al., 1999), and curve fitting was performed in Origin(OriginLabs).

62 Chapter 3

Table 3.3. Data collection and refinement statistics for OppA* complexed with SLSQSLSQS.

Data collectionSpace group P1Cell dimensionsxxxa, b, c (A ) 42.4, 58.9, 61.4xxxα, β, γ (◦) 100.6, 101.3, 103.4Wavelength (A ) 0.872Resolution range (A) 37.2-1.5Unique reflections 84,393Completeness (%) 95.9 (93.0)Rmeas 0.05 (0.39)I/σ (I) 14.3 (2.9)Redundancy 2.0RefinementResolution range (A) 29-1.5Number of reflections 82,534Rwork/R f ree 0.16/0.19No. atomsProtein 4510Water 1169Average B-factors (A 2)Protein 16.4Water 28.4R.m.s deviationsBond lengths (A ) 0.007Bond angles (◦) 1.120

a The number in parenthesis correspondsto the highest resolution shell

3.5.4 Isothermal titration calorimetry

Peptide binding to wild-type and mutant OppA* was measured by microcalorime-try on a ITC200 calorimeter (MicroCal) at 25 ◦C, using samples of 200 µL of protein(typically 20 µM of protein in 20 mM MES, pH 6.0, 150 mM NaCl). Peptide (stocksolutions of 200 - 1000 µM in the same buffer as the protein) was added stepwiseto the cell; 20 injections were made in 2 µL steps with an interval of 120 seconds.The first titration of each experiment was 0.5 µL of peptide instead of 2 µL, and wassubsequently deleted in the data analysis, as was outlier peaks. Data was analyzedusing the MicroCal software provided (Wiseman et al., 1989).

Contribution of a hydrophobic pocket to peptide binding in lactococcal OppA 63

3.5.5 Differential scanning calorimetry

The stability of OppA*, with and without bradykinin bound, was measured byDSC on a VP-DSC (MicroCal) at 25 ◦C and at 25 psi. 600 µL of OppA* wild-typeor mutant (3-4 µM of protein in 20 mM MES, pH 6.0, 150 mM NaCl) was added tothe cell. Protein samples and buffers were degassed before injection into the cell,to prevent the formation of air bubbles. For temperature scans in the presence ofbradykinin, the ligand was added to a final concentration of 50 times the KD of theparticular mutant, except for OppA*(F493K) and OppA*(F493R) when bradykininwas present at 20 times the KD; the samples were equilibrated for 10 min at 25µC, prior to the temperature ramps. A scan rate of 60 ◦C/hour was used anda temperature range of 25-55 ◦C. Data was analyzed by the MicroCal software(Wiseman et al., 1989)