The Protein Kinase C Inhibitor Bisindolyl Maleimide 2 Bindswith Reversed Orientations to Different Conformations ofProtein Kinase A*

Received for publication, December 23, 2003, and in revised form, March 1, 2004Published, JBC Papers in Press, March 1, 2004, DOI 10.1074/jbc.M314082200

Michael Gassel‡, Christine B. Breitenlechner§, Norbert Konig‡, Robert Huber§,Richard A. Engh§¶�, and Dirk Bossemeyer‡**From the ‡Department of Pathochemistry, German Cancer Research Center, 69120 Heidelberg, Germany,the §Abteilung Strukturforschung, Max-Planck-Institut fur Biochemie, 82152 Martinsried, Germany,and the ¶Department of Medicinal Chemistry, Roche Diagnostics GmbH, 82372 Penzberg, Germany

As the key mediators of eukaryotic signal transduc-tion, the protein kinases often cause disease, and inparticular cancer, when disregulated. Appropriately se-lective protein kinase inhibitors are sought after as re-search tools and as therapeutic drugs; several have al-ready proven valuable in clinical use. The AGCsubfamily protein kinase C (PKC) was identified early asa cause of cancer, leading to the discovery of a variety ofPKC inhibitors. Despite its importance and early discov-ery, no crystal structure for PKC has yet been reported.Therefore, we have co-crystallized PKC inhibitor bisin-dolyl maleimide 2 (BIM2) with PKA variants to study itsbinding interactions. BIM2 co-crystallized as an asym-metric pair of kinase-inhibitor complexes. In this asym-metric unit, the two kinase domains have different lobeconfigurations, and two different inhibitor conformersbind in different orientations. One kinase molecule (A)is partially open with respect to the catalytic conforma-tion, the other (B) represents the most open conforma-tion of PKA reported so far. In monomer A, the BIM2inhibitor binds tightly via an induced fit in the ATPpocket. The indole moieties are rotated out of the planewith respect to the chemically related but planar inhib-itor staurosporine. In molecule B a different conformerof BIM2 binds in a reversed orientation relative to theequivalent maleimide atoms in molecule A. Also, a crit-ical active site salt bridge is disrupted, usually indicat-ing the induction of an inactive conformation. Molecu-lar modeling of the clinical phase III PKC inhibitorLY333531 into the electron density of BIM2 reveals theprobable binding mechanism and explains selectivityproperties of the inhibitor.

Deregulated protein kinase activity causes a wide variety ofhuman diseases, usually by producing an overactive kinase.

This is consistent with the fact that most protein kinases in thecell are inactivated most of the time to ensure the integrity ofsignal transduction. Thus, the many diseases that are corre-lated with protein kinase deregulation, including the majorityof all cancers, usually arise from mutations or other events thatactivate kinases, cause their overexpression, or disable theirintracellular inhibition. The prevalence of kinase deregulationin disease clearly demonstrates the need for therapeutic pro-tein kinase inhibitors, whereas the ubiquity and variety ofprotein kinases (collectively, the “kinome”) necessitate precisetarget selectivity. Despite this seeming difficulty, several pro-tein kinase inhibitors have been approved for human treat-ment or are in advanced clinical trials.

Crystal structure analyses of protein kinase inhibitor com-plexes reveal the intermolecular interactions responsible forligand binding, and have thereby enabled structure-based ra-tional design and optimization of kinase inhibitors. To date,crystal structures have been determined for some 30 proteinkinases, representing some 6% of the 518 protein kinases in thehuman genome (1). Many of these structures have been com-plexes with protein kinase inhibitors, but most have shown aninactivated state often incompatible with inhibitor binding.Inactivity is associated most often with displacements of helixC, the major � helix of the kinase N-lobe, with concomitantdisruption of the active site salt bridge in the active site be-tween a conserved lysine residue and a conserved glutamatelocated in the middle of helix C. Similarly often, the activationloop shows unproductive conformations, either blocking theactive site, or locking helix C into an inactive conformation, orcausing other structural states of the kinase incompatible withkinase activity. Often, these structures are “open” with respectto the “closed” conformation of kinases in catalytic conforma-tions. In more than half of all protein kinase structures inac-tivity is associated with a steric block in the ATP-binding site(2–4).

Despite the variations in sequence, the fold of the activeprotein kinase catalytic domain is well conserved. Inactivatedprotein kinase structures differ more, but cluster into proteinkinase subfamilies that reflect different inactivation mecha-nisms. As a consequence of the conservation of the active struc-ture, many properties can be analyzed with respect to rela-tively few sequence positions that define that property. Acentrally important example of such a property is the selectiv-ity of binding at the ATP binding pocket. Protein kinases sharea common bi-lobal catalytic domain structure that forms theATP-binding site at the lobal interface. ATP binds at thisinterface via interactions with some 15 residues of the protein,including about 10 side chain interactions that therefore areespecially important as potential determinants of ATP site

* This work was supported in part by the Bayerische Wirtschaftsmin-isterium. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

The atomic coordinates and structure factors (code ISZM) have beendeposited in the Protein Data Bank, Research Collaboratory for Struc-tural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

� To whom correspondence may be addressed: Abteilung Strukturfor-schung, Max-Planck-Institut fur Biochemie, 82152 Martinsried, Ger-many. Tel.: 49-89-8578-2629; Fax: 49-89-8578-3516; E-mail: engh@biochem.mpg.de.

** To whom correspondence may be addressed: Dept. of Pathochem-istry, German Cancer Research Center, 69120 Heidelberg, Germany.Tel.: 49-6221-423266; Fax: 49-6221-423249; E-mail: d.bossemeyer@dkfz-heidelberg.de.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 22, Issue of May 28, pp. 23679–23690, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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inhibitor selectivity. Thus, the essential binding properties ofATP and other ATP site ligands can in many cases be simu-lated for a particular protein kinase target by a limited set ofpoint mutations of a closely related protein kinase. The con-struction of such hybrids has been demonstrated for kinases(5–7). Even though subtle differences in structure or flexibilitycan lead to quite different overall reaction kinetics, for practicalligand design purposes, most or all protein-ligand interactionswill be revealed or can be modeled using the surrogate kinaseapproach. That a single residue can be identified as the prin-cipal selectivity determinant for an inhibitor type by mutationof a series of kinases verifies this approach (8).

Along these lines, the cAMP-dependent protein kinase(PKA)1 has been used as a surrogate kinase for co-crystalliza-tion with several protein kinase inhibitors, such as H7, H8, andH89 (9), staurosporine (10), balanol (11), and recently the Rho-kinase inhibitors Y27632, H1152P, and Fasudil (12). The com-plexes with staurosporine, balanol, and Fasudil are in partiallyopen conformations, in contrast to the other inhibitor/sub-strate-PKA complexes that are in closed conformations. Inthese three structures the hydrogen bond between the activa-tion loop Thr197 phosphoryl group and His87 from helix C of theN-lobe is not formed as a result of the partial opening of thecleft via rotation of the N-lobe with respect to the C-lobe.

