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• W2047875304 abstract "As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions. BIM2 co-crystallized as an asymmetric pair of kinase-inhibitor complexes. In this asymmetric unit, the two kinase domains have different lobe configurations, and two different inhibitor conformers bind in different orientations. One kinase molecule (A) is partially open with respect to the catalytic conformation, the other (B) represents the most open conformation of PKA reported so far. In monomer A, the BIM2 inhibitor binds tightly via an induced fit in the ATP pocket. The indole moieties are rotated out of the plane with respect to the chemically related but planar inhibitor staurosporine. In molecule B a different conformer of BIM2 binds in a reversed orientation relative to the equivalent maleimide atoms in molecule A. Also, a critical active site salt bridge is disrupted, usually indicating the induction of an inactive conformation. Molecular modeling of the clinical phase III PKC inhibitor LY333531 into the electron density of BIM2 reveals the probable binding mechanism and explains selectivity properties of the inhibitor. As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions. BIM2 co-crystallized as an asymmetric pair of kinase-inhibitor complexes. In this asymmetric unit, the two kinase domains have different lobe configurations, and two different inhibitor conformers bind in different orientations. One kinase molecule (A) is partially open with respect to the catalytic conformation, the other (B) represents the most open conformation of PKA reported so far. In monomer A, the BIM2 inhibitor binds tightly via an induced fit in the ATP pocket. The indole moieties are rotated out of the plane with respect to the chemically related but planar inhibitor staurosporine. In molecule B a different conformer of BIM2 binds in a reversed orientation relative to the equivalent maleimide atoms in molecule A. Also, a critical active site salt bridge is disrupted, usually indicating the induction of an inactive conformation. Molecular modeling of the clinical phase III PKC inhibitor LY333531 into the electron density of BIM2 reveals the probable binding mechanism and explains selectivity properties of the inhibitor. Deregulated protein kinase activity causes a wide variety of human diseases, usually by producing an overactive kinase. This is consistent with the fact that most protein kinases in the cell are inactivated most of the time to ensure the integrity of signal transduction. Thus, the many diseases that are correlated with protein kinase deregulation, including the majority of all cancers, usually arise from mutations or other events that activate kinases, cause their overexpression, or disable their intracellular inhibition. The prevalence of kinase deregulation in disease clearly demonstrates the need for therapeutic protein kinase inhibitors, whereas the ubiquity and variety of protein kinases (collectively, the “kinome”) necessitate precise target selectivity. Despite this seeming difficulty, several protein kinase inhibitors have been approved for human treatment or are in advanced clinical trials. Crystal structure analyses of protein kinase inhibitor complexes reveal the intermolecular interactions responsible for ligand binding, and have thereby enabled structure-based rational design and optimization of kinase inhibitors. To date, crystal structures have been determined for some 30 protein kinases, representing some 6% of the 518 protein kinases in the human genome (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6240) Google Scholar). Many of these structures have been complexes with protein kinase inhibitors, but most have shown an inactivated state often incompatible with inhibitor binding. Inactivity is associated most often with displacements of helix C, the major α helix of the kinase N-lobe, with concomitant disruption of the active site salt bridge in the active site between a conserved lysine residue and a conserved glutamate located in the middle of helix C. Similarly often, the activation loop shows unproductive conformations, either blocking the active site, or locking helix C into an inactive conformation, or causing other structural states of the kinase incompatible with kinase activity. Often, these structures are “open” with respect to the “closed” conformation of kinases in catalytic conformations. In more than half of all protein kinase structures inactivity is associated with a steric block in the ATP-binding site (2Engh R.A. Bossemeyer D. Pharmacol. Ther. 2002; 93: 99-111Crossref PubMed Scopus (87) Google Scholar, 3Engh R.A. Bossemeyer D. Adv. Enzyme Regul. 