Conformational diversity of catalytic cores of protein kinases.

X-ray crystallography of the protein kinase family has provided an impressive array of crystal structures, setting the stage for rational design of specific inhibitors of these vitally important regulators of the signaling pathways of the cell. Initial work on the first crystal structure of a protein kinase, cyclic AMP-dependent protein kinase, has provided evidence of conformational changes suggested to be critical for the common catalytic event of transferring the gamma phosphate from ATP onto the targeted protein. This review updates the current status of the extent of conformational diversity of the protein kinase family and suggests that both the nature and the extent of those changes can provide a rationale for the increased occurrence of specific protein kinase inhibitors targeted at the ATP-binding site. It focuses on the fact that in addition to the sequence diversities in ATP binding clefts reported recently, there is conformational diversity in the beta sheets of the upper domains of the catalytic cores. This difference is directly related to the regulation of kinases by multiple mechanisms.

Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure.

The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vivo at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain.

Detergent binding to unmyristylated protein kinase A--structural implications for the role of myristate.

Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knighton et al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlsson et al., 1993; Zheng et al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zheng et al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This "inside out" motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60v-src.

Modelling study of protein kinase inhibitors: binding mode of staurosporine and origin of the selectivity of CGP 52411.

A model for the binding mode of the potent protein kinase inhibitor staurosporine is proposed. Using the information provided by the crystal structure of the cyclic-AMP-dependent protein kinase, it is suggested that staurosporine, despite a seemingly unrelated chemical structure, exploits the same key hydrogen-bond interactions as ATP, the cofactor of the protein kinases, in its binding mode. The structure-activity relationship of the inhibitor and a docking analysis give strong support to this hypothesis. The selectivity of the dianilinophthalimide inhibitor CGP 52411 towards the EGF-receptor protein tyrosine kinase is rationalized on the basis of the model. It is proposed that this selectivity originates in the occupancy, by one of the anilino moieties of the inhibitor, of the region of the enzyme cleft that normally binds the ribose ring of ATP, which appears to possess a marked lipophilic character in this kinase.

Catalytic activity and transformation potential of v-Src require arginine 385 in the substrate binding pocket.

Tyrosine kinase are important mediators of signal transduction in eukaryotic cells. In order to better understand the mechanism of catalysis we studied a set of mutants of the prototype tyrosine kinase, the c-Src protein, a homologue of the Rous Sarcoma virus oncogene. Based on an X-ray structure of cAMP-dependent protein kinase (cAPK) we mutated an arginine residue conserved in subdomain VI of all known kinases to a non-charged residue. This residue coordinates phosphate of the autophosphorylation site located in subdomain VII of cAPK and this interaction has been proposed to be crucial for substrate binding. The mutant R385A of c-Src had low kinase activity towards exogenous substrates yet was able to autophosphorylate at tyrosine 416. When introduced into an activated v-src gene the R385A mutation totally blocked cell transformation. Our data suggest that the function of the conserved arginine 385 is to coordinate the phosphate of the autophosphorylation site and to provide in this way a stable template for substrate binding.

Structural basis for chromosome X-linked agammaglobulinemia: a tyrosine kinase disease.

X-linked agammaglobulinemia (XLA) is a hereditary defect of B-cell differentiation in man caused by deficiency of Bruton tyrosine kinase (BTK). A three-dimensional model for the BTK kinase domain, based on the core structure of cAMP-dependent protein kinase, was used to interpret the structural basis for disease in eight independent point mutations in patients with XLA. As Arg-525 of BTK has been thought to functionally substitute for a critical lysine residue in protein-serine kinases, the mutation Arg-525-->Gln was studied and found to abrogate the tyrosine kinase activity of BTK. All of the eight mutations (Lys-430-->Glu, Arg-520-->Glu, Arg-525-->Gln, Arg-562-->Pro, Ala-582-->Val, Glu-589-->Gly, Gly-594-->Glu, and Gly-613-->Asp) were located on one face of the BTK kinase domain, indicating structural clustering of functionally important residues.