Mutants of PKA have also been designed to improve its valueas surrogate kinase for PKB inhibitors (7). Because of its closerelationship to PKC and its well established crystallizationconditions, PKA is currently also the best model system forstudying PKC inhibitors. So far, co-crystallization attemptswith PKA and the flexible bisindolyl maleimide (BIM) cognatesof staurosporine, described as PKC inhibitors (13, 14–17), havefailed to produce crystal structures. However, the triple mutantV123A,L173M,Q181K of PKA� (PKAB3), originally designedas a model for PKB (7), has formed high quality crystals of aBIM inhibitor complex. A sequence alignment of PKA� andPKB� with PKC isoforms (Table I) shows how the PKAB3triple mutant is similar to PKA as a surrogate for PKC with theadditional ability to model inhibitor-methionine interactionsfor three conventional PKC isoforms.

Three distinct subfamilies of PKC isoforms can be definedaccording to their essential activators: conventional PKCs (�,�I/II, and �) require phosphatidylserine, diacylglycerol, andCa2�; novel PKCs (�, �, �, and �) need phosphatidylserine anddiacylglycerol but not Ca2�; atypical PKCs (� and ) are insen-sitive to both diacylglycerol and Ca2� although phosphatidyl-serine regulates activity (for reviews, see Refs. 18 and 19 andcitations therein). Furthermore, additional lipid mediators,like fatty acids and lysophospholipids, have been shown toinfluence the catalytic activity of PKCs (reviewed in Ref. 20). Ingeneral, interaction of PKCs with the activators leads to phos-phorylation of a threonine residue on the activation loop of allPKC isoforms and additionally of a serine or threonine residuein the hydrophobic motif of the conventional and novel PKCs.The atypical PKCs possess a glutamate at the hydrophobicmotif phosphorylation position that intrinsically performs the

activation function (for reviews, see Refs. 18 and 21). PKCisoforms are involved in nearly all essential cell processes,PKC�, for example, is involved in regulation of proliferation,apoptosis, differentiation, cell migration, adhesion, amongother cellular and pathogenic processes (for a review, see Ref.22).

Despite its early identification and importance in cancerresearch, no PKC crystal structure has been reported to date.The only available co-crystal structures of PKC inhibitors witha kinase target are those of the relatively unselective stauro-sporine (IC50, PKC 5 nM (23)) and its closely related derivativeUCN01 (IC50, PKC� 29 nM (24)). Besides co-crystallization withPKA (10) (Protein Data Bank code 1STC), the extended andrigid planar staurosporine has been crystallized with Cdk2 (25)(Protein Data Bank code 1AQ1), CSK (26) (Protein Data Bankcode 1BYG), and others. UCN01 (7-hydroxystaurosporine) hasbeen co-crystallized with Cdk2 (27) (Protein Data Bank code1PKD) and Chk1 (28) (Protein Data Bank code 1NVQ). Thebisindolyl maleimide class of PKC inhibitors is derived fromstaurosporine by elimination of a single bond that converts theextended planar aromatic group into the three aromats of thecompound names with corresponding additional degrees of flex-ibility. One PKC inhibitor of this class, LY333531, shows PKCisoform specificity (e.g. 80- and 60-fold selectivity for PKC� Iand PKC� II over PKC� (29) and is in phase III clinical trialsfor diabetic retinopathy and diabetic macular edema (Ref. 30and citations therein). The effects of the additional flexibility ofBIM inhibitors on the structural binding modes, and the meansby which this alteration can introduce selectivity to the inhib-itor have not been explained.

Here we present the crystal structure of bisindolyl maleim-ide 2 (BIM2) in a complex with the triple mutantV123A,L173M,Q181K (PKAB3) of PKA. By means of this sur-rogate kinase approach, we identify the key binding modes andcan evaluate their significance for PKC. The asymmetric unit ofthe crystal structure consists of two protein-inhibitor com-plexes, with different conformations of the two kinase mole-cules, bound with opposite orientations of different conformersof BIM2. One kinase monomer has an intermediate open statelike in the staurosporine structure; the other is in the mostopen conformation observed for PKA so far. In the open confor-mation, the whole N-terminal lobe including helix C is rotatedby more than 20° compared with the closed form. The active

1 The abbreviations used are: PKA, cAMP-dependent protein kinase;PKAB3, triple mutant of cAMP-dependent protein kinase(V123A,L173M,Q181K); PKB, protein kinase B; PKC, protein kinase C;BIM2, bisindolyl maleimide 2 (2-[1-[2-(1-methylpyrrolidino)ethyl]-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide); BIM2MolA and BIM2MolB,the two BIM2�PKAB3 complexes of the crystallographic asymmetricunit; LY333531, (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetra-hydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]-oxadiazacyclohexadecene-1,3(2H)-dione; Mops, 4-morpholinepropane-sulfonic acid; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; AMP-PNP, adenosine 5�-(�,�-imino)triphosphate;Mes, 4-morpholineethanesulfonic acid.

TABLE IAmino acids residues of different protein kinases

The numbering is according to PKA. Amino acids, which interact inmolecule A and molecule B with BIM2 are in bold, whereas amino acidsthat interact only in molecule A with BIM2 are shaded. Amino acid 181does not interact with BIM2 but has been exchanged to prevent arotation of the glutamine towards the ATP-binding site (7). Phe327 doesnot interact with Bim2, but probably with LY333531. PKC�I andPKC�II are merged because they do not differ within the kinasedomain.

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site salt bridge between Lys72 and Glu91 is disrupted, a char-acteristic of inactive kinase conformations. The two differentinhibitor binding modes are enabled by the inherent symmetryof the maleimide moiety and the rotational freedom available tothe indole moieties. The similarity of BIM2 and LY333531 (Fig.1) allows modeling of LY333531-PKC interactions and suggestsan explanation for the selectivity properties of the inhibitors.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—Recombinant mutated bovineC� catalytic subunit of the cAMP-dependent protein kinase (PKAB3 (7))was solubly expressed in Escherichia coli BL21(DE3) cells and thenpurified via affinity chromatography and ion exchange chromatographyas described earlier (9). Two positions distinguish bovine (Asn32 andMet63) from human PKA (Ser32 and Lys63). 4-Fold phosphorylated pro-tein was used for crystallization of BIM2.

Activity Tests—The determination of enzyme activity was accom-plished by an ATP regenerative NADH consuming assay according toRef. 31. After the addition of 0.42 mM MEGA 8 (ensures solubility of theinhibitor) to the assay mixture (100 mM Mops, pH 6.8, 100 mM KCl, 10mM MgCl2, 1 mM phosphoenolpyruvate, 0.1 mM Kemptide, 1 mM �-mer-captoethanol, 15 units/ml lactate dehydrogenase (Sigma), 8 units/mlpyruvate kinase (Sigma), 0.21 mM NADH) we have added the successiveMe2SO/inhibitor solution and the enzyme and started the reaction withATP. The decrease of NADH was measured as time-dependent at �340 nm with three independent measurements per data point.

