The Structure of Lombricine Kinase
2011; Elsevier BV; Volume: 286; Issue: 11 Linguagem: Inglês
10.1074/jbc.m110.202796
ISSN1083-351X
AutoresD. Jeffrey. Bush, Olga Kirillova, S.A. Clark, Omar Davulcu, Felcy Fabiola, Qing Xie, Thayumanasamy Somasundaram, W. Ross Ellington, Michael S. Chapman,
Tópico(s)Tardigrade Biology and Ecology
ResumoLombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309–317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His178. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309–317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His178. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. IntroductionLombricine, arginine, and creatine kinases (EC 2.7.3) are homologous phosphagen kinases that catalyze the buffering of cellular ATP levels through phosphoryl transfer to/from their respective guanidino-containing substrates. The reaction is central to short-term temporal energy buffering (1Schlattner U. Forstner M. Eder M. Stachowiak O. Fritz-Wolf K. Wallimann T. Mol. Cell. Biochem. 1998; 184: 125-140Crossref PubMed Google Scholar, 2Ellington W.R. Annu. Rev. Physiol. 2001; 63: 289-325Crossref PubMed Scopus (406) Google Scholar) as well as in spatial shuttling of energy from production to consumption sites (3Tombes R.M. Shapiro B.M. Cell. 1985; 41: 325-334Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 4Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Biochem. J. 1992; 281: 21-40Crossref PubMed Scopus (1583) Google Scholar, 5Ellington W.R. Kinsey S.T. Biol. Bull. 1998; 195: 264-272Crossref PubMed Scopus (18) Google Scholar). A wide array of endergonic processes is driven by nucleotide hydrolysis, from motion in molecular motors, active transport, and synthetic metabolism to signal transduction. Thus, the maintenance of a constant ATP/ADP ratio, displaced far from thermodynamic equilibrium in the face of high and variable rates of ATP turnover, is crucial for cellular homeostasis (2Ellington W.R. Annu. Rev. Physiol. 2001; 63: 289-325Crossref PubMed Scopus (406) Google Scholar).Different organisms use different phosphagen substrates, usually only one and each with its own specific phosphagen kinase (Fig. 1) (2Ellington W.R. Annu. Rev. Physiol. 2001; 63: 289-325Crossref PubMed Scopus (406) Google Scholar). Lombricine kinase, as well as taurocyamine kinase and glycocyamine kinase, is found exclusively in annelids and allied groups (2Ellington W.R. Annu. Rev. Physiol. 2001; 63: 289-325Crossref PubMed Scopus (406) Google Scholar). Phylogenetic analyses and studies of the intron/exon organization of the genes of these phosphagen kinases unique to annelids have shown that they are more closely related to creatine kinases (with which they share 50–60% sequence identity) than typical monomeric arginine kinases such as that from the horseshoe crab (6Suzuki T. Uda K. Adachi M. Sanada H. Tanaka K. Mizuta C. Ishida K. Ellington W.R. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009; 152: 60-66Crossref PubMed Scopus (31) Google Scholar) (40% sequence identity). Annelids are more diverse in their choice of phosphagen, and the substrate specificities of the corresponding kinases are often lower (6Suzuki T. Uda K. Adachi M. Sanada H. Tanaka K. Mizuta C. Ishida K. Ellington W.R. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009; 152: 60-66Crossref PubMed Scopus (31) Google Scholar).Structural work has concentrated on the presumptive ancestral arginine kinase and the vertebrate creatine kinase (7Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (260) Google Scholar, 8Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8449-8454Crossref PubMed Scopus (232) Google Scholar), but sequence alignment suggests that subunit fold, if not quaternary structure, is conserved across the family (2Ellington W.R. Annu. Rev. Physiol. 