Intermolecular contacts in various crystal forms related to the open and closed conformational states of the catalytic subunit of cAMP-dependent protein kinase.

All hitherto solved crystal structures of the catalytic (C) subunit of cAMP-dependent protein kinase can be classified into two groups, those with a closed and those with an open conformation of the ATP-binding lobe. The molecules with the closed conformation are all related by a crystallographic 2(1) axis that connects them into an infinite-chain motif. The motif has only one large contact region that involves many residues, several of them in the ATP-binding lobe, embedded in an extensive network of water molecules. The dominant feature of this region is the hydrophobic interaction between Trp196 and Arg133, Arg134. This motif has been found so far in three different crystal forms, two correspond to ternary enzyme-inhibitor-ATP complexes with mammalian and recombinant C, and one to a binary enzyme-inhibitor complex with recombinant C. The open conformation has been found in two closely related crystal structures, both of cubic symmetry, of the apoenzyme and a binary complex of the mammalian catalytic subunit. In this cubic structure of the binary complex, the hydrogen-bonded intramolecular contacts between Arg18 of the inhibitor and the ATP-binding lobe of the binary and ternary complexes of the recombinant enzyme are missing due to a strong hydrophobic intermolecular contact involving the diiodinated Tyr7. In solution, no crystal contacts prevent these hydrogen bonds involving Arg18 from forming so that it is likely that the binary complex with Tyr7 of the peptide inhibitor iodinated or not, can assume the closed conformation in solution. While the closed structure very likely represents a stable conformation in solution, there is no evidence to suggest that the open conformation represents a unique stable conformational state of the enzyme in solution.

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.

Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.

Structure of the mammalian catalytic subunit of cAMP-dependent protein kinase and an inhibitor peptide displays an open conformation.

The crystal structure of a binary complex of the porcine heart catalytic (C) subunit of cAMP-dependent protein kinase (space group P4(1)32; a = 171.5 A) complexed with a di-iodinated peptide inhibitor, PKI(5-24), has been solved and refined to 2.9 A resolution with an overall R of 21.1%. The r.m.s. deviations from ideal bond lengths and angles are 0.022 A and 4.3 degrees. A single isotropic B of 17 A(2) was used for all atoms. The structure solution was carried out initially by molecular replacement of electron density followed by refinement against atomic coordinates from orthorhombic crystals of a binary complex of the mouse recombinant enzyme previously described [Knighton, Zheng, Ten Eyck, Ashford, Xuong, Taylor & Sowadski (1991). Science, 253, 407-414]. The most striking difference between the two crystal structures is a large displacement of the small lobe of the enzyme. In the cubic crystal, the beta-sheet of the small lobe is rotated by 15 degrees and translated by 1.9 A with respect to the orthorhombic crystal. Possible explanations for why this binary complex crystallized in an open conformation in contrast to a similar binary complex of the recombinant enzyme are discussed. This study demonstrates that considerable information about parts of a crystal structure can be obtained without a complete crystal structure analysis. Specifically, the six rigid-group parameters of a poly alanine model of the beta-structure were obtained satisfactorily from a crystal structure by refinement of difference Fourier coefficients based on an approximate partial structure model.

cAMP-dependent protein kinase defines a family of enzymes.

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E. coli, the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thr197 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.

Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor.

To identify structural features that distinguish protein-tyrosine kinases from protein-serine kinases, a molecular model of the kinase domain of epidermal growth factor receptor was constructed by substituting its amino acid sequence for the amino acid sequence of the catalytic subunit of cAMP-dependent protein kinase in a 2.7-A refined crystallographic model. General folding was conserved as was the configuration of invariant residues at the active site. Two sequence motifs that distinguish the two families correspond to loops that converge at the active site of the enzyme. A conserved arginine in the catalytic loop is proposed to interact with the gamma phosphate of ATP. The second loop provides a binding surface that positions the tyrosine of the substrate. A positively charged surface provides additional sites for substrate recognition.