Crystallization—BIM2 were purchased from Calbiochem and co-crystallized with PKAB3 in the presence of PKI(5–24) at 75 mM LiCl, 25mM Mes/Bistris, pH 6.4. The hanging drop vapor diffusion methodagainst 15% methanol as precipitant was used to obtain a 100 � 100 �300-�m crystal.

Data Collection and Structure Determination—Diffraction data weremeasured at the Deutsches Elektronen Synchrotron (DESY, Hamburg)from frozen crystals on a CCD detector (Mar research) at 1.05 Å wave-length. The data were processed with the programs MOSFLM andSCALA. The crystals have orthorhombic symmetry (P212121) with cellconstants 82.06, 89.00, and 116.38 in a crystal packing arrangement notpreviously reported (Table II). The structure was determined by molec-ular replacement using MOLREP from the CCP4 program suite.2 Asstarting model we chose a PKA-PKI-(5–24)-staurosporine complex(1STC (10). Calculation of Matthews coefficient and solvent contentsuggested two molecules in the asymmetric unit with 52.3% solvent.Indeed, very good monomer rotation and translation function solutionwas found, enabling determination of a second molecule with an R-factor of 47.5. A first map calculated after rigid body refinement (R-factor 45%) showed that PKI was not present in the complex, despite itspresence in crystallization solutions as usual. Furthermore, difference

densities in the B molecule showed that large segments of the moleculeneeded to be remodeled. We deleted the segments (the entire N-termi-nal lobe up to 123 and the C-terminal residues from 315 onwards) ofabout 150 amino acids together and calculated new maps. After severalcircles of model building and refinement the R-factor fell to 28.8%(R-free 33.9%) and most parts of molecule B were replaced. In moleculeA only residues 315–350 were omitted and rebuilt. The segment 317–332 (330 for molecule B) remained undefined. Phosphorylation siteswere found at Ser139, Thr197, and Ser338. Ser10 is not resolved. Watermolecules were automatically inserted using CCP4 programs PEAK-MAX and WATPEAK and visually inspected. Finally, the inhibitormolecules were built and the whole complex was further refined. Ref-mac 5.1.24 was used for refinement, and MOLOC3 was used for modelbuilding and graphical modeling. For data and refinement statistics,see Table II.

Superimposition, Calculation of Secondary Elements, and Angle De-termination—Superimpositions were performed using the programs In-sight II or MOLOC. In the case of MOLOC, amino acid residues 150–300 were used for superimposition of structures 1STC, 1CTP, 1CMK,and 1J3H and both molecules of the here presented structure on struc-ture 1CDK as basis. The secondary elements were calculated with theprogram InsightII using the Kabsch-Sander algorithm. The angledetermination was performed in the following way. The backbone ofeach first and last two amino acids of the helices were taken to calculatethe center of masses using the gromacs package.4 These center ofmasses defined the top and bottom of the helix axes. The coordinates ofthe top and bottom were used to create vectors in the R3 and withcos� � x�y/�x���y� (x and y are the vectors defining the helix axes). Theangle between these vectors of two different structures was calculated.

Sequence Alignment—Sequence alignments were performed usingClustalW.5

RESULTS AND DISCUSSION

PKAB3 as Model for the PKC-ATP-binding Pocket—Thestructural and sequence similarities of AGC kinase subgroupmembers PKA and PKB (Ref. 32, Protein Data Bank code1CDK; Ref. 33, Protein Data Bank code 1O6K) imply a corre-spondingly similar structure for the kinase domain of PKC.Thus, the clearest determinant of the selectivity of proteinkinase inhibitors is the amino acid composition of the catalyticATP-binding site. It follows that amino acid exchanges in thecatalytic site of one kinase can be useful as a surrogate foranother kinase, reported, for example, for PKA mutants thatmimic PKB (7). For this study we used the triple mutant

2 www.ccp4.ac.uk/main/html.

3 www.moloc.ch.4 www.gromacs.org.5 www.ebi.ac.uk/clustalw.

FIG. 1. The compounds relevant to this paper. A, BIM2 bound to the PKA mutant structure; B, LY333531, the PKC� specific compound; C,staurosporine, whose binding mode has been determined crystallographically with several kinases, including PKA (Protein Data Bank code 1STC(10)).

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PKAB3 (V123A,L173M,Q181K), which was originally con-structed to model the adenine-binding site of PKB in PKA.Sequence alignment of PKA, PKB, and PKC shows that one ofthese mutations, namely L173M (numbering of PKA), intro-duces a residue that is conserved in PKB and the three classicalPKC isoforms �, �I/II, and �. The other PKC isoforms have, likePKA, a leucine residue in the corresponding position. The sec-ond exchange, V123A, does not occur among PKC isoforms, butthe important contacts of residue 123 to inhibitors are back-bone contacts and presumably relatively unaffected by the sidechain. The third exchange, Q181K, was chosen only after ob-servation that the double mutant (V123A,L173M) lead to a newrotamer conformation of the glutamine of PKA that placed theamide group into the ATP binding pocket (7). This mutationintroduces a residue conserved as a lysine among all PKCisoforms.

Overall Structure—The triple mutated recombinant catalyticsubunit of cyclic AMP-dependent PKA and the PKC inhibitorBIM2 formed crystals notably lacking the pseudo-substratepeptide PKI(5–24), although the peptide was present under thestandard crystallization conditions. The 4-fold phosphorylatedPKA is disordered at the N terminus including the phospho-rylation position of (p)Ser10. The region between residues 317and 332 is also not visible. The corresponding dimer formed theasymmetric unit in an orthorhombic space group P212121 (Ta-ble II) with cell constants and a packing arrangement previ-ously unobserved for PKA (a � 82.1, b � 89.0, c � 116.4). Thisarrangement is apparently induced by BIM2 binding andarises from the two new N- and C-domain conformations ofPKA in the crystal. One of the PKA complexes of the asymmet-ric unit (molecule A) is in an intermediate open conformation,similar to that observed for the PKA complex with stauros-porine (1STC (10)) and also HA-1077 (1Q8W (12)), whereasthe other, molecule B, is in the most open conformation de-scribed so far for PKA (Fig. 2). The inhibitors in both molecules

A and B occupy the ATP-binding site, but different conforma-tions of the inhibitor bind with different orientations in the twomolecules (Fig. 3).

