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• W1978558133 abstract "The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp250-Glu252). In addition, the glycine-rich loop of the protein, analogous to the “Gly triad” in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp233 of the catalytic HGD motif, a novel twin arginine motif (Arg340/Arg341), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism. The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp250-Glu252). In addition, the glycine-rich loop of the protein, analogous to the “Gly triad” in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp233 of the catalytic HGD motif, a novel twin arginine motif (Arg340/Arg341), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism. Methionine is indispensable for cellular survival and is in high demand in proliferating cells. This essential amino acid plays critical roles in many ubiquitous cellular functions including protein synthesis, biological methylation, polyamine biosynthesis, as well as in the biosynthesis of the plant hormone ethylene and in some bacteria, quorum sensing. The biosynthesis of methionine is energetically costly, and the need for sufficient methionine has driven the evolution of methionine salvage pathways (1Sufrin J.R. Meshnick S.R. Spiess A.J. Garofalo-Hannan J. Pan X.Q. Bacchi C.J. Antimicrob. Agents Chemother. 1995; 39: 2511-2515Crossref PubMed Scopus (57) Google Scholar). Although the pathway is ubiquitous in almost all organisms, some metabolic variations are found in the pathways between mammals, plants, microbes, and certain parasitic protozoa. A key metabolic difference between mammals and prokaryotic pathogens is the absolute requirement for mtnK (2Sekowska A. Denervaud V. Ashida H. Michoud K. Haas D. Yokota A. Danchin A. BMC Microbiol. 2004; 4: 9Crossref PubMed Scopus (124) Google Scholar), which encodes 5-methylthioribose (MTR) 3The abbreviations used are: MTR, 5-methylthioribose; MTA, 5′-methylthioadenosine; APH(3′)-IIIa, 3′,5″-aminogylcoside phosphotransferase type IIIa; Ho-MAD, holmium multi-wavelength anomalous dispersion; AMPPNP, adenosine 5′-(β,γ-imido)triphosphate; AMPPCP, β,γ-methyleneadenosine 5′-triphosphate; PKA, cAMP-dependent protein kinase A.3The abbreviations used are: MTR, 5-methylthioribose; MTA, 5′-methylthioadenosine; APH(3′)-IIIa, 3′,5″-aminogylcoside phosphotransferase type IIIa; Ho-MAD, holmium multi-wavelength anomalous dispersion; AMPPNP, adenosine 5′-(β,γ-imido)triphosphate; AMPPCP, β,γ-methyleneadenosine 5′-triphosphate; PKA, cAMP-dependent protein kinase A. kinase (EC 2.7.1.100) (3Ferro A.J. Barrett A. Shapiro S.K. J. Biol. Chem. 1978; 253: 6021-6025Abstract Full Text PDF PubMed Google Scholar) for bacterial methionine salvage. MTR kinase is regulated by the environmental methionine level (4Tower P.A. Alexander D.B. Johnson L.L. Riscoe M.K. J. Gen. Microbiol. 1993; 139: 1027-1031Crossref PubMed Scopus (7) Google Scholar) and its expression enables organisms to grow on non-methionine sulfur sources such as MTR or 5′-methylthioadenosine (MTA) (5Riscoe M.K. Ferro A.J. Fitchen J.H. Antimicrob. Agents Chemother. 1988; 32: 1904-1906Crossref PubMed Scopus (29) Google Scholar, 6Sekowska A. Mulard L. Krogh S. Tse J.K. Danchin A. BMC Microbiol. 2001; 1: 15Crossref PubMed Scopus (26) Google Scholar, 7Sauter M. Cornell K.A. Beszteri S. Rzewuski G. Plant Physiol. 2004; 136: 4061-4071Crossref PubMed Scopus (43) Google Scholar). MTA is a byproduct and inhibitor of polyamine synthesis (8Sekowska A. Danchin A. BMC Microbiol. 2002; 2: 8Crossref PubMed Scopus (78) Google Scholar) and hence is toxic to cells and must be rapidly degraded. In various microbes, plants, and certain protozoa, MTA is degraded by MTA nucleosidase into MTR and adenine. MTR kinase then catalyzes the phosphorylation of MTR to MTR 1-phosphate, which is subsequently converted to methionine via a series of intermediates (8Sekowska A. Danchin A. BMC Microbiol. 2002; 2: 8Crossref PubMed Scopus (78) Google Scholar, 9Murphy B.A. Grundy F.J. Henkin T.M. J. Bacteriol. 