2.0 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and detergent.

. A mutant (Serl39Ala) of the mouse recombinant catalytic (C) subunit of cAMP-dependent protein kinase was co-crystallized with a peptide inhibitor, PKI(5-24), and MEGA-8 (octanoyl-N-methylglucamide) detergent. This structure was refined using all observed data (30 248 reflections) between 30 and 1.95 A resolution to an R factor of 0.186. R.m.s. deviations of bond lengths and bond angles are 0.013 A and 2.3 degrees, respectively. The final model has 3075 atoms (207 solvent) with a mean B factor of 31.9 A(2). The placement of invariant protein-kinase residues and most C:PKI(5-24) interactions were confirmed, but register errors affecting residues 55-64 and 309-339 were corrected during refinement by shifting the affected sequences toward the C terminus along the previously determined backbone path. New details of C:PKI(5-24) interactions and the Ser338 autophosphorylation site are described, and the acyl group binding site near the catalytic subunit NH(2) terminus is identified.

2.2 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor.

. The crystal structure of a ternary complex containing the catalytic subunit of cAMP-dependent protein kinase, ATP and a 20-residue inhibitor peptide was refined at a resolution of 2.2 A to an R value of 0.177. In order to identify the metal binding sites, the crystals, originally grown in the presence of low concentrations of Mg(2+), were soaked in Mn(2+). Two Mn(2+) ions were identified using an anomalous Fourier map. One Mn(2+) ion bridges the gamma- and beta-phosphates and interacts with Asp184 and two water molecules. The second Mn(2+) ion interacts with the side chains of Asn171 and Asp l84 as well as with a water molecule. Modeling a serine into the P site of the inhibitor peptide suggests a mechanism for phosphotransfer.

Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor.

The structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase, MgATP, and a 20-residue inhibitor peptide was determined at a resolution of 2.7 A using the difference Fourier technique starting from the model of the binary complex (Knighton et al., 1991a). The model of the ternary complex was refined using both X-PLOR and TNT to an R factor of 0.212 and 0.224, respectively. The orientation of the nucleotide and the interactions of MgATP with numerous conserved residues at the active site of the enzyme are clearly defined. The unique protein kinase nucleotide binding site consists of a five-stranded antiparallel beta-sheet with the base buried in a hydrophobic site along beta-strands 1 and 2 and fixed by hydrogen bonds to the N6 amino and N7 nitrogens. The small lobe secures the nucleotide via a glycine-rich loop and by ion pairing with Lys72 and Glu91. While the small lobe fixes the nontransferable alpha- and beta-phosphates in this inhibitor complex, the gamma-phosphate is secured by two Mg2+ ions and interacts both directly and indirectly with several residues in the large lobe--Asp184, Asn171, Lys168. Asp166 is positioned to serve as a catalytic base. The structure is correlated with previous chemical evidence, and the features that distinguish this nucleotide binding motif from other nucleotide binding proteins are delineated.

A template for the protein kinase family.

The crystal structure of the catalytic subunit of cAMP-dependent protein kinase, complexed with ATP and a 20-residue inhibitor peptide, is reviewed and correlated with chemical and genetic data. The striking convergence of the structure with the biochemistry and genetics provides for the first time a molecular basis for understanding how this enzyme functions, as well as an explanation for the highly conserved residues that are scattered throughout the molecule. Because these residues probably serve a common role in all eukaryotic protein kinases, this first protein kinase structure serves as a general template for the entire family of enzymes.

Crystal structures of the catalytic subunit of cAMP-dependent protein kinase reveal general features of the protein kinase family.

The crystal structure of the catalytic subunit of cAMP-dependent protein kinase serves as a template for the catalytic core of all eukaryotic protein kinases. The various crystal structures are reviewed with particular emphasis on the numerous conserved residues that converge at the active site. The structures also reveal the importance of posttranslational modifications, including myristylation and phosphorylation.

Structural basis of the intrasteric regulation of myosin light chain kinases.

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.