Many different relative N- and C-lobe orientations have beenobserved for PKA. One measure to identify the closed confor-mations is the existence of an H-bond between the imidazole ofHis87 from helix C and the phosphoryl group of Thr197 from theactivation loop. Structures for which this His87-(p)Thr197 con-tact is broken because of the opening movement of the N-lobeexhibit a variety of open conformations (Fig. 2). The hingingmovements of PKA that cause the opening occur primarilywithin the dipeptide segment of Gly125 and Gly126 positionedbetween the ATP binding residues and helix D, as definedoriginally by Olah and co-workers (34). Rotations of helix C canbe taken as a measure of the opening of PKA structures, andranks structures from the most closed in structure 1CDKthrough other open conformations of PKA such as staurospo-rine bound (1STC (10), or the apoenzyme structures (1CTP (35,36) and 1J3H (37)) to the most open conformation of BIM2MolBfrom this study (Table III and Fig. 3). The helix C tilts ofBIM2MolA and 1STC (intermediate open) are 8.1° and 8.3°with respect to 1CDK. The greatest rotations occur in thestructures 1J3H-molecule A (14.2°) and BIM2MolB (14.6°) (Ta-ble III). In addition to the hinge movement shared by all struc-tures, BIM2MolB shows a rotation of its N-lobe by 15° whencompared with molecule A of 1J3H. This rotation is clockwisefor the N-lobe from a viewpoint above the N-lobe toward theC-lobe. The axis of this rotation is parallel to the viewing axis.The C terminus of the helix is fixed to the C-domain, whichresults in a rotation of helix C of one-half the total rotationrelative to both domains, similar to the motion of a piston. Therotation of helix C relative to the N-domain contributes to thedisruption of the salt bridge between Lys72 and Glu91, a char-acteristic of inactive kinase structures (2–4). Another strikingconformational difference of BIM2MolB to any other PKAstructure is the displacement of the hinge region that connectsthe N- and C-lobes (amino acids Glu121-Pro125) and forms themost important inhibitor and ATP binding interactions. Thisdisplacement amounts to 3.38 Å at the C� of Ala/Val123 and

TABLE IIData collection and refinement statistics

PKAB3-BIM2

Data collectionSpace group P212121Cell (a, b, c) (Å) 82.06 89.00 116.39Resolution range (Å) 24.77–2.5Observations read 279090Unique reflections 31110Completeness (%) 99.8I/sigma 5.2Rsym (%) 9.9Multiplicity 5.2

RefinementNumber of Non-hydrogen atoms usedin refinement

5411

R factor (%) 23.3Free R factor (%) 29.1Free R value test size (%) 5.1Resolution range 70–2.5Used reflections 28539

Standard deviation from ideal valuesBond length (Å) 0.016Bond angles (°) 1.610

Temperature factorsAll atoms 52.4Main chain atoms PKAB3, Mol A 58.0Side chain atoms PKAB3, Mol A 58.5Inhibitor atoms BIM2, Mol A 42.6Main chain atoms PKAB3, Mol B 46.3Side chain atoms PKAB3, Mol B 47.2Inhibitor atoms BIM2, Mol B 54.8Solvent molecules 47.8

FIG. 2. Root mean square differences of the C� positions of thefour PKA structures relative to closed conformation PKA-PKI-AMP-PNP structure 1CDK. The C-terminal lobes of the moleculeswere superimposed to assess the extent of motion of the N-terminal lobe(and hydrophobic motif binding C-terminal strand) relative to the C-terminal lobe.

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5.05 Å at the carbonyl of Pro/Ala124 in comparison to moleculeA of mouse PKA structure 1J3H (37) (Fig. 4).

The Bisindolyl Maleimides, Cognates of Staurosporine—Many cognates of staurosporine have been identified as poten-tial therapeutic kinase inhibitors; some are in clinical trials.Target complex structures available for them include the rigidplanar inhibitor staurosporine itself (e.g. Refs. 10 and 38) andthe closely related UCN01 (28, 39). Here we report a kinasecomplex structure for a non-planar, flexible staurosporine cog-nate of the bisindolyl maleimide class (15–17). The structure ofPKAB3 and bisindolyl maleimide 2 demonstrates the similar-ities and differences of binding modi between the planar stau-rosporine and the flexible bisindolyl maleimide 2, indicatingthe factors that govern selectivity in this inhibitor class. It isunique in that two inhibitor conformations bind the same siteof the same enzyme in two different orientations, and further-more, in a single crystal. Enzyme inhibitors that bind withdifferent orientations have been observed previously, for exam-ple, serine proteinase inhibitors that as a function of pH bindwith opposite orientations (46), or kinase inhibitors that binddifferent kinases with unequal orientations (40).

The chemical structures of the 3 inhibitors discussed in thisarticle are shown in Fig. 1. They originate from a single chem-ical class and share the symmetric basic architecture of amaleimide flanked by two indole rings. In the case of theindolocarbazole staurosporine, the entire structure is rigidifiedby an additional C-C bond (C7–C8) that links the indoles andmaleimide into an extended aromatic and planar system, andadditionally via cyclic bonding of the sugar moiety from the twoindole nitrogen atoms (Fig. 1C). This extended and rigid pla-narity of staurosporine is shown in many crystal structures(with PKA and PKI (10), LCK (41), and CDK2 (27)) to induce anideal planar fit in its many kinase hosts, which may cause itsrelatively low specificity. Lacking the C7–C8 bond, and with a

single bond connecting a pyrrolidine tail, BIM2 possesses de-grees of freedom that allow rotation of the indole rings out of asingle plane and that allow a large number of conformations forthe pyrrolidine tail (Fig. 1A). The crystal structure shows howthese degrees of freedom are utilized (Fig. 5, a and b). Thesmall molecule crystal structure of BIM4, a derivative consist-ing only of the maleimide head group and the two indoles,shows a comparable rotation of one indole as found in ourstructures, indicating an energetically preferred conformation(15). LY333531, also known as ruboxystaurin, seems to repre-sent an intermediate between staurosporine and BIM2 withrespect to flexibility of the indole moieties. Although the in-doles are not directly linked as in staurosporine, the 6-atomether linker connecting the N-atoms of both indoles forms acycle that restricts the orientations available to the indole rings(Fig. 1B).

Binding of BIM2, Molecule A—In the partially open struc-ture of PKAB3 in molecule A, BIM2 forms 4 H-bonds with theenzyme in molecule A. The symmetrical maleimide makes twohydrogen bonds to the hinge region between N:19 and Glu121:Oand O32 and Val123:N (Fig. 6a). A third contact of the maleim-ide is formed from O33 to Thr183:OG1, whereas the N30 of thepyrrolidine group connects to Glu170:OE2 (Table V). The con-tact to the side chain of Glu170 is possible, because Glu170 is ina different rotamer conformation. Another residue, Glu127, alsoshows a new rotamer, because its original position is occupiedby the pyrrolidine group. Glu170, together with Glu127, areessential residues for the recognition of the substrate consen-sus arginine residues, including those of PKI. The interactionof BIM2 with both residues in different rotamer conformationsmight explain the absence of PKI(5–24) in the structures.

