Title: The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition
Abstract: Article15 November 1997free access The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition E.D. Lowe E.D. Lowe Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author M.E.M. Noble M.E.M. Noble Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author V.T. Skamnaki V.T. Skamnaki National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48, Vas Constantinou Avenue, Athens, 116 35 Greece Search for more papers by this author N.G. Oikonomakos N.G. Oikonomakos National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48, Vas Constantinou Avenue, Athens, 116 35 Greece Search for more papers by this author D.J. Owen D.J. Owen MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author L.N. Johnson Corresponding Author L.N. Johnson Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author E.D. Lowe E.D. Lowe Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author M.E.M. Noble M.E.M. Noble Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author V.T. Skamnaki V.T. Skamnaki National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48, Vas Constantinou Avenue, Athens, 116 35 Greece Search for more papers by this author N.G. Oikonomakos N.G. Oikonomakos National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48, Vas Constantinou Avenue, Athens, 116 35 Greece Search for more papers by this author D.J. Owen D.J. Owen MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author L.N. Johnson Corresponding Author L.N. Johnson Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Author Information E.D. Lowe1, M.E.M. Noble1, V.T. Skamnaki2, N.G. Oikonomakos2, D.J. Owen3 and L.N. Johnson 1 1Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK 2National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48, Vas Constantinou Avenue, Athens, 116 35 Greece 3MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6646-6658https://doi.org/10.1093/emboj/16.22.6646 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The structure of a truncated form of the γ-subunit of phosphorylase kinase (PHKγt) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel β-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the γ-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered. Introduction Phosphorylase kinase (PHK) is a key enzyme involved in the control of glycogen degradation. The enzyme integrates extracellular signals arising from hormone receptor interactions and from neuronal impulses mediated through calcium with those arising from intracellular events, to provide a tightly controlled kinase activity which regulates glycogen phosphorylase. PHK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (αβγδ)4, and a total mol. wt of 1.3×106 Da. Activity is regulated by cyclic AMP-dependent protein kinase phosphorylation, autophosphorylation, allosteric effectors (e.g. ADP), metal ion concentration (Ca2+ and Mg2+), proteolysis and pH (Pickett-Gies and Walsh, 1986). The α and β subunits are regulatory and are the targets for control by phosphorylation. The δ subunit is essentially identical to calmodulin and confers Ca2+ sensitivity. The 386 amino acid γ subunit is the catalytic subunit which comprises an N-terminal kinase domain (residues 1–298) and a regulatory calmodulin-binding domain (residues 299–386). In muscle and liver, PHK catalyses the Ca2+-dependent phosphorylation of inactive glycogen phosphorylase b (GPb) to active glycogen phosphorylase a (GPa). Although in vivo the only definite substrate for PHK is glycogen phosphorylase, in vitro the enzyme will also phosphorylate, with lower activity, glycogen synthase, troponin I, troponin T, PHK α and β subunits and several other proteins (Pickett-Gies and Walsh, 1986). The site phosphorylated in GPb is Ser14. Analysis of sequences surrounding this and other sites recognized has led to a consensus sequence of Arg/Lys-X-X-Ser-Val/Ile-Y for possible substrates, where X and Y are any amino acids (Pearson and Kemp, 1991). Activity is considerably increased if residue Y is arginine as in phosphorylase (Graves, 1983). In brain, there is a high activity ratio of PHK to phosphorylase, an observation which has led to suggestions of possible alternative functions for PHK in neuronal tissue. Neuronal-specific protein B-50 (GAP-43), neurogranin and the microtubule-associated protein tau have been shown to be targets for PHK (Paudel et al., 1993; Paudel, 1997). As a first step towards understanding the structure and function