2001; 41: 121-149Crossref PubMed Scopus (38) Google Scholar, 4Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1361) Google Scholar). Despite the variations in sequence, the fold of the active protein kinase catalytic domain is well conserved. Inactivated protein kinase structures differ more, but cluster into protein kinase subfamilies that reflect different inactivation mechanisms. As a consequence of the conservation of the active structure, many properties can be analyzed with respect to relatively few sequence positions that define that property. A centrally important example of such a property is the selectivity of binding at the ATP binding pocket. Protein kinases share a common bi-lobal catalytic domain structure that forms the ATP-binding site at the lobal interface. ATP binds at this interface via interactions with some 15 residues of the protein, including about 10 side chain interactions that therefore are especially important as potential determinants of ATP site inhibitor selectivity. Thus, the essential binding properties of ATP and other ATP site ligands can in many cases be simulated for a particular protein kinase target by a limited set of point mutations of a closely related protein kinase. The construction of such hybrids has been demonstrated for kinases (5Fox T. Coll J.T. Xie X.L. Ford P.J. Germann U.A. Porter M.D. Pazhanisamy S. Fleming M.A. Galullo V. Su M.S. Wilson K.P. Protein Sci. 1998; 7: 2249-2255Crossref PubMed Scopus (129) Google Scholar, 6Bishop A.C. Shah K. Liu Y. Witucki L. Kung C. Shokat K.M. Curr. Biol. 1998; 8: 257-266Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 7Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar). Even though subtle differences in structure or flexibility can lead to quite different overall reaction kinetics, for practical ligand design purposes, most or all protein-ligand interactions will be revealed or can be modeled using the surrogate kinase approach. That a single residue can be identified as the principal selectivity determinant for an inhibitor type by mutation of a series of kinases verifies this approach (8Liu Y. Bishop A. Witucki L. Kraybill B. Shimizu E. Tsien J. Ubersax J. Blethrow J. Morgan D.O. Shokat K.M. Chem. Biol. 1999; 6: 671-678Abstract Full Text PDF PubMed Scopus (232) Google Scholar). Along these lines, the cAMP-dependent protein kinase (PKA) 1The 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 asymmetric unit; LY333531, (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-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′-(β,γ-imino)triphosphate; Mes, 4-morpholineethanesulfonic acid. has been used as a surrogate kinase for co-crystallization with several protein kinase inhibitors, such as H7, H8, and H89 (9Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. J. Biol. Chem. 1996; 271: 26157-26164Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), staurosporine (10Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure. 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), balanol (11Narayana N. Diller T.C. Koide K. Bunnage M.E. Nicolaou K.C. Brunton L.L. Xuong N.H. Ten Eyck L.F. Taylor S.S. Biochemistry. 1999; 38: 2367-2376Crossref PubMed Scopus (93) Google Scholar), and recently the Rhokinase inhibitors Y27632, H1152P, and Fasudil (12Breitenlechner C.B. Gaßel M. Hidaka H. Kinzel V. Huber R. Engh R.A. Bossemeyer D. Structure. 2003; 11: 1595-1607Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The complexes with staurosporine, balanol, and Fasudil are in partially open conformations, in contrast to the other inhibitor/substrate-PKA complexes that are in closed conformations. In these three structures the hydrogen bond between the activation loop Thr197 phosphoryl group and His87 from helix C of the N-lobe is not formed as a result of the partial opening of the cleft via rotation of the N-lobe with respect to the C-lobe. Mutants of PKA have also been designed to improve its value as surrogate kinase for PKB inhibitors (7Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar). Because of its close relationship to PKC and its well established crystallization conditions, PKA is currently also the best model system for studying