The total number of van der Waals contacts between inhib-itor and enzyme is 127. In detail, the maleimide is coordinatedby the hinge region (Glu121-Ala123), Ala70, Met120, Tyr122, andThr183. The main interacting amino acid residues to indole Iare Lys72 and Thr183, whereas the peripheral glycine loop res-idues Leu49 and Val57 coordinate indole II. The pyrrolidinemoiety interacts mainly with Phe54, leading to a distortedconformation of the glycine loop, and makes side chain contactsto Glu127 and Glu170. Met173 is centered directly underthe BIM2 and has contacts to all moieties of the inhibitor(Table IV).

Binding of BIM2, Molecule B—In the open enzyme structureof molecule B, BIM2 binds with an opposite orientation. De-spite the displacement of the hinge region in this structure, itforms 2 H-bonds to the same hinge region atoms as in moleculeA, between N19 and Glu121:O, respectively, and O33 and

TABLE IIIAngles of the movement of helix C during the tilting of the N-terminal

lobe of different structures in comparison with 1CDK

Tilting of helix C

degree

BIM2MolA 8.11STC 8.31J3H-B 8.81CTP 10.81CMK 11.31J3H-A 14.2BIM2MoIB 14.6

FIG. 3. Superposition of structures1CDK, BIM2MolA, and BIM2MolB.Superposition of 1CDK (gray), BIM2MolA(red), and BIM2MolB (blue) are shown.

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Val123:N (Fig. 6b). This is possible because of the symmetricalnature of the maleimide head group, and because the BIM2inhibitor occupies a different location with respect to moleculeA. First, BIM2 follows the outwards directed hinge regiondisplacement to keep the contact for these hydrogen bonds.Second, BIM2(B) is rotated in the plane of its maleimide groupso that in an overlay of molecules A and B, the indole II groupof BIM2(B) comes close to the indole II moiety of BIM2(A).Apart from this, no further hydrogen bonds to the enzyme areestablished. The total number of inhibitor to protein van derWaals contacts are 91 in this conformation. The interactions ofBIM2 in molecule B are limited to contacts to the N-terminallobe and the hinge region. No contacts to the C-terminal lobeare established, a consequence of the open conformation of thekinase, the displaced position of the hinge region, and theshifting rotation of the inhibitor. In detail, the maleimide is

coordinated by the hinge region (Glu121-Gly125), Ala70 andMet120. Indole I is tightly packed by Leu49, Tyr122, and Pro124,whereas indole II interacts with Val57 and Lys72. Most strikingis the extensive contact of indole I to Pro124, which makes 16van der Waals contacts, one-sixth of the total number of con-tacts of this inhibitor with the enzyme. We are not aware thatany other protein kinase inhibitor interacts with the side chainof a residue in the homologous position. This unusual contactappears to be a consequence of the inwardly rotated conforma-tion of indole II, which can only be accommodated when theBIM2(B) molecule and the N-terminal lobe are rotated, whichwedges the inhibitor between the side chains of Pro124 andVal57.

Comparison of the Two Binding Modes—A comparison of thebinding modes of BIM2 in molecule A and molecule B high-lights the similarities but especially the significant differences.

FIG. 4. Superposition of the struc-tures for 1J3H molecule A andBIM2MolB. Superposition of 1J3H mole-cule A (orange) and BIM2MolB (blue) areshown.

FIG. 5. Stereo view of the differencedensity maps prior to inhibitor mod-eling, contoured at 2 � for BIM2bound to molecule A (a) and moleculeB (b). In molecule A, the entire inhibitorgeometry is unambiguously defined,whereas in molecule B, there is evidenceof some disorder especially at the pyrrol-idine moiety. Therefore, a second contour-ing at 1.5 � is added. Building the unam-biguously defined portions of the inhibitoridentified the major binding mode with nomodel bias.

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The principal similarities involve the maleimide groups, withtheir identical contacts to the hinge (Table V) and extensivecontacts to Ala70, a conserved residue from �-strand 3. Assum-ing that these identical interactions represent the essentialbinding interactions, the differences in binding modes observedin molecules A and B result from the different lobe configura-tions of the two kinase monomers. A particularly notable dif-ference between the two binding modes is the reversal of theoverall inhibitor orientation (Fig. 7). Although the bisindolylmaleimide scaffold itself is symmetric, the pyrrolidine substi-tution on indole I breaks the symmetry and defines an overallorientation. Thus, whereas the hydrogen bonding pattern ofthe maleimide and hinge loop remains the same between thetwo binding modes, the hinge hydrogen bond acceptor of theinhibitor is alternately the oxygen distal or proximal to thesubstituted indole I in molecules A and B, respectively (Fig. 6,a and b). The electron densities of the inhibitors (Fig. 5, a andb) confirm these two orientations, although there is evidencefor some disorder in molecule B, most likely associated withdifferent pyrrolidine binding geometries of low occupancy. Thereversed orientation is accompanied by a different arrange-ment of the indole moieties (Figs. 5, a and b, and 7a). Viewedfrom the N-lobe (as “up”), the indole moieties all rotate up-wards out of the plane of the maleimide and maintain intramo-lecular contact. In molecule A, however, they are oriented tothe “right” or toward the bulk of the kinase domain, whereas inmolecule B, they are oriented to the “left” or away from thekinase domain. This leads to generally different sets of inter-actions between the inhibitor and kinase residues. An excep-tion is Val57, which is in both orientations of inhibitor one of theresidues that contacts indole II. Both orientations of the pyr-rolidine form a face to face set of van der Waals interactionswith indole I. The pyrrolidine orientation in molecule B seemsto be stabilized only by these intramolecular interactions, be-cause no contacts to the kinase are established. In summary,

BIM2 adopts two very different conformations, with differentrotamers for pyrrolidine-tethered indole I (� 90°) and theuntethered indole II (� 30°) (Fig. 7a).

Because of its symmetry, the maleimides bind similarly inboth molecule A and molecule B; the indoles, pyrrolidines, andlinkers bind very differently. The maleimides form functionallyidentical contacts to the same hinge region atoms and six andseven van der Waals contacts to Ala70, a conserved residuefrom �-strand 3. The reversal of the BIM2 orientation, asdefined and constrained by the asymmetric pyrrolidine linker,leads to very different conformational adaptations to the bind-ing site. The conservation of interactions between indole II andboth indole I and pyrrolidine leads to an inwardly rotatedconformation of indole II and an outwardly rotated indole I.The reversal of the inhibitor orientation replaces the outwardconformation of indole I with an inward conformation of indoleII in the same binding site, and vice versa at the other indolebinding site. In molecule A, indole II is rotated out of themaleimide plane toward the glycine flap, interacting withLeu49 and Val57 from the adjacent N- and C-terminal anchor-ing ends of the glycine flap, respectively. These close interac-tions of indole II with Val57 replace the more distributed inter-actions that are observed in planar structures such asstaurosporine. In molecule B, indole II also interacts withVal57, but in this case, indole II is positioned at the inner orsolvent shielded site of the ATP pocket. Thus, the interactionsbetween indole II and Val57 require rotations of both the inhib-itor and the N-lobe �-sheet relative to their positions in mole-cule A, in opposite directions, to bring the interacting partnerstogether. Also associated with the effective rotation of the in-hibitor toward solution in molecule B is a reorientation of Lys72

that leads to occupancy of the volume occupied by the inhibitorin molecule A. The effective rotation of BIM2 in molecule B alsoinvolves new contacts with Pro124 from the hinge region, whichalso undergoes a conformational change apparently in response

FIG. 6. Stereo views of the bindingpattern of BIM2 in its two orienta-tions in PKA molecules A (intermedi-ate open geometry) and B (wide opengeometry). All interacting residues arehighlighted). a, molecule A. The hinge re-gion H-bonds Ala123:N-O32 and Glu121:O-N19 are supplemented by an additionalThr183:OG1-O33 H-bond. b, molecule B.Here only the hinge region H-bondsAla123:N-O33 and Glu121:O-N19 areformed.