relationships for this complex enzyme, we have determined the crystal structure of the kinase domain (residues 1–298) of the catalytic γ-subunit of phosphorylase kinase (PHKγt) in complex with the non-hydrolysable substrate analogue adenylyl imidodiphosphate (AMPPNP) (Owen et al., 1995a) at a resolution of 2.5 Å. Subsequent higher resolution data collection on an XRIICCD detector, described here, has allowed determination of the conformations of some regions of the protein which were undefined in the original structure. Attempts to co-crystallize with a peptide substrate [residues 9–18 of GPb, Lys(−5)-Arg(−4)-Lys(−3)-Gln(−2)-Ile(−1)-Ser*(0)-Val(+1)-Arg(+2)-Gly(+3)-Leu(+4)] were not successful [Ser*(0) indicates the phosphorylatable serine: residues N-terminal and C-terminal to this residue are numbered −1, −2 etc. and +1, +2 etc. respectively]. Peptide substrates exhibit Km values that are ∼50-fold higher than the corresponding Km values for phosphorylase itself (Graves, 1983), suggesting that structural features of the phosphorylase molecule may play a role in recognition. Indeed only one out of the 29 serines in the 842 amino acid glycogen phosphorylase molecule is phosphorylated, although some other serines are surrounded by a consensus sequence motif. In order to elaborate on protein kinase specificity, Songyang et al. (1996) studied primary sequence specificity using an oriented degenerate peptide library and identified a so-called optimal peptide substrate (sequence Lys-Arg-Met-Met-Ser*-Phe-Phe-Leu-Phe). On the basis of the crystal structure and crystal lattice contacts for the PHKγt structure, we designed a modified version of this peptide (the 'modified Cantley' or MC-peptide), with a sequence closer to that of glycogen phosphorylase. In order to fit the available space, the peptide was truncated to seven residues, the minimum length peptide that is recognized without significant loss of activity (Graves, 1983). The methionine in the −2 position was replaced by a glutamine and the phenylalanine in position +2 was replaced by arginine, a residue which is known to be important in phosphorylase specificity (Graves, 1983). We have also synthesized a peptide of the same length, with a sequence identical to the phosphorylated site of phosphorylase (the 'natural substrate', or NS-peptide), for which we have determined the kinetic parameters as a substrate for PHKγt. We report the crystal structure of the ternary complex of PHKγt co-crystallized with the MC-peptide (sequence Ac-Arg-Gln-Met-Ser*-Phe-Arg-Leu) and AMPPNP. The results allow identification of the specific interactions that determine substrate specificity and also demonstrate an unexpected dimerization in which the catalytic sites of the kinase are separated by only 14 Å. Results Kinetics The Michaelis–Menten kinetic parameters for PHKγt with two different peptide substrates are summarized in Table I. Of the two peptides, the higher affinity, as deduced from the Km, is displayed by the modified Cantley peptide (MC-peptide, Km = 0.4 mM), compared with the natural substrate peptide (NS-peptide, Km = 1.8 mM). The factor of 25 in relative catalytic efficiency (kcat/Km) is consistent with the anticipated preference calculated on the basis of the position by position preferences observed by Songyang et al. (1996) at the different peptide subsites, which suggests that the MC-peptide would be a better substrate by a factor of at least 12.1. As previously observed, peptide substrates were poorer substrates than glycogen phosphorylase, for which Km = 8.9 μM and kcat = 4000/min. The MC-peptide behaves as a competitive inhibitor with respect to GPb, with a Ki of 0.6 mM (data not shown). Table 1. Peptides and kinetic characterization Subsite number: −3 −2 −1 0 1 2 3 Totalb kcat (min−1) Km (mM) kcat/Km (min−1 mM−1) Natural substrate Lys Gln Ile Ser Val Arg Gly (Preferencea): 2.5 1.7 1.5 1.5c 2.0 1.5c 28.8 70 1.8 40 Cantley peptide Arg Met Met Ser Phe Phe Leu (Preferencea): 5.7 2.0 2.2 3.9 2.5 2.1 513.5 MC-peptide Arg Gln Met Ser Phe Arg Leu (Preferencea): 5.7 1.7 2.2 3.9 2.0 2.1 349.2 400 0.4 1000 a Preferences are the reported relative abundances of the given residue at the specific subsite position in a degenerate library of peptides phosphorylated by PHK, according to Songyang et al. (1996). b The total preference value is the product of the preferences for each of the subsites of the given peptide. c Songyang et al. do not report the relative abundance values for residues with a relative