2001; 63: 289-325Crossref PubMed Scopus (406) Google Scholar). Common structural hallmarks include a small N-terminal α-helical domain connected by a flexible linker to a larger C-terminal domain of β-sheet flanked by α-helices (7Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (260) Google Scholar, 8Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8449-8454Crossref PubMed Scopus (232) Google Scholar). There is also a conserved "essential" cysteine (Cys271 in the horseshoe crab (Limulus polyphemus) arginine kinase (LpAK)) 2The abbreviations used are: LpAK, Limulus polyphemus (Atlantic horseshoe crab) arginine kinase; AK, arginine kinase; CK, creatine kinase; GK, glycocyamine kinase; LK, lombricine kinase; GgCKmit, Gallus gallus (chicken) mitochondrial CK; HsCKBB, Homo sapiens (human) brain CK; HsCKmit, H. sapiens sarcomeric mitochondrial CK; OcCKMM, Oryctolagus cuniculus (rabbit) CK muscle dimer; SjAK, Stichopus japonicus (sea cucumber) AK; TcCK, Torpedo californica (Pacific electric ray) CK; TSA, transition state analog; TSAC, transition state analog complex; UcLK, Urechis caupo (Innkeeper worm) LK; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s., root mean square; r.m.s.d., root mean square deviation; AMPPNP, adenosine 5′-(β,γ-imino)triphosphate. that may be involved in substrate binding synergy and/or electrostatic catalysis as a thiolate (9Furter R. Furter-Graves E.M. Wallimann T. Biochemistry. 1993; 32: 7022-7029Crossref PubMed Scopus (111) Google Scholar, 10Gattis J.L. Ruben E. Fenley M.O. Ellington W.R. Chapman M.S. Biochemistry. 2004; 43: 8680-8689Crossref PubMed Scopus (34) Google Scholar), as well as a highly conserved NEEDH motif (11Ellington W.R. Bush J. Biochem. Biophys. Res. Commun. 2002; 291: 939-944Crossref PubMed Scopus (11) Google Scholar), of which the second glutamate (LpAK Glu225) is implicated as the base that catalyzes phosphagen proton abstraction (12Pruett P.S. Azzi A. Clark S.A. Yousef M.S. Gattis J.L. Somasundaram T. Ellington W.R. Chapman M.S. J. Biol. Chem. 2003; 29: 26952-26957Abstract Full Text Full Text PDF Scopus (45) Google Scholar). Differences between the paralogs are due, in part, to the varying quaternary structures among isoforms (monomer, dimer, or octamer) and adaptations to different cellular targeting (cytoplasmic or mitochondrial intermembrane space).Lombricine kinase (LK) is a biological homodimer with relaxed substrate specificity relative to the highly specific arginine and creatine kinases (6Suzuki T. Uda K. Adachi M. Sanada H. Tanaka K. Mizuta C. Ishida K. Ellington W.R. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009; 152: 60-66Crossref PubMed Scopus (31) Google Scholar). LK catalyzes reactions with either lombricine or taurocyamine substrates but not arginine (Fig. 1). Lombricine (guanidinoethyl phosphoserine) is found as the d-serine isomer in most annelids but as the l-serine form in echiuroid worms (2Ellington W.R. Annu. Rev. Physiol. 2001; 63: 289-325Crossref PubMed Scopus (406) Google Scholar), suggesting that close homologs tolerate some stereochemical variation. The apparent dissociation constant and specificity index of Eisenia LK for lombricine are Km = 5.33 mm and kcat/Km = 3.37 s−1mm−1 and are modestly weaker for taurocyamine (Km = 15.31 mm and kcat/Km = 0.48 s−1mm−1, respectively (13Tanaka K. Suzuki T. FEBS Lett. 2004; 573: 78-82Crossref PubMed Scopus (38) Google Scholar)). Our decision to work with taurocyamine in substrate complexes was predicated on the scarcity of lombricine, which must be isolated from kilogram quantities of worms.The two variable loops have been implicated in phosphagen kinase substrate specificity (8Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8449-8454Crossref PubMed Scopus (232) Google Scholar, 14Suzuki T. Fukuta H. Nagato H. Umekawa M. J. Biol. Chem. 2000; 275: 23884-23890Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 15Azzi A. Clark S.A. Ellington W.R. Chapman M.S. Protein Sci. 2004; 13: 575-585Crossref PubMed Scopus (56) Google Scholar, 16Novak W.R. Wang P.F. McLeish M.J. Kenyon G.L. Babbitt P.C. Biochemistry. 2004; 43: 13766-13774Crossref PubMed Scopus (26) Google Scholar, 17Jourden M.J. Clarke C.N. Palmer A.K. Barth E.J. Prada R.C. Hale R.N. Fraga D. Snider M.J. Edmiston P.L. Biochim. Biophys. Acta. 2007; 1774: 1519-1527Crossref PubMed Scopus (12) Google Scholar). One set (LpAK loop 59–64) and Urechis caupo lombricine kinase (UcLK) loop 53–58) is located in the small N-terminal α-helical domain and facilitates binding of the phosphagen substrate through backbone hydrogen bonds to the carboxylate of the substrate. With loop length inversely correlated to substrate size, the mechanism of the small domain specificity loop has been rationalized in terms of lock-and-key steric hindrance (15Azzi A. Clark S.A. Ellington W.R. Chapman M.S. Protein Sci. 2004; 13: 575-585Crossref PubMed Scopus (56) Google Scholar, 17Jourden M.J. Clarke C.N. Palmer A.K. Barth E.J. Prada R.C. Hale R.N. Fraga D. Snider M.J. Edmiston P.L. Biochim. Biophys. Acta. 2007; 1774: 1519-1527Crossref PubMed Scopus (12) Google Scholar, 18Suzuki T. Kamidochi M. Inoue N. Kawamichi H. Yazawa Y. Furukohri T. Ellington W.R. Biochem. J. 1999; 340: 671-675Crossref PubMed Scopus (99) Google Scholar, 19Lim K. Pullalarevu S. Surabian K.T. Howard A. Suzuki T. Moult J. Herzberg O. Biochemistry. 2010; 49: 2031-2041Crossref PubMed Scopus (21) Google Scholar).The other loop is in the large domain (LpAK loop 311–319 and UcLK loop 309–317) and serves to align and position the guanidinium of the substrate for optimal nucleophilic attack on ATP (8Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8449-8454Crossref PubMed Scopus (232) Google Scholar, 20Yousef M.S. Fabiola F. Gattis J.L. Somasundaram T. Chapman M.S. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 2009-2017Crossref PubMed Scopus (62) Google Scholar). In arginine kinase, interactions between Glu312 and the guanidinium are prominent in this alignment. Creatine kinases have a valine at the corresponding position, which forms a hydrophobic mini-pocket accommodating the methyl group distinctive for creatine (16Novak W.R. Wang P.F. McLeish M.J. Kenyon G.L. Babbitt P.C. Biochemistry. 2004; 43: 13766-13774Crossref PubMed Scopus (26) Google Scholar, 17Jourden M.J. Clarke C.N. Palmer A.K. Barth E.J. Prada R.C. Hale R.N. Fraga D. Snider M.J. Edmiston P.L. Biochim. Biophys. Acta. 2007; 1774: 1519-1527Crossref PubMed Scopus (12) Google Scholar, 21Lahiri S.D. Wang P.F. Babbitt P.C. McLeish M.J. Kenyon G.L. Allen K.N. Biochemistry. 2002; 41: 13861-13867Crossref PubMed Scopus (118) Google Scholar). However, mediation of specificity is more complex than lock-and-key. In a chimeric construct with the CK specificity determinants in an LpAK background, it is possible to regain AK activity with additional mutations (15Azzi A. Clark S.A. Ellington W.R. Chapman M.S. Protein Sci. 2004; 13: 575-585Crossref PubMed Scopus (56) Google Scholar). Furthermore, a creatine-LpAK structure shows that creatine is not excluded from the active site but is imperfectly aligned with the nucleotide (15Azzi A. Clark S.A. Ellington W.R. Chapman M.S. Protein Sci. 2004; 13: 575-585Crossref PubMed Scopus (56) Google Scholar). In crystal structures, the loop has been fully resolved only in the presence of substrates. Consistent with disorder in the substrate-free states, NMR-based Lipari-Szabo analysis has identified inherent nanosecond dynamics in this region (22Davulcu O. Flynn P.F. Chapman M.S. Skalicky J.J. Structure. 2009; 17: 1356-1367Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It appears that substrates either induce an ordering or select from an ensemble of loop conformations (23Boehr D.D. Dyson H.J. Wright P.E. Chem. Rev. 2006; 106: 3055-3079Crossref PubMed Scopus (371) Google Scholar, 24Boehr D.D. Wright P.E. Science. 2008; 320: 1429-1430Crossref PubMed Scopus (164) Google Scholar, 25Ma B. Kumar S. Tsai C.J. Nussinov R. Protein Eng. 1999; 12: 713-720Crossref PubMed Scopus (479) Google Scholar) to achieve a substrate-bound conformation that is catalytically competent for the cognate phosphagen substrate. The loop was fully resolved in one of the two subunits in the substrate-free UcLK structure reported here, providing an improved basis for understanding substrate-associated conformational changes in phosphagen kinases.Past NMR and kinetic studies have shown that creatine and arginine kinases share the same rapid equilibrium, random order, bimolecular-bimolecular mechanism with direct, partially associative, in-line phosphoryl transfer (26Hansen D.E. Knowles J.R. J. Biol. Chem. 1981; 256: 5967-5969Abstract Full Text PDF PubMed Google Scholar, 27Murali N. Jarori G.K. Landy S.B. Rao B.D. Biochemistry. 1993; 21: 12941-12948Crossref Scopus (51) Google Scholar, 28Murali N. Jarori G.K. Rao B.D. 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Paired substrate-free and transition state analog complex (TSAC) structures are available for both arginine kinase and creatine kinase; the TSAC has bound phosphagen, ADP, and a nitrate mimicking the γ-phosphoryl in transit (21Lahiri S.D. Wang P.F. Babbitt P.C. McLeish M.J. Kenyon G.L. Allen K.N. Biochemistry. 2002; 41: 13861-13867Crossref PubMed Scopus (118) Google Scholar, 35Yousef M.S. Clark S.A. Pruett P.K. Somasundaram T. Ellington W.R. Chapman M.S. Protein Sci. 2003; 12: 103-111Crossref PubMed Scopus (80) Google Scholar). Upon substrate binding there are domain reorientations up to 18°, together with the ordering and/or reconfiguring of two flexible loops over the substrates. The reconfiguration of the active site, which likely removes solvent water from where it could participate in a wasteful side reaction, appears to be critical in the alignment of enzyme catalytic groups and in the precise alignment of reactive substrate atoms with each other (20Yousef M.S. Fabiola F. Gattis J.L. Somasundaram T. Chapman M.S. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 2009-2017Crossref PubMed Scopus (62) Google Scholar).NMR relaxation dispersion measurements have identified intrinsic collective motions in the interface between the N- and C-terminal domains and in LpAK active site loop 182–209 (corresponding to UcLK loop 175–203) (22Davulcu O. Flynn P.F. Chapman M.S. Skalicky J.J. Structure. 2009; 17: 1356-1367Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These motions, measured in the absence of substrates, occur at turnover-commensurate rates (22Davulcu O. Flynn P.F. Chapman M.S. Skalicky J.J. Structure. 2009; 17: 1356-1367Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) at sites that are also implicated in the substrate-associated conformation changes (36Niu X. Brüschweiler-Li L. Davulcu O. Skalicky J.J. Brüschweiler R. Chapman M.S. J. Mol. Biol. 2011; 405: 479-496Crossref PubMed Scopus (31) Google Scholar). These observations suggest that substrate-associated changes take advantage of modes of flexibility intrinsic to the enzyme and that some of the intrinsic motions may be rate-limiting on turnover. The comparison of the nucleotide-bound and -free UcLK structures reported here adds to our understanding of the dependence of domain configuration upon the presence of substrates.RESULTS AND DISCUSSIONStructure DeterminationStructures were determined successfully for two crystal forms: the ADP form, with a single subunit in the asymmetric unit; and the ADP form, with a dimer (Table 1). Substrates were not included in the initial phasing models for the ADP form. Electron density for the nucleotide phosphates was stronger than the average protein density, whereas ribose and base were weaker. However, there was no doubt as to its identity when the active sites of LpAK TSAC and a CK nucleotide complex (8Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8449-8454Crossref PubMed Scopus (232) Google Scholar, 21Lahiri S.D. Wang P.F. Babbitt P.C. McLeish M.J. Kenyon G.L. Allen K.N. Biochemistry. 2002; 41: 13861-13867Crossref PubMed Scopus (118) Google Scholar) were superimposed (Fig. 2). No density was visible for either the taurocyamine or the nitrate, which were also present in the crystallization solutions. Thus, attempts to crystallize UcLK as TSAC yielded only the binary nucleotide complex. Analogously, crystals of multimeric CK have been reported with substrates bound to some subunits but not others (21Lahiri S.D. Wang P.F. Babbitt P.C. McLeish M.J. Kenyon G.L. Allen K.N. Biochemistry. 2002; 41: 13861-13867Crossref PubMed Scopus (118) Google Scholar, 60Bong S.M. Moon J.H. Nam K.H. Lee K.S. Chi Y.M. Hwang K.Y. FEBS Lett. 2008; 582: 3959-3965Crossref PubMed Scopus (60) Google Scholar, 61Ohren J.F. Kundracik M.L. Borders Jr., C.L. Edmiston P. Viola R.E. Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 381-389Crossref PubMed Scopus (27) Google Scholar). (Such asymmetry was cited as supporting negative cooperativity (61Ohren J.F. Kundracik M.L. Borders Jr., C.L. Edmiston P. Viola R.E. Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 381-389Crossref PubMed Scopus (27) Google Scholar, 62Wu X. Ye S. Guo S. Yan W. Bartlam M. Rao Z. FASEB J. 2010; 24: 242-252Crossref PubMed Scopus (26) Google Scholar), but this argument has been undercut by a glycocyamine kinase structure with both subunits in the closed substrate-bound configuration (19Lim K. Pullalarevu S. Surabian K.T. Howard A. Suzuki T. Moult J. Herzberg O. Biochemistry. 2010; 49: 2031-2041Crossref PubMed Scopus (21) Google Scholar).) Lombricine was not available, and the concentration of the alternative substrate, taurocyamine, limited by solubility to 3× Km, may not have been sufficient to obtain TSAC crystals, even though LpAK has been crystallized with a wide variety of weakly binding substrate analogs (15Azzi A. Clark S.A. Ellington W.R. Chapman M.S. Protein Sci. 2004; 13: 575-585Crossref PubMed Scopus (56) Google Scholar). 3S. A. Clark, E. A. Ruben, M. S. Yousef, J. D. Bush, M. O. Fenley, J. D. Evanseck, W. R. Ellington, and M. S. Chapman, manuscript in preparation.FIGURE 2Electron density for points of interest in the substrate-free and ADP-bound crystal structures. A, an overview of the substrate-free subunit of UcLK colored by subdomain (as determined in LpAK); gray indicates unassigned. The inset shows the TSA structure of LpAK and the location of substrates (stick model) in the active site (20Yousef M.S. Fabiola F. Gattis J.L. Somasundaram T. Chapman M.S. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 2009-2017Crossref PubMed Scopus (62) Google Scholar). B and C, stereographic pairs showing maps calculated with coefficients of 2mFo − DFc following maximum likelihood atomic refinement (73Read R.J. Acta Crystallogr. A. 1986; 42: 140-149Crossref Scopus (2035) Google Scholar). B, the substrate-free UcLK-(308–317) flexible loop of subunit A is colored by atom type and its map contoured at 1.1 σ. In other phosphagen kinases this substrate specificity loop is highly disordered. The LK conformation is unique among substrate-free structures but is related to the nucleotide-bound structure (orange) by a quasi-rigid 7° rotation. C, the UcLK-ADP complex is colored by atom type. Density for part of subdomain 3 is contoured at 1.25 σ and at 0.7 σ for the ADP. The protein structure is similar to that of substrate-free UcLK (cyan), except for His178, which has distinctly different side chain density. Although the ADP is similar to that in the LpAK-TSA complex (dark brown) (20Yousef M.S. Fabiola F. Gattis J.L. Somasundaram T. Chapman M.S. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 2009-2017Crossref PubMed Scopus (62) Google Scholar), with mostly similar interactions, subdomain 3 is much more like substrate-free LpAK (light brown) (35Yousef M.S. Clark S.A. Pruett P.K. Somasundaram T. Ellington W.R. Chapman M.S. Protein Sci. 2003; 12: 103-111Crossref PubMed Scopus (80) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Subunit Structure and Comparison with HomologsOf the known phosphagen kinase structures, UcLK is structurally most similar to that of the chicken mitochondrial creatine kinase (GgCKmit; r.m.s.d.Cα = 0.96 Å) with which it has the highest sequence identity (57%). Other substrate-free vertebrate CKs follow shortly thereafter. Like the other phosphagen kinases, LK has a 100-residue N-terminal α-helical domain followed by a 250-residue mostly β-sheet domain.The overall structures of the substrate-free and ADP complex UcLK are surprisingly similar (r.m.s.d.Cα = 0.54 Å), considering the conventional wisdom that most of the >2 Å r.m.s. conformational changes (21Lahiri S.D. Wang P.F. Babbitt P.C. McLeish M.J. Kenyon G.L. Allen K.N. Biochemistry. 2002; 41: 13861-13867Crossref PubMed Scopus (118) Google Scholar, 35Yousef M.S. Clark S.A. Pruett P.K. Somasundaram T. Ellington W.R. Chapman M.S. Protein Sci. 2003; 12: 103-111Crossref PubMed Scopus (80) Google Scholar) between open and closed state phosphagen kinases are thought to be nucleotide-induced (65Dumas C. Janin J. FEBS Lett. 1983; 153: 128-130Crossref Scopus (28) Google Scholar, 66Forstner M. Kriechbaum M. Laggner P. Wallimann T. Biophys J. 1998; 75: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In fact, the overall difference (0.54 Å) is little more than between the two subunits in the dimeric crystal form (r.m.s.d.Cα = 0.40 Å), so systematic differences are barely observable above experimental error. With such modest differences between ADP-bound and substrate-free annelid UcLK, the ADP-bound LK is much closer to substrate-free chicken GgCKmit (r.m.s.d.Cα = 0.9 Å) than to the most similar transition state CK, human brain (HsCKBB; r.m.s.d.Cα = 1.9 Å) (60Bong S.M. Moon J.H. Nam K.H. Lee K.S. Chi Y.M. Hwang K.Y. FEBS Lett. 2008; 582: 3959-3965Crossref PubMed Scopus (60) Google Scholar). The annelid UcLK-ADP complex shows little of the substrate-bound character described for other phosphagen kinases.With this in mind, both LK structures were compared with a larger group of phosphagen kinases, including representatives of substrate-free, nucleotide, and transition state complexes (Table 2). Table 2 is readily ordered to show that the overall magnitude of conformational changes between the substrate-free and TSA states varies systematically: CK (2.7 Å Cα r.m.s.) > GK (2.4 Å) > CK (2.0 Å). The substrate-free and TSA states have been regarded as the open and closed forms, respectively, but the homologs actually exhibit varying degrees of closure. The majority of binary ADP complexes have hitherto been regarded as closed form, a good first approximation, but one that will be revisited (see below). One of the subunits in the rabbit muscle OcCKMM structure was seen as an exception in open form (61Ohren J.F. Kundracik M.L. Borders Jr., C.L. Edmiston P. Viola R.E. Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 381-389Crossref PubMed Scopus (27) Google Scholar), but with the addition of the annelid UcLK structures, the binary complexes can be interpreted in a new light.TABLE 2Cα r.m.s. differences between superimposed subunits of lombricine kinase and representative creatine and glycocyamine kinases, following superimposition with SSM (56Krissinel E. Henrick K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3102) Google Scholar) Open table in a new tab Within the difference matrix (Table 2), the most consistent placement of both the substrate-free and nucleotide-bound structures of annelid UcLK is between the ADP complex of rabbit muscle OcCKMM and substrate-free chicken mitochondrial GgCKmit. The open form taken by one of the subunits of the rabbit OcCKMM-ADP complex has been rationalized in terms of cooperativity (61Ohren J.F. Kundracik M.L. Borders Jr., C.L. Edmiston P. Viola R.E. Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 381-389Crossref PubMed Scopus (27) Google Scholar). Such rationalization cannot explain the even more open annelid UcLK-ADP structure (Fig. 3, C and D), which is from a crystallographically symmetric dimer and therefore does not exhibit any cooperativity that could inhibit enzyme closure. With the addition of the annelid UcLK-ADP structure, it is now clear that in addition to the majority "closed form," there is a second group of nucleotide complexes that are more open than those first characterized. The ADP complexes of TcCK (21Lahiri S.D. Wang P.F. Babbitt P.C. McLeish M.J. Kenyon G.L. Allen K.N. Biochemistry. 2002; 41: 13861-13867Crossref PubMed Scopus (118) Google Scholar) and most subunits of human mitochondrial HsCKmit (Protein Data Bank ID 2GL6) are more comparable to the closed form TSA CKs (0.7–1.1 Å Cα r.m.s.) than they are to the open, substrate-free forms (1.4–1.9 Å). This is mirrored by the binary complexes of rabbit muscle OcCKMM and annelid UcLK that appear closer to the substrate-free forms (∼0.9 Å) than transition state CKs (1.7–2.1 Å). Furthermore, Table 2 shows rabbit OcCKMM-ADP to be slightly more closed like than annelid UcLK-ADP. Table 2 also makes apparent the differences in human mitochondrial HsCKmit (Protein Data Bank ID 2GL6) between the mostly closed D subunit (like TcCK-ADP) and the other subunits, which are more similar to the fully closed TSA states. In summary, this analysis shows that: 1) corresponding states of homologous phosphagen kinases (and even of the same enzyme) exhibit varying degrees of openness/closure; and 2) the greatest variation is in the nucleotide-bound state, for which a full spectrum of conformers from nearly fully open to fully closed is observed. The determinants of the extent of closure on ADP binding are unknown and could be specific for each homolog. Alternatively, in solution, the ADP-bound enzyme may exist as an equilibrium between multiple states of near equal energy, with minor environmental factors selecting the conformer seen in each crystal structure. The latter becomes more plausible with NMR evidence that, in solution, substrate-free LpAK contains a minor fraction in the closed form of the enzyme (36Niu X. Brüschweiler-Li L. Davulcu O. Skalicky J.J. Brüschweiler R. Chapman M.S. J. Mol. Biol. 2011; 405: 479-496Crossref PubMed Scopus (31) Google Scholar), likely in dynamic equilibrium with the predominant open form, suggesting that the energetic difference between open and closed forms is small.FIGURE 3His178 and subdomain 3 configurations. A, ADP interactions of UcLK His178 and corresponding residues from representative homologs. Four TSA structures (LpAK, TcCK, rabbit muscle OcCKMM, and huma
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