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to BIM2 binding in this orientation. In summary, the reversedorientation of BIM2 in molecule B is associated with a rotationof inhibitor and opposite rotations of glycine loop and hingeregion atoms that, together with a conformer change of Lys72,embedded the inhibitor aromats in a new set of interactions.

Comparison of the BIM2-Molecule A Complex and 1STC—The kinase lobe configuration of the PKAB3 molecule A-BIM2complex resembles that of PKA-staurosporine (Fig. 2), facili-tating the comparison of the two inhibitors. The most strikingdifference between staurosporine and BIM2 is the flexibility ofthe latter. Staurosporine, entirely rigid (Figs. 1 and 7b), wouldbe expected to induce any observed conformational changes ofthe enzyme. Indeed, almost all side chains in the vicinity ofstaurosporine move to expand the binding site, as do the pep-tide backbones at Thr183, and Phe327. In addition, Phe54 fromthe glycine flap moves toward the inhibitor molecule (Fig. 7a(10)). One consequence of the staurosporine architecture is thata large number of atoms are in close proximity available toform van der Waals interactions with suitable protein residues.This is notable especially for inhibitor interactions with resi-dues Leu49, Met120, and Glu127 (Table IV). However, the corol-lary expectation that a more flexible molecule might induceless change in the protein because of flexibility and greaterpropensity to be changed is clearly not confirmed for BIM2. Inmolecule A, and more dramatically in molecule B, several con-formational changes are observed that are more pronouncedand far reaching than in the PKA/staurosporine structure. Thisis vivid confirmation of an understanding that the proteinkinase adopts a variety of conformations that is not readilyapparent by a limited set of crystal structures (with low ligandvariation and/or few crystal packing arrangements), at least inresponse to various small molecule ligands, and thus presum-ably also in response to a much greater variety of influences ina physiological environment.

Induced fit displacement of the glycine loop has been ob-served since the first PKA-inhibitor complexes (9). A strikingfeature of the molecule A structure in comparison to other PKAstructures is a tilting of the glycine loop toward BIM2 (Fig. 8b).Especially the � turn of residue Phe54 at the tip of the glycineloop forms a number of hydrophobic contacts to the methylgroup (C31, 5 of 7 contacts) substituent of the pyrrolidine ring.The interactions between staurosporine and Phe54 occurs viathe methyl group (C35, 2 of 2 contacts) substituent of the pyranring (Fig. 8a). Superposition of the complex structures of 1STCand molecule A reveals that these two inhibitor contact atomsare displaced by some 3 Å with respect to each other. Similarly,the Phe54:CZ atoms of the superimposed structures are dis-placed by 2.8 Å. Thus, as in 1STC, BIM2 binding induces amotion of Phe54 toward the inhibitor. However, unlike stauro-sporine, this causes the entire glycine loop � turn to tilt down-wards, thereby displacing residues 49–59 by up to 5.5 Å (Phe54:O). PKA binds elongated ligands such as AMP-PNP (1CDK) or

TABLE IVContacts of staurosporine and BIM2 to PKA/PKAB3

Residue inPKA�

1STC BIM2MolA BIM2MolBResidue in PKC isoforms

vdW H-bonds vdW H-bonds vdW H-bonds

Leu49 19 –a 13 – 12 – Leu/IleGly50 5 – 2 – 3 –Thr51 – – – – 6 – Lys/ArgGly52 – – – – 1 –Phe54 2 – 7 – – – Phe/TyrVal57 24 – 5 – 16 –Ala70 6 – 6 – 7 –Lys72 4 – 2 – 3 –Val104 1 – 5 – – – Val/ThrMet120 5 – 2 – 3 Met/IleGlu121 4 1 4 1 2 1Tyr122 13 – 5 10 – Tyr/PheVal123 10 (�3)b 1 8b 1 7b 1Pro124 – – – – 16 – AsnGly125 – – – – 3 –Gly126 3 – – – – –Glu127 9 1 5 – – – AspGlu170 7 1 12 1 – – AspAsn171 4 – 1 – – –Leu173 7c – 13c – – – Leu/MetThr183 18 – 28 1 – – Thr/AlaAsp184 9 – 9 – – –Phe327 7 – NRd NR NR NRH2O 5 (2)e – – – 2 (2)e –Total 165f 4 127 4 91 2

a –; no contact.b The PKAB3 structure has an alanine at this position. For a better comparison of the van der Waals (vdW) contacts the number in parentheses

count the interaction of the CG1 and CG2 of the valine of PKA, whereas the first number corresponds to the interaction up to the CB.c The PKAB3 has, like classical PKC isoforms, a methionine at this position and is therefore not directly comparable.d NR, no resolution.e The number in parentheses represent the number of different water molecules.f Includes the contacts mentioned in b.

TABLE VH-bonds between PKA/PKAB3 and inhibitors

Staurosporine BIM2-MolA BIM2-MolB

Glu121:O — N19 Glu121:O — N19 Glu121:O — N19bba 3.10 bb 2.80 bb 3.35Val123:N — O30 Val123:N — O32 Val123:N — O33bb 2.61 bb 2.81 bb 2.61Glu127:OE2 — N31 –b –scc 2.93Glu170:O — N31 Glu170:OE2 — N30 –bb 2.53 sc 2.86

– Thr183:OG1 — O33 –sc 2.72

a bb, backbone.b —, no contact.c sc, side chain.

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balanol (1BX6) also via direct interactions with Phe54, butthese ligand geometries do not induce the glycine loop motions.Thus, this highly conserved phenylalanine seems to play a keyrole in inhibitor binding; mutant studies are required to eval-uate the energetic consequences although glycine loop flexibili-ties will complicate their interpretation. The many interactions(24) of Val57 from the glycine loop with the planar surface ofstaurosporine (Table IV) are restricted to five contacts withindole II in BIM2(A) (Table IV).