abundance below 1.5. Residues of the natural substrate peptide falling into this category are indicated. In order to obtain a maximum expected overall preference for the natural substrate peptide, these residues are assigned a preference of 1.5 in this analysis. Higher resolution binary complex Crystals of the binary complex between PHKγt and AMPPNP diffracted to a resolution of 2.1 Å. This diffraction limit had been observed previously, although attempts to collect a complete dataset to this resolution had been hindered by radiation-induced crystal decay, even under cryo-crystallographic conditions. The XRIICCD detector on BL4 of the ESRF allowed 180° of data to be collected in a period of ∼2 h, compared with a typical period of 7 h for an equivalent data collection on a detector system with a longer read out cycle, such as an imaging plate. Over the course of this faster data collection, crystal decay was not apparent. The higher overall resolution thus obtained allowed the refinement of a more accurate model, which in turn allowed the positioning of several loops, notably the loop connecting strand β3 with the C-helix, which had previously not been traceable. The resulting model is complete from residue 14 through to residue 291. Statistics of the refined 2.1 Å model are given in Table II. Table 2. Data collection and refinement Complex Beamline Binary complex BL4, ESRF Ternary complex Super ESCA, Elletra Ternary complex D2AM, ESRF Space group P212121 P3221 P3221 Cell a = 47.7 Å, b = 67.7 Å, c = 110.8 Å a = b = 64.1 Å, c = 144.4 Å a = b = 65.3 Å, c = 145.8 Å Maximum resolution 2.1 Å 3.45 Å 2.6 Å Observations 137 663 14 119 21 644 Unique reflections 20 569 6927 10 062 Rmergea 0.047 0.188 0.098 Completeness 95.5% 89.9% 86.3% Highest shell 2.21–2.10 Å 3.72–3.45 Å 2.74–2.60 Å Rmergea 0.36 0.37 0.36 Completeness 79.1% 87.5% 78.1% Refinement: Non-hydrogen atoms 2243 protein, 31 AMPPNP, 2 Mn2+, 153 water 2092 protein, 65 peptide 31 AMPPNP, 2 Mn2+ 2243 protein, 65 peptide, 31 AMPPNP, 2 Mn2+, 88 water, 6 glycerol Rconvb 19.8% (6.0–2.1 Å) 35.3% (25.0–3.5 Å) 23.6% (25.0–2.6 Å) Rfreec 28.8% (6.0–2.1 Å) 35.3% (25.0–3.5 Å) 30.0% (25.0–2.6 Å) R.m.s.ds from ideal geometry: Bondsd 0.006 Å 0.014 Å 0.005 Å Anglesd 1.7° 1.5° 1.8° Ramachandran plote 89.6% 89.1% 90.4% a Where Ihj is the intensity of the jth observation of unique reflection h. b Where Foh and Fch are the observed and calculated structure factor amplitudes for relection h. c Rfree is equivalent to Rconv for a randomly selected 5% subset of reflections not used in structure refinement. d As calculated by REFMAC (Murshudov et al., 1997). e Residues in the most favoured regions of the Ramachandran plot as calculated by PROCHECK (Laskowski et al., 1993). Ternary complex PHKγt was crystallized in complex with the non-hydrolysable ATP analogue AMPPNP and the MC-peptide in the presence of manganese, and the structure solved at a resolution of 2.6 Å by molecular replacement. The refined 2.1 Å structure of the binary complex was used as the initial search model. Electron density is visible for residues 14–292 of PHKγt, for the whole of the substrate peptide and for AMPPNP with two manganese ions. Figure 1A shows electron density defining the conformation of the peptide in the refined 2mFo−DFc map. Although crystals with the MC-peptide complex grew readily and reproducibly, no crystals of the equivalent complex with the NS-peptide were obtained despite extensive trials. Figure 1.Binding of the MC-peptide to PHKγt. (A) Electron density from the final refined 2Fo−Fc map defining the conformation of the peptide. The map was calculated using 2mFo−DFc coefficients output on the final cycle of REFMAC refinement, and is contoured at 0.2 e−/Å3 (= 0.9 σ), with contours further than 2.0 Å from peptide atoms deleted for clarity. (B) Location of the peptide-binding site with respect to the kinase catalytic domain. The PHKγt fold is shown with the N-terminal domain in white, the glycine-rich hairpin (residues 26–33) in pale magenta, the hinge region (residues 108 and109) in magenta, the C-terminal domain in salmon pink and the activation segment (residues 167–193) in cyan. AMPPNP is shown in individual atomic colours, with two manganese ions in cyan and the peptide shown in green, forming a short anti-parallel β-sheet with a part of the activation segment. (C) GRASP (Nicholls and Honig, 1991) representation of the surface to which peptide is bound. The molecular surface of PHKγt is coloured according to