PKC inhibitors. So far, co-crystallization attempts with PKA and the flexible bisindolyl maleimide (BIM) cognates of staurosporine, described as PKC inhibitors (13Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 14Davis P.D. Hill C.H. Keech E. Lawton G. Nixon J.S. Sedgwick A.D. Wadsworth J. Westmacott D. Wilkinson S.E. FEBS Lett. 1989; 259: 61-63Crossref PubMed Scopus (439) Google Scholar, 15Davis P.D. Elliott L.H. Harris W. Hill C.H. Hurst S.A. Keech E. Kumar M.K. Lawton G. Nixon J.S. Wilkinson S.E. J. Med. Chem. 1992; 35: 994-1001Crossref PubMed Scopus (217) Google Scholar, 16Davis P.D. Hill C.H. Lawton G. Nixon J.S. Wilkinson S.E. Hurst S.A. Keech E. Turner S.E. J. Med. Chem. 1992; 35: 177-184Crossref PubMed Scopus (180) Google Scholar, 17Bit R.A. Davis P.D. Elliott L.H. Harris W. Hill C.H. Keech E. Kumar H. Lawton G. Maw A. Nixon J.S. Vesey D.R. Wadsworth J. Wilkinson S.E. J. Med. Chem. 1993; 36: 21-29Crossref PubMed Scopus (161) Google Scholar), have failed to produce crystal structures. However, the triple mutant V123A,L173M,Q181K of PKAα (PKAB3), originally designed as a model for PKB (7Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar), has formed high quality crystals of a BIM inhibitor complex. A sequence alignment of PKAα and PKBα with PKC isoforms (Table I) shows how the PKAB3 triple mutant is similar to PKA as a surrogate for PKC with the additional ability to model inhibitor-methionine interactions for three conventional PKC isoforms.Table IAmino acids residues of different protein kinases Three distinct subfamilies of PKC isoforms can be defined according to their essential activators: conventional PKCs (α, βI/II, and γ) require phosphatidylserine, diacylglycerol, and Ca2+; novel PKCs (δ, ϵ, η, and θ) need phosphatidylserine and diacylglycerol but not Ca2+; atypical PKCs (ζ and Ι) are insensitive to both diacylglycerol and Ca2+ although phosphatidylserine regulates activity (for reviews, see Refs. 18Newton A.C. Biochem. J. 2003; 370: 361-371Crossref PubMed Scopus (663) Google Scholar and 19Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar and citations therein). Furthermore, additional lipid mediators, like fatty acids and lysophospholipids, have been shown to influence the catalytic activity of PKCs (reviewed in Ref. 20Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2363) Google Scholar). In general, interaction of PKCs with the activators leads to phosphorylation of a threonine residue on the activation loop of all PKC isoforms and additionally of a serine or threonine residue in the hydrophobic motif of the conventional and novel PKCs. The atypical PKCs possess a glutamate at the hydrophobic motif phosphorylation position that intrinsically performs the activation function (for reviews, see Refs. 18Newton A.C. Biochem. J. 2003; 370: 361-371Crossref PubMed Scopus (663) Google Scholar and 21Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (835) Google Scholar). PKC isoforms are involved in nearly all essential cell processes, PKCα, for example, is involved in regulation of proliferation, apoptosis, differentiation, cell migration, adhesion, among other cellular and pathogenic processes (for a review, see Ref. 22Nakashima S. J. Biochem. (Tokyo). 2002; 132: 669-675Crossref PubMed Scopus (201) Google Scholar). Despite its early identification and importance in cancer research, no PKC crystal structure has been reported to date. The only available co-crystal structures of PKC inhibitors with a kinase target are those of the relatively unselective staurosporine (IC50, PKC 5 nm (23Meggio F. Deana A.D. Ruzzene M. Brunati A.M. Cesaro L. Guerra B. Meyer T. Mett H. Fabbro D. Furet P. Dobrowolska G. Pinna L.A. Eur. J. Biochem. 1995; 234: 317-322Crossref PubMed Scopus (251) Google Scholar)) and its closely related derivative UCN01 (IC50, PKCα 29 nm (24Seynaeve C.M. Kazanietz M.G. Blumberg P.M. Sausville E.A. Worland P.J. Mol. Pharmacol. 1994; 45: 1207-1214PubMed Google Scholar)). Besides co-crystallization with PKA (10Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure. 