Other notable differences in the binding mode of BIM2 andstaurosporine concern the number of van der Waals contactsbetween BIM2 and Tyr122 (Table IV). A comparison with thestaurosporine structure reveals a loss of 6 contacts because ofthe inwardly rotated position of the indole II of BIM2 (Fig. 6).In addition, an interaction between Phe327 and the inhibitor isnot established, whereas staurosporine exhibits seven contactsto this amino acid. In the PKA-BIM2 structure described here,Phe327 is flexible (with no apparent electron density, but acomparison with the PKA/staurosporine structure suggeststhat the distance between Phe327 and indole II of BIM2(A)would be too far for a contact). The contact between the inhib-itor and Phe327 may be the major reason why this stretch isordered in the partially open structures such as 1STC (10),1BX6 (11), and 1Q8W (12). In the open apoenzyme structures1CTP (42, 43) and 1J3H (both molecules (37)), the regionaround Phe327 is also unresolved. In the closed apoenzymestructures (e.g. 1Q61 und 1Q62 (7)), the whole enzyme archi-

tecture is more compact and the structures show this entirepolypeptide to be ordered.

PKAB3–BIM2 Binding Versus Hypothetical PKA–BIM2Binding—An evaluation of the likely inhibitor binding interac-tions in PKA requires an assessment of the effects of the threepoint mutations of PKAB3 relative to PKA. As described above,the exchange of Q181K (PKA position PKAB3) was introducedto prevent an artificial rotamer conformation of the Gln181 thatapparently resulted from the V123A exchange (7). As such,neither Gln181 nor Lys181 is expected to directly affect inhibitorbinding. The mutation V123A expands the volume of the ade-nine binding cavity, an alteration that potentially could alterthe character of the hinge hydrogen bonding interactions atthis position, although none has been evident from crystalstructures so far. BIM2 in molecules A and B has 8 to 7 van derWaals contacts to Ala123 (out of totals of 127 or 91, respec-tively). In silico exchange to valine reveals the formation of upto three van der Waals contacts in molecule A and no additionalvan der Waals contact in molecule B. In comparison, stau-rosporine has 13 hydrophobic contacts to the correspondingvaline (Table IV); in silico exchange to alanine removes onlythree of them. The most interesting exchange is L173M. Inmolecule A this methionine is surrounded by the aromaticmaleimide and indole core of the inhibitor, forming 13 van derWaals contacts mostly via the terminal methyl group. In silicomutation back to leucine basically preserves the side chainvolume, but shortens its extension, thereby removing most

FIG. 7. a, stereo view to compare theconformations of the two BIM2 molecules(yellow, BIM2 molecule A; green, BIM2molecule B). The compounds are superim-posed using the chemically (not spatially)equivalent atoms of the maleimide moiety(e.g. mapping O33 molecule A to O33 ofmolecule B). The two orientations ofBIM2 are related by a 2-fold symmetryaxis through the maleimide moiety. Theindole orientations are not related by thissymmetry transformation, however, asthey are rotated in each case toward theN-terminal lobe. b, comparison of thebinding conformers of the two BIM2 mol-ecules (yellow, BIM2 molecule A; green,BIM2 molecule B) and the staurosporine(color by atom). The compounds are fittedusing spatially (not chemically) equiva-lent atoms of the maleimide moiety (e.g.O33 molecule A on O32 of molecule B).Compared with staurosporine, the rota-tion of the indole rings out of the plane isstriking, as is the overall equivalence ofthe spatial volumes occupied by theinhibitors.

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hydrophobic contacts under the assumption that the BIM2 inPKA shows the same indole rotamers. Determination of theIC50 of BIM2 to PKA and PKAB3 reveals that PKAB3 (IC50,6.35 �M) is less sensitive to the inhibitor than PKA (IC50, 2.94�M) (Table VI). Thus, in PKA, BIM2 might be shifted somewhatto optimize contacts here.

However, previous efforts to co-crystallize wild type PKAtogether with BIM2 have failed, attempts with PKAB3 havesucceeded. The three residues that differ between PKA andPKAB3 seem at first glance unlikely to cause a difference incrystallization properties. The exchange Q181K is at the sur-face of the protein and could theoretically alter crystal contacts,but, there are no crystal contacts to either Gln181 or Lys181 oradjacent residues in the relevant crystals.

The exchanges of V123A and L173M are clearly shieldedwithin the ATP pocket and should not directly affect crystalli-zation. Because the crystal form described here involves differ-ent kinase configurations and lacks PKI binding, and presum-ably competes with the standard crystal form duringcrystallization, even rather subtle differences between PKAB3and PKA might lead to the differences in crystallization.

Proposed Binding Mode of BIM2 in PKC—Based on thecomplex structure of molecule A, and given the similaritiesamong AGC group protein kinases, the key binding mecha-nisms of BIM2 in PKC can be proposed. An alignment of PKAand PKC isoforms reveals that PKC isoforms � and � show thehighest degree of variability of amino acid residues involved inBIM2 interaction (Tables I and IV, in comparison to PKA).Deduced from the BIM2-molecule A complex and an analysis ofneighboring amino acids (defined by proximity to within 6.5 Å)five residues need to be considered to predict the binding mech-anism of BIM2 in PKC isoforms. The first of these residues isMet173, which has many van der Waals interactions with allmoieties of BIM2 (Table IV). An equivalent methionine is pres-ent in the three classic isoforms PKC�, PKC�I/II, and PKC�;

the other PKC isoforms have a leucine, like PKA (Table I). Thenext two residues are the PKA (and PKB) glutamic acid resi-dues of Glu127 and Glu170, replaced by aspartic acid residues inall PKC isoforms. Glu127 and Glu170 form contacts with thepyrrolidine ring, and in silico exchanges to aspartate show thatminor adjustments can re-establish a comparable number ofvan der Waals contacts (Asp127, 5; Glu127, 6; Asp170, 10; Glu170,13) to the pyrrolidine ring.

The final two variable residues in contact with BIM2 (A) areVal104 and Thr183, either likewise conserved in PKC or presentas Thr or Ala, respectively (Table I). The exchange V104Toccurs for all isoforms except PKC� and PKC. T183A is presentin six of the nine isoforms, whereas PKC�, PKC�, and PKChave a threonine as in PKA. In PKAB3, a hydrogen bond existsbetween BIM2:O33 and Thr183 (Fig. 6a); this interaction is ofcourse lost in the six PKC isoforms with alanine at this posi-tion. The van der Waals contacts to Val104 can be equivalentlyestablished to a threonine at this position, possibly supple-mented by a new hydrogen bond. This potential new hydrogenbond could compensate for the loss of the hydrogen bond atposition 183 in several PKC isoforms. Only PKC� lacks a hy-drogen bonding possibility at both positions.

The combination of the side chains at the three positions 104,173, and 183 are predicted to be the principal determinants ofBIM2 selectivity among PKC isoforms. (Because residues 127and 170 are conserved across PKC isoforms they cannot berelevant for isoform specificity.) Seven different combinationsof residues at positions 104, 173, and 183 occur among the nine

TABLE VIKm/IC50 values of PKA and mutants

PKA PKAB2 PKAB3

KmATP (�M) 12 128 30.6IC50 BIM2 (�M) 2.94 36.1 6.35

FIG. 8. Phe54-inhibitor interaction.a, interaction between Phe54 and C35 ofstaurosporine. b, interaction betweenPhe54 and C31 of BIM2. The flexibility ofPhe54 and the glycine loop enable it toadapt to different inhibitor configurationsand maintain apparently favorablecontacts.