electrostatic potential such that deep blue corresponds to a potential of 30 kT, and deep red corresponds to a potential of −30 kT. The MC-peptide is shown as bonded atoms. Download figure Download PowerPoint The overall fold of PHKγt in the ternary complex resembles that of the binary complex described previously (Owen et al., 1995a). A smaller N-terminal domain (residues 14–107), formed principally from β-sheet, is connected to a larger C-terminal domain (residues 110–292), formed mainly from α-helix. The two domains are joined by a hinge region around residues Lys108 and Gly109. The nucleotide binds at the cleft between the two domains, sandwiched between the glycine-rich hairpin formed by strands β1 and β2, and a surface from the C-terminal domain. The C-terminal domain contains the 'activation segment', a stretch of residues between the conserved DFG and APE protein kinase motifs (Hanks and Quinn, 1991) which contains the site of activatory phosphorylation of many protein kinases (but not PHK) that are controlled by phosphorylation of the catalytic domain (Johnson et al., 1996). The MC-peptide binds in the active site groove of PHKγt (Figure 1B), following a path very similar to that of the equivalent residues of the protein kinase inhibitor PKI bound to cAPK. This similarity of binding breaks down, however, over the C-terminal two residues of the MC-peptide. Binding of the peptide is achieved through marked complementarity of shape, hydropathy and potential, as suggested from the GRASP image in Figure 1C. Contacts of the substrate peptide to PHKγt Polar contacts involving the MC-peptide in complex with PHKγt are summarized in Figure 2A. Arg(−3) forms an ion pair with Glu110 from the hinge region, which interacts in turn with the O2′ and O3′ ribose oxygens of the nucleotide substrate. The involvement of this residue in binding basic residues at the P-3 site had been anticipated by site-directed mutagenesis (Huang et al., 1995). The amide oxygen of Gln(−2) is hydrogen bonded with the main chain nitrogen of Ser188, and the amide nitrogen is close (3.75 Å) to the OG atom of the same residue. Ser188 is part of a single turn of α-helix close to the end of the activation segment. The peptide oxygen of Gln(−2) is 3.4 Å from the NZ atom of Lys151, and hydrogen bonded to a water. No hydrogen bonds are observed for Met(−1). Figure 2.Contacts of the MC-peptide to PHKγt. (A) (Stereo) Polar contacts (<3.4 Å) involved in binding nucleotide and peptide substrates to PHKγt are shown, with the residues involved labelled at their CA position. The backbone of the PHKγt molecule is shown as a faint worm representation, with atoms of protein residues shown as lines. Atoms of AMPPNP and the MC-peptide are shown in thicker ball and stick representation. Labelled residues from the protein are Ser31, Lys48, Met106, Glu110, Lys151, Glu153, Asn154, Asp167, Glu182, Val183, Cys184, Gly185 and Ser188. (B) Interactions of the MC-peptide with residues at the end of the activation segment. The MC-peptide is shown with green carbon atoms, while the activation segment is shown with cyan carbon atoms. Download figure Download PowerPoint The OG atom of Ser(0), the nucleophile in the phosphotransfer reaction, is poised 3.6 Å from the phosphorus atom of the imidophosphate group of the nucleotide substrate analogue. The closest contact of the OG atom is a distance of 2.6 Å to an imidophosphate terminal oxygen. The other polar side chain contact involves Arg(+2), which appears from the electron density to have some conformational variability. The best defined conformation for this side chain positions it to interact with the side chain of Glu182, the residue in PHK which plays an analogous role to the phosphorylated residues Thr197 (in cAPK) or Thr160 (in CDK2). The most striking aspect of the polar interactions between the MC-peptide and PHKγt is the existence of a short stretch of anti-parallel β-sheet formed by Phe(+1) and Leu(+3) peptide which hydrogen-bonds with Gly185 and Val183 from the enzyme. This interaction is consistent with the extended conformation of this end of the peptide substrate, and of these residues from the protein. The mutation of the residue corresponding to Gly185 to Glu in the testis/liver isoform of phosphorylase kinase causes autosomal liver glycogenosis (Maichele et al., 1996). The structure suggests that it would be difficult to accommodate a glutamic acid at position 185. The phosphorylase kinase deficiency results support the notion of the crucial role in the correct conformation of the activation segment for substrate recognition. Apolar contacts exist between the aliphatic portion of Arg(−3) and Phe112 from PHKγt, and between the side chain of Met(−1) and atoms of the glycine-rich loop. The most extensive apolar interaction, however, involves Phe(+1) which sits in a hydrophobic pocket formed from Val183, Pro187 and Leu190 from the enzyme, together with Leu(+3) from the peptide substrate (Figure 2B). The side chain of Leu(+3) itself interacts with this same apolar pocket, as well as with residue Met235′ from a crystallographic 2-fold related molecule. This interaction is one of the contacts defining a dimer formed within this crystal form, which also involves the carboxyl group of the peptide interacting with the peptide nitrogen of Met235′, and the peptide oxygen of Arg(+2) interacting with the peptide nitrogen of Lys234′. Comparison with other conformations of kinase-binding peptides The conformation of the MC-peptide, closely related to the naturally occurring substrate of PHK, can be compared with the conformations of two other relevant peptides: firstly, with that of the tight binding inhibitor PKI in complex with cAPK (Bossmeyer et al., 1993; Zheng et al., 1993a), and secondly with the naturally occurring substrate of PHK in conformations which it adopts when not bound to PHK. These comparisons are presented in Figure 3. Figure 3.Conformations of kinase inhibitor and substrate peptides. (A) Conformation of residues 11–17 of GPa. (B) Conformation of residues 11–17 of GPb. (C) Conformation of the MC-peptide, as observed in complex with PHKγt. (D) Conformation of the equivalent part of the protein kinase inhibitor peptide, as observed in complex with cAPK. Download figure Download PowerPoint As noted, the binding of the MC-peptide to PHKγt resembles closely the binding of PKI to cAPK. Both the conformation and the contacts of Arg(−3) are common to the two proteins. Gln(−2) differs from PKI, which has an arginine in the equivalent locus. Whereas the arginine of PKI interacts with Glu230 of cAPK, Gln(−2) of the MC-peptide interacts with Ser188. PHK has a threonine residue (Thr221) in place of Glu230 of cAPK. The conformation and contacts of Met(−1) resemble those of the equivalent glutamine residue of PKI, and the main chain conformation of Ser*(0) is close to that of the equivalent Ala in cAPK. Indeed, Ser*(0) of the MC-peptide can simply be constructed from Ala(0) of PKI by attachment of the OG atom with a χ1 value of 61°. Phe(+1) is bound in a similar fashion to the equivalent isoleucine residue of PKI, but there is a significant difference in the conformation and contacts of Arg(+2) and Leu(+3) from those of the equivalent histidine and aspartate residues in PKI. Arg(+2) interacts with Glu182, the residue equivalent to the phosphorylated Thr197 of cAPK. This interaction site is not available to the histidine of PKI, since it is occupied by a histidine residue (His87) from the N-terminal domain of cAPK. His(+2) of PKI, by contrast, is not involved in any direct polar interactions with the kinase. The conformation observed for the C-terminal two residues of the MC-peptide is necessary to allow the short stretch of anti-parallel β-sheet which exists between the substrate and the kinase activation loop in the complex described here, which is not apparent in the PKI–cAPK interaction (Figure 2B). The conformations of residues 11–17 in GPa and GPb differ from each other (Barford et al., 1991) and from the conformation of the MC-peptide in the ternary complex. In GPb, the N-terminal residues adopt an irregular conformation, with Ser14 and Val15 nearly α-helical. In GPa, the conformation around Ser14 is extended, although residues 15–17 have an α-helical conformation, which allows the Ser14 phosphate group to make hydrogen bonds with the main chain nitrogens of residues 15 and 16. The conformation of the MC-peptide bound to PHKγt is all β. The N-terminal residues 11–19 in GPb exhibit high B-factors and are mobile. Mobility in the region around the site of phosphorylation appears to be essential to allow the substrate to be accommodated in the defined conformation when bound to the kinase, and may be a common feature of sites of phosphorylation in other proteins. Dimerization Although crystallized with only one molecule in the crystallographic asymmetric unit, two PHKγt molecules associate across a crystallographic 2-fold axis to form an intimate dimer. Dimerization occurs in a head-to-tail fashion, such that residues at the N-terminus of the C-helix of each molecule interact with residues at the N-terminus of the G-helix of the other. Figure 4A and B shows this dimerization, with residues that undergo a change in surface area upon dimerization listed. The total change in accessible surface area due to dimerization is 2068 Å2 (considering only kinase surface area), or 3160 Å2 (considering protein + peptide surface area). The molecular surface formed by the resulting dimer is presented in Figure 4C and D. Interestingly, the MC-peptide is also involved in contacts across this 2-fold axis, with the contacts listed above from the last two residues of the MC-peptide to residues Lys234 and Met235 of the 2-fold related kinase molecule. These contacts involve the terminal leucine of the MC-peptide. In the natural substrate peptide, this residue is replaced by a glycine. It is possible that the lattice contacts mediated by the leucine contribute to the fact that crystals of the MC-peptide ternary complex, but not the natural substrate peptide ternary complex, have been grown, although the interactions of Leu(+3) <4.0 Å in length involve its main chain atoms only. Figure 4.Dimerization of PHKγt in this crystal form. Orthogonal views (A) perpendicular to the molecular 2-fold axis and (B) along the molecular 2-fold axis. Two crystallographically related PHKγt molecules are shown, coloured according to Figure 1B, with one molecule given partial transparency for distinction. (C) As (A), but with the molecular surface rendered. (D) As (B), but with the molecular surface rendered. Residues which undergo a change in accessible surface area upon dimerization (considering only changes caused by protein–protein contacts) are as follows: 24, 27, 29, 30, 54–59, 62, 65, 66, 69, 112, 113, 169, 170, 224–226, 230–233, 236, 247, 249, 250 and 252. Download figure Download PowerPoint The mode of dimerization does not correspond to the dimerization described for the fibroblast growth factor (FGF) receptor kinase (Mohammadi et al., 1996), but does appear to bring the activation segment of each kinase monomer into relatively close apposition with the catalytic site of its partner, and for this reason may reflect the approach of two kinase molecules involved in transactivation (Figure 4B). The distance between the active sites of the two monomers is ∼14 Å, which differs considerably from the GPb Ser14–Ca–Ser14′–Ca distance of 61.6 Å, but is closer to the GPa Ser14–Ca–Ser14′–Ca distance of 28.8 Å. PHK is active as a hexadecameric entity containing four copies of the catalytic γ-subunit arranged as a dimer of dimers (Norcum et al., 1994), and acts upon a dimeric GPb substrate. This gives rise to the speculation that the dimerization observed in these crystals may reflect a functional state of PHK. Comparison of protein, nucleotide conformation and metal ions in different ternary complexes The conformations of the PHKγt ternary complex with the MC-peptide, and the cAPK ternary complex with PKI resemble each other very closely (Figure 5A). The only structural differences close to the active site involve the relative position of the glycine-rich nucleotide-binding hairpin, which is somewhat more open in the PHKγt complex as a result of a serine residue in place of the third glycine (Owen et al., 1995a). At a more remote position, there is a marked difference in the orientation of the C- and G-helices of the two complexes. The C-helix is known to adopt a variety of positions in different kinase structures (e.g. Knighton et al., 1991; De Bondt et al., 1993; Jeffrey et al., 1995; Sicheri et al., 1997; Xu et al., 1997). The structure presented here provides evidence that its orientation can also differ between two serine/threonine kinases in fully activated conformation. Figure 5.Comparison of the PHKγt ternary complex protein conformation and flexibility with cAPK and the PHKγt binary complex. (A) Superposition of the catalytic cores of PHKγt ternary complex (green) and cAPK ternary complex (cyan). (B) Superposition of the PHKγt ternary complex (green) and the PHKγt binary complex (magenta). (C) Colour ramped fold of the PHKγt binary complex, such that blue corresponds to a CA atomic temperature factor of 10 Å2 and red corresponds to a CA atomic temperature factor of 60 Å2. (D) Colour ramped fold of the PHKγt ternary complex, such that blue corresponds to a CA atomic temperature factor of 10 Å2 and red corresponds to a CA atomic temperature factor of 60 Å2.