2002; 184: 2314-2318Crossref PubMed Scopus (42) Google Scholar). In mammalian cells, however, the degradation of MTA and its conversion to MTR 1-phosphate is achieved in a single step by MTA phosphorylase (10Pegg A.E. Williams-Ashman H.G. Biochem. J. 1969; 115: 241-247Crossref PubMed Scopus (141) Google Scholar). This metabolic difference in the way MTA is removed has been explored and analogs of MTR, synthesized as pro-drugs, have been shown to selectively kill MTR kinase-containing organisms with little effect on mammalian cells (5Riscoe M.K. Ferro A.J. Fitchen J.H. Antimicrob. Agents Chemother. 1988; 32: 1904-1906Crossref PubMed Scopus (29) Google Scholar, 11Gianotti A.J. Tower P.A. Sheley J.H. Conte P.A. Spiro C. Ferro A.J. Fitchen J.H. Riscoe M.K. J. Biol. Chem. 1990; 265: 831-837Abstract Full Text PDF PubMed Google Scholar). The absence of a mammalian homolog makes MTR kinase a good target for the design of novel antibiotics. The modern rational approach to new drug discovery includes structure-based drug design, which requires a detailed understanding of the structure of the target enzyme and its catalytic mechanism and substrate specificity. Thus, three-dimensional structures of MTR kinase with and without substrates are indispensable for the development of specific antibiotics targeting this enzyme. To this end, we present the first structural analysis of MTR kinase in its apo form and in complex with ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR. Structure Determination of MTR Kinase Using Ho-MAD Phasing—Bacillus subtilis MTR kinase (gi:37999472) was expressed, purified, and crystallized as described previously (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar). The crystals were derivatized by soaking the crystals for 3 days in 2 mm HoCl3, 25% (w/v) PEG2000MME, 25% (v/v) ethylene glycol, 0.3 m sodium acetate, and 0.1 m Tris-HCl, pH 7.8. The holmium derivative was flash frozen in a nitrogen stream at 110 K, and a MAD dataset was collected around the LIII edge of Ho to 2.5 Å resolution at Station X8C, National Synchrotron Light Source (NSLS), Brookhaven National Laboratory. The peak and inflection data were processed using HKL2000 (13Otwinowski Z. Minor W. Methods in Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar), and the remote data were reprocessed at home using d*TREK (14Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). Two pairs of Ho atoms in the asymmetric unit were readily located using SnB (15Weeks C.M. Blessing R.H. Miller R. Mungee R. Potter S.A. Rappleye J. Smith G.D. Xu H. Furey W. Z. Kristallographie. 2002; 217: 686-693Google Scholar). The CNS suite of programs was used for heavy atom refinement, MAD phasing, and density modification (16Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). The data and phasing statistics are summarized in Table 1.TABLE 1Diffraction data, MAD phasing, and refinement statistics Open table in a new tab An initial model was built using RESOLVE (17Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1628) Google Scholar, 18Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar), and cycles of structure refinement were iterated between CNS (16Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) and manual rebuilding in Xfit (19McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2016) Google Scholar) with 5% cross-validated data (20Brunger A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 24-36Crossref PubMed Google Scholar). While the structure was being refined, unambiguous density for ADP was identified in the active site in complex with Ho ions (21Ku S.Y. Smith G.D. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007; 63: 493-499Crossref PubMed Scopus (5) Google Scholar). The MTRK-ADP-Ho complex (21Ku S.Y. Smith G.D. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007; 63: 493-499Crossref PubMed Scopus (5) Google Scholar) was used as the initial model for the refinement of other MTR kinase structures: the apo-MTR kinase (MTRK), the ADP-magnesium complex (MTRK-ADP), the ADP-magnesium phosphate complex (MTRK-ADP-PO4), the AMPPCP-magnesium complex (MTRK-AMPPCP), and the AMPPCP-magnesium, substrate MTR complex (MTRK-AMPPCP-MTR). Structure Determination of Apo-MTR Kinase and Its Substrate Complexes—Crystals of apo-MTRK were grown as described previously (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar). The MTRK-ADP and the MTRK-AMPPCP complexes were prepared by overnight soaking of MTRK crystals in 25% (w/v) PEG2000MME, 0.3 m sodium acetate, 20 mm magnesium acetate, and either 4 mm ADP or AMPPCP (sodium salt). The MTRK-AMPPCP-MTR complex was prepared by soaking the MTRK crystals in 25% (w/v) PEG2000MME, 0.3 m sodium acetate, 20 mm magnesium acetate, 4 mm AMPPCP, and 2 mm MTR for 2 h. The substrate MTR was synthesized by hydrolysis of MTA as described previously (22Schlenk F. Zydek-Cwick C.R. Dainko J.L. Biochim. Biophys. Acta. 1973; 320: 357-362Crossref PubMed Scopus (34) Google Scholar). We also attempted, by co-crystallization, to determine the structure of an MTRK-ATP complex. In this case 10 mm ATP (sodium salt) and 2 mm MgCl2 were added to the apo-MTRK protein solution prior to crystallizing the mixture as described for apo-MTRK (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar). Due to hydrolysis of the ATP by the enzyme, ADP, rather than ATP, was observed in each of the active sites (see below). Crystals of the complexes were soaked in 25% (v/v) ethylene glycol, 25% (w/v) PEG2000MME, 0.3 m sodium acetate for 30 s and flash frozen at 110 K. All data were collected using CuKα x-radiation from an RU-H3R rotating anode generator, Osmic optics, and R-AXIS IV++ image plate detector, and processed using d*TREK (14Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). The refinement and model building was initiated using the iterative procedures implemented in ARP/ wARP (23Morris R.J. Perrakis A. Lamzin V.S. Methods Enzymol. 2003; 374: 229-244Crossref PubMed Scopus (469) Google Scholar) with the structure of the MTRK-ADP-Ho complex as the starting model. Cycles of structure refinement were then alternated between Refmac (24Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar) and manual rebuilding using Coot (25Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22534) Google Scholar) before a final round of TLS (translation, rotation/liberation, screw-rotation) refinement. Two TLS groups per subunit (residues 1-120 and 121-397) were defined and the TLS tensor was refined using Refmac in the CCP4 program suite (26Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1639) Google Scholar, 27Winn M.D. Murshudov G.N. Papiz M.Z. Methods Enzymol. 2003; 374: 300-321Crossref PubMed Scopus (668) Google Scholar, 28Collaborative Computational Project, N.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar). The final B. subtilis structural models of apo-MTRK, MTRK-ADP, MTRK-AMPPCP, and MTRK-AMPPCP-MTR have R/Rfree factors of 20.0/25.2% (2.1 Å), 19.9/24.9% (2.2 Å), 20.4/23.8% (2.0 Å), and 20.2/25.5% (2.3 Å), respectively. Co-crystallization of ATP with the protein resulted in electron density compatible with ADP molecules rather than ATP in both active sites of the protein. In both monomers spherical density was also observed in the σA weighted Fo - Fc map just below the position that the γ-phosphate of ATP would have occupied if ATP was present. A water molecule was initially modeled in both monomers but in monomer B, the B-factor for the water refined to a very small value while still showing positive density of >3σ in the Fo - Fc difference map. A phosphate ion was therefore modeled in place of this water molecule. The phosphate has higher B-factors than its neighboring atoms suggesting that it is not present at full occupancy. Reduced occupancy for the phosphate is supported by the observation that when its occupancy was set to 0.5, its B-factor refined to about half of those of the neighboring atoms. During the refinement of the phosphate we have fixed the occupancy at 1 and have refined its B-factor. A water molecule rather than a phosphate ion is modeled in monomer A because, although visible, the density for a phosphate ion