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human PKC isoforms (PKC�1 and PKC�2 do not differ in thekinase core domain). This leads to the prediction of similarspecificities for isoform pairs PKC� and PKC�I/II, and PKC�and PKC�, as corroborated by the reported activity of therelated bisindolyl maleimide 1 toward different PKC isoforms.BIM1 is a potent inhibitor of PKC� (8.4 nM) and PKC�1 (18nM), a medium inhibitor of PKC� (210 nM) and PKC� (132 nM)and shows low inhibition toward PKC� (5.8 �M) (19, 44).

Proposed Binding of LY333531 in PKAB3/PKC—LY333531(Fig. 1B) is a PKC�I/II isoform-specific bisindolyl maleimideinhibitor (29). It inhibits PKC�I/II with IC50 values of 5–6 nM

and is 80–60-fold selective for these isoforms over PKC� (29).Other kinases like PKA and SRC are not inhibited byLY333531 (IC50 � 100 �M). LY333531 is in phase III clinicaltrials and has been shown to be effective against diabetic ret-inopathy and diabetic macular edema (Ref. 30 and citationstherein). Because of the cyclic linkage of both indoles,LY333531 has fewer conformations available to it comparedwith BIM2; it lacks, however, the strict planarity of staurospo-rine. Superposition of LY333531 onto the BIM2 inhibitor ofmolecule A shows that LY333531 can occupy the same vol-umes, with, however, a different indole configuration (Fig. 9).The maleimide can remain unchanged with respect to BIM2.The indole arrangements of LY333531 are restricted by thecyclic linkage, however, so that the indole rings are orientedoutwards, in contrast to the inwards rotation of indole II of bothBIM2 conformations. In this respect, LY333531 is more similarto staurosporine. One consequence of this is that van der Waalscontacts to Phe327, which is conserved in all PKC isoforms, maybecome established as in 1STC, and the number of contacts toTyr122 will increase. The cyclic linker can be modeled in alter-nate conformations (Fig. 9), in each conformation the dimeth-ylamine remains near the N30 position of the pyrrolidine ofBIM2 and van der Waals contacts to Phe54 of the glycine loopand Glu/Asp127 and Glu/Asp170 can be established.

To evaluate these features for LY333531/PKC inhibition, wemodeled the inhibitor into the molecule A structure after insilico exchanges V104T, A123V, P124N, E127D, E170D, andT183A to mimic PKC�I/II. Furthermore, missing amino acidresidues 317–332 were modeled based on the structure of PKA/staurosporine. The greatest uncertainties in the model lie incontacts with Val57 and with the glycine loop in general, be-cause of its flexibility. The isoform selectivity profile for thebisindolyl maleimide LY333531 includes a significant prefer-ence for PKC�I/II over PKC�, which cannot be explained bythis model, and no significant differences in amino acid com-position exist within 6.5 Å of the inhibitor. The explanationprobably involves either a new rotamer conformation to bring anew side chain into inhibitor proximity (as previously observedby PKAB2, the precursor of PKAB3 (38), Table VI) or alterna-tively involves factors more subtle than the side chain compo-

sition of the binding pocket, such as protein backbone shifts.This question can be clarified by crystal structures of differentPKC isoforms and LY333531.

Conclusions—Crystallographic studies of enzyme-inhibitorcomplex structures provide information directly relevant fordrug design, particularly with respect to potency and selectiv-ity. For protein kinases, and their typically ATP competitiveinhibitors, the mechanisms by which kinases can be selectivelyinhibited lie mainly in variations among the residues that linethe ATP-binding site, or by kinase specific flexibilities. Thelatter may be related to functionally relevant activity modula-tion mechanisms, or may be sequence-specific ligand induciblestructural modifications.

The structures we describe here show aspects of both phe-nomena, uniquely showing extreme variability within a singlecrystal. Using a mutant form of PKA as a surrogate for PKC wesucceeded in the co-crystallization of the inhibitor BIM2. Theinhibitor binds in two different conformations to two differentconformations of the same kinase. Each binding mode shows arange of apparent ligand-induced binding modes. Simulta-neously, the different kinase lobe orientations, including themost open form of PKA observed so far, may conversely deter-mine the overall inhibitor binding mode.

Although one of the structures (molecule A) resembles inmany aspects the structure of PKA with staurosporine, theglycine loop is distorted in a new way, there are no contactswith the AGC-specific residue Phe327, and there are new con-tacts to the carboxyl group of Glu170 that could affect thebinding of substrate peptides. Even more unusual is the othermolecule (B) in the asymmetric unit, with its extreme openconformation and additional rotations of inhibitor N-lobe anddisplacement of the hinge region peptide. Here, the glycine looploses its antiparallel �-sheet character (45) and as in moleculeA no contacts are found to Phe327 or to the C-terminal lobe ofthe kinase. The structural similarities of BIM2 and LY333531,an inhibitor against diabetic retinopathy currently in phase IIIclinical trials, enable structure-based modeling of the bindingmode of the drug. As a kind of intermediate between stauros-porine and BIM2, LY333531 would be able to bind in the samevolume and hinge region interactions as BIM2, but probablywould additionally make van der Waals contacts to Phe327.Similar to both BIM2 and staurosporine, one methyl group ofthe free rotatable dimethylamine group is positioned appropri-ately to establish a contact to Phe54 on the tip of the glycineloop. Also, it is likely that LY333531 is able to make at least onehydrogen contact to either the Val104 or Thr183 homologues inthe various PKC isoforms. The combination of a valine with analanine in PKC� might explain the lowest affinity of LY333531for PKC� from all conventional and novel PKC isoforms tested(29).

FIG. 9. Modeling the binding modeof the PKC selective inhibitorLY333531 currently undergoingphase III clinical trials in PKAB3.Because of the cyclic linkage of the twoindole rings, the ring configurations arerestricted to relatively symmetric confor-mations that place the six memberedrings of each indole at the periphery of theinhibitor, a configuration not seen for theBIM2 geometries.

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Acknowledgment—We thank Prof. Volker Kinzel for constant support.

Note Addid in Proof—Our suggestions for the binding mode ofLY333531 in PKC are corroborated by the just published structure ofLY333531 in PDK1 from Komander et al. (47).

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Dual Binding Mode of BIM2 to PKA23690

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Engh and Dirk BossemeyerMichael Gassel, Christine B. Breitenlechner, Norbert König, Robert Huber, Richard A.

Orientations to Different Conformations of Protein Kinase AThe Protein Kinase C Inhibitor Bisindolyl Maleimide 2 Binds with Reversed

doi: 10.1074/jbc.M314082200 originally published online March 1, 20042004, 279:23679-23690.J. Biol. Chem. 

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