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) (Protein Data Bank code 1STC), the extended and rigid planar staurosporine has been crystallized with Cdk2 (25Lawrie A.M. Noble M.E. Tunnah P. Brown N.R. Johnson L.N. Endicott J.A. Nat. Struct. Biol. 1997; 4: 796-801Crossref PubMed Scopus (242) Google Scholar) (Protein Data Bank code 1AQ1), CSK (26Lamers M.B. Antson A.A. Hubbard R.E. Scott R.K. Williams D.H. J. Mol. Biol. 1999; 285: 713-725Crossref PubMed Scopus (138) Google Scholar) (Protein Data Bank code 1BYG), and others. UCN01 (7-hydroxystaurosporine) has been co-crystallized with Cdk2 (27Johnson L.N. De Moliner E. Brown N.R. Song H. Barford D. Endicott J.A. Noble M.E. Pharmacol. Ther. 2002; 93: 113-124Crossref PubMed Scopus (54) Google Scholar) (Protein Data Bank code 1PKD) and Chk1 (28Zhao B. Bower M.J. McDevitt P.J. Zhao H.Z. Davis S.T. Johanson K.O. Green S.M. Concha N.O. Zhou B.B.S. J. Biol. Chem. 2002; 277: 46609-46615Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) (Protein Data Bank code 1NVQ). The bisindolyl maleimide class of PKC inhibitors is derived from staurosporine by elimination of a single bond that converts the extended planar aromatic group into the three aromats of the compound names with corresponding additional degrees of flexibility. One PKC inhibitor of this class, LY333531, shows PKC isoform specificity (e.g. 80- and 60-fold selectivity for PKCβ I and PKCβ II over PKCα (29Jirousek M.R. Gillig J.R. Gonzalez C.M. Heath W.F. McDonald III, J.H. Neel D.A. Rito C.J. Singh U. Stramm L.E. Melikian-Badalian A. Baevsky M. Ballas L.M. Hall S.E. Winneroski L.L. Faul M.M. J. Med. Chem. 1996; 39: 2664-2671Crossref PubMed Scopus (325) Google Scholar) and is in phase III clinical trials for diabetic retinopathy and diabetic macular edema (Ref. 30Frank R.N. Am. J. Ophthalmol. 2002; 133: 693-698Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar and citations therein). The effects of the additional flexibility of BIM inhibitors on the structural binding modes, and the means by which this alteration can introduce selectivity to the inhibitor have not been explained. Here we present the crystal structure of bisindolyl maleimide 2 (BIM2) in a complex with the triple mutant V123A,L173M,Q181K (PKAB3) of PKA. By means of this surrogate kinase approach, we identify the key binding modes and can evaluate their significance for PKC. The asymmetric unit of the crystal structure consists of two protein-inhibitor complexes, with different conformations of the two kinase molecules, bound with opposite orientations of different conformers of BIM2. One kinase monomer has an intermediate open state like in the staurosporine structure; the other is in the most open conformation observed for PKA so far. In the open conformation, the whole N-terminal lobe including helix C is rotated by more than 20° compared with the closed form. The active site salt bridge between Lys72 and Glu91 is disrupted, a characteristic of inactive kinase conformations. The two different inhibitor binding modes are enabled by the inherent symmetry of the maleimide moiety and the rotational freedom available to the indole moieties. The similarity of BIM2 and LY333531 (Fig. 1) allows modeling of LY333531-PKC interactions and suggests an explanation for the selectivity properties of the inhibitors. Protein Expression and Purification—Recombinant mutated bovine Cα catalytic subunit of the cAMP-dependent protein kinase (PKAB3 (7Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar)) was solubly expressed in Escherichia coli BL21(DE3) cells and then purified via affinity chromatography and ion exchange chromatography as described earlier (9Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. J. Biol. Chem. 1996; 271: 26157-26164Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Two positions distinguish bovine (Asn32 and Met63) from human PKA (Ser32 and Lys63). 4-Fold phosphorylated protein was used for crystallization of BIM2. Activity Tests—The determination of enzyme activity was accomplished by an ATP regenerative NADH consuming assay according to Ref. 31Cook P.F. Neville Jr., M.E. Vrana K.E. Hartl F.T. Roskoski Jr., R. Biochemistry. 1982; 21: 5794-5799Crossref PubMed Scopus (346) Google Scholar. After the addition of 0.42 mm MEGA 8 (ensures solubility of the inhibitor) to the assay mixture (100 mm Mops, pH 6.8, 100 mm KCl, 10 mm MgCl2, 1 mm phosphoenolpyruvate, 0.1 mm Kemptide, 1 mm β-mercaptoethanol, 15 units/ml lactate dehydrogenase (Sigma), 8 units/ml pyruvate kinase (Sigma), 0.21 mm NADH) we have added the successive Me2SO/inhibitor solution and the enzyme and started the reaction with ATP. The decrease of NADH was measured as time-dependent at λ = 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, 25 mm Mes/Bistris, pH 6.4. The hanging drop vapor diffusion method against 15% methanol as precipitant was used to obtain a 100 × 100 × 300-μm crystal. Data Collection and Structure Determination—Diffraction data were measured at the Deutsches Elektronen Synchrotron (DESY, Hamburg) from frozen crystals on a CCD detector (Mar research) at 1.05 Å wavelength. The data were processed with the programs MOSFLM and SCALA. The crystals have orthorhombic symmetry (P212121) with cell constants 82.06, 89.00, and 116.38 in a crystal packing arrangement not previously reported (Table II). The structure was determined by molecular replacement using MOLREP from the CCP4 program suite. 2www.ccp4.ac.uk/main/html. As starting model we chose a PKA-PKI-(5-24)-staurosporine complex (1STC (10Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure. 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Calculation of Matthews coefficient and solvent content suggested two molecules in the asymmetric unit with 52.3% solvent. Indeed, very good monomer rotation and translation function solution was 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 its presence in crystallization solutions as usual. Furthermore, difference densities in the B molecule showed that large segments of the molecule needed to be remodeled. We deleted the segments (the entire N-terminal lobe up to 123 and the C-terminal residues from 315 onwards) of about 150 amino acids together and calculated new maps. After several circles 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 molecule A only residues 315-350 were omitted and rebuilt. The segment 317-332 (330 for molecule B) remained undefined. Phosphorylation sites were found at Ser139, Thr197, and Ser338. Ser10 is not resolved. Water molecules were automatically inserted using CCP4 programs PEAK-MAX and WATPEAK and visually inspected. Finally, the inhibitor molecules were built and the whole complex was further refined. Refmac 5.1.24 was used for refinement, and MOLOC 3www.moloc.ch. was used for model building and graphical modeling. For data and refinement statistics, see Table II.Table IIData collection and refinement statisticsPKAB3-BIM2Data collectionSpace groupP212121Cell (a, b, c) (Å)82.06 89.00 116.39Resolution range (Å)24.77—2.5Observations read279090Unique reflections31110Completeness (%)99.8I/sigma5.2Rsym (%)9.9Multiplicity5.2RefinementNumber of Non-hydrogen atoms used5411in refinementR factor (%)23.3Free R factor (%)29.1Free R value test size (%)5.1Resolution range70—2.5Used reflections28539Standard deviation from ideal valuesBond length (Å)0.016Bond angles (°)1.610Temperature factorsAll atoms52.4Main chain atoms PKAB3, Mol A58.0Side chain atoms PKAB3, Mol A58.5Inhibitor atoms BIM2, Mol A42.6Main chain atoms PKAB3, Mol B46.3Side chain atoms PKAB3, Mol B47.2Inhibitor atoms BIM2, Mol B54.8Solvent molecules47.8 Open table in a new tab Superimposition, Calculation of Secondary Elements, and Angle Determination—Superimpositions were performed using the programs Insight 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 structure 1CDK as basis. The secondary elements were calculated with the program InsightII using the Kabsch-Sander algorithm. The angle determination was performed in the following way. The backbone of each first and last two amino acids of the helices were taken to calculate the center of masses using the gromacs package. 4www.gromacs.org. These center of masses defined the top and bottom of the helix axes. The coordinates of the top and bottom were used to create vectors in the R3 and with cosβ = x·y/|x|·|y| (x and y are the vectors defining the helix axes). The angle between these vectors of two different structures was calculated. Sequence Alignment—Sequence alignments were performed using ClustalW. 5www.ebi.ac.uk/clustalw. PKAB3 as Model for the PKC-ATP-binding Pocket—The structural and sequence similarities of AGC kinase subgroup members PKA and PKB (Ref. 32Bossemeyer D. Engh R.A. Kinzel V. Ponstingl H. Huber R. EMBO J. 1993; 12: 849-859Crossref PubMed Scopus (372) Google Scholar, Protein Data Bank code 1CDK; Ref. 33Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (432) Google Scholar, Protein Data Bank code 1O6K) imply a correspondingly similar structure for the kinase domain of PKC. Thus, the clearest determinant of the selectivity of protein kinase inhibitors is the amino acid composition of the catalytic ATP-binding site. It follows that amino acid exchanges in the catalytic site of one kinase can be useful as a surrogate for another kinase, reported, for example, for PKA mutants that mimic PKB (7Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar). For this study we used the triple mutant PKAB3 (V123A,L173M,Q181K), which was originally constructed to model the adenine-binding site of PKB in PKA. Sequence alignment of PKA, PKB, and PKC shows that one of these mutations, namely L173M (numbering of PKA), introduces a residue that is conserved in PKB and the three classical PKC isoforms α, βI/II, and γ. The other PKC isoforms have, like PKA, a leucine residue in the corresponding position. The second exchange, V123A, does not occur among PKC isoforms, but the important contacts of residue 123 to inhibitors are backbone contacts and presumably relatively unaffected by the side chain. The third exchange, Q181K, was chosen only after observation that the double mutant (V123A,L173M) lead to a new rotamer conformation of the glutamine of PKA that placed the amide group into the ATP binding pocket (7Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar). This mutation introduces a residue conserved as a lysine among all PKC isoforms. Overall Structure—The triple mutated recombinant catalytic subunit of cyclic AMP-dependent PKA and the PKC inhibitor BIM2 formed crystals notably lacking the pseudo-substrate peptide PKI(5-24), although the peptide was present under the standard crystallization conditions. The 4-fold phosphorylated PKA is disordered at the N terminus including the phosphorylation position of (p)Ser10. The region between residues 317 and 332 is also not visible. The corresponding dimer formed the asymmetric unit in an orthorhombic space group P212121 (Table II) with cell constants and a packing arrangement previously unobserved for PKA (a = 82.1, b = 89.0, c = 116.4). This arrangement is apparently induced by BIM2 binding and arises from the two new N- and C-domain conformations of PKA in the crystal. One of the PKA complexes of the asymmetric unit (molecule A) is in an intermediate open conformation, similar to that observed for the PKA complex with staurosporine (1STC (10Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure. 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar)) and also HA-1077 (1Q8W (12Breitenlechner C.B. Gaßel M. Hidaka H. Kinzel V. Huber R. Engh R.A. Bossemeyer D. Structure. 2003; 11: 1595-1607Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar)), whereas the other, molecule B, is in the most open conformation described so far for PKA (Fig. 2). The inhibitors in both molecules A and B occupy the ATP-binding site, but different conformations of the inhibitor bind with different orientations in the two molecules (Fig. 3).Fig. 3Superposition of structures 1CDK, BIM2MolA, and BIM2MolB. Superposition of 1CDK (gray), BIM2MolA (red), and BIM2MolB (blue) are shown.View Large Image Figure ViewerDownload (PPT) Many different relative N- and C-lobe orientations have been observed for PKA. One measure to identify the closed conformations is the existence of an H-bond between the imidazole of His87 from helix C and the phosphoryl group of Thr197 from the activation loop. Structures for which this His87-(p)Thr197 contact is broken because of the opening movement of the N-lobe exhibit a variety of open conformations (Fig. 2). The hinging movements of PKA that cause the opening occur primarily within the dipeptide segment of Gly125 and Gly126 positioned between the ATP binding residues and helix D, as defined originally by Olah and co-workers (34Olah G.A. Mitchell R.D. Sosnick T.R. Walsh D.A. Trewhella J. Biochemistry. 1993; 32: 3649-3657Crossref PubMed Scopus (73) Google Scholar). Rotations of helix C can be taken as a measure of the opening of PKA structures, and ranks structures from the most closed in structure 1CDK through other open conformations of PKA such as staurosporine bound (1STC (10Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure. 1997; 5: 162" @default.
• W2047875304 created "2016-06-24" @default.
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• W2047875304 creator A5034573042 @default.
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• W2047875304 date "2004-05-01" @default.
• W2047875304 modified "2023-09-30" @default.
• W2047875304 title "The Protein Kinase C Inhibitor Bisindolyl Maleimide 2 Binds with Reversed Orientations to Different Conformations of Protein Kinase A" @default.
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