is less convincing than that observed in monomer B. The final co-crystallized MTRK-ADP-PO4 complex has R/Rfree factors of 21.1/27.4% (2.6 Å). Data, refinement, and structure validation statistics for the five MTR kinase structures are summarized in Table 1. A list of the disordered residues and disordered side chains in the five structures is provided in the supplementary material (Table S1). All figures were prepared using PyMOL (29DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Overall Fold and Quaternary Structure—The structure of B. subtilis MTR kinase has a bilobal architecture consisting of a smaller N-terminal lobe (N-lobe) and a larger C-terminal MTR-binding lobe (C-lobe) connected by a linker region (residue 115-120) (Fig. 1A). Although about 30% of the N-lobe is disordered in the structures and could not be modeled (see supplementary Table S1), the backbone density clearly reveals the same fold as the N-lobe of a protein kinase. The N-lobe of MTR kinase is composed of 5 twisted anti-parallel β strands (β1-β5) flanked by two α helices (α1 and α2) in a αβββαββ topology. The larger C-lobe is predominantly α helical, with 12 α helices (α3-14) and four short 310 helices (η1-η4) along with four short β strands (β7-β10) between helices α8 and α9, and a short strand β6 at the C-terminal end of the linker region. Strand β8 forms an anti-parallel β turn with β9 and also interacts with the tip of strand β6, whereas strands β7 and β10 form a second twisted anti-parallel sheet. Together with the N-lobe, the topology profile of these anti-parallel β strands in a helical environment is also consistent with that of a protein kinase. Thus, although MTR kinase is only found in bacteria, plants, and other lower organisms, the protein has the same bilobal fold seen in eukaryotic protein kinases. The crystal structure of B. subtilis MTR kinase has two subunits in the asymmetric unit related by 2-fold non-crystallographic symmetry (Fig. 1B). The inter-subunit interactions in the dimer interface are symmetrical: there are six inter-subunit salt bridges between Lys162 and Glu178, Lys217 and Asp321, and Lys220 and Glu227; four inter-subunit main chain interactions between Thr224 and its non-crystallographic symmetry equivalent, and Ala226 and its non-crystallographic symmetry equivalent; and two inter-subunit hydrogen bonds between the carbonyl oxygen of Phe182 and the Oη oxygen of Tyr312 (Fig. 1C). Upon dimerization of MTR kinase, about 1670 Å2 of surface area is buried (30Fraternali F. Cavallo L. Nucleic Acids Res. 2002; 30: 2950-2960Crossref PubMed Scopus (90) Google Scholar). Examination of the protein quaternary structure data base (31Henrick K. Thornton J.M. Trends Biochem. Sci. 1998; 23: 358-361Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar) reveals that this interface is larger than the interfaces found in 75% of all homodimeric kinases. The dimeric structure of B. subtilis MTR kinase is also consistent with the gel filtration profile of the 45-kDa enzyme showing an apparent molecular mass of ∼100 kDa in solution (data not shown) and previous in vitro characterization of MTR kinases from other species (32Guranowski A. Plant Physiol. 1983; 71: 932-935Crossref PubMed Google Scholar, 33Cornell K.A. Winter R.W. Tower P.A. Riscoe M.K. Biochem. J. 1996; 317: 285-290Crossref PubMed Scopus (32) Google Scholar), as well as with the cooperative kinetics observed for Oryza sativa MTR kinase (7Sauter M. Cornell K.A. Beszteri S. Rzewuski G. Plant Physiol. 2004; 136: 4061-4071Crossref PubMed Scopus (43) Google Scholar). Structural Neighbors of MTR Kinase—Tertiary structural comparison of MTR kinase to the Protein Data Bank using Dali (34Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3545) Google Scholar) or FATCAT (35Ye Y. Godzik A. Bioinformatics. 2003; 19: II246-II255Crossref PubMed Scopus (463) Google Scholar) reveals structural similarity to choline kinase (36Peisach D. Gee P. Kent C. Xu Z. Structure (Camb.). 2003; 11: 703-713Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) (1NW1) (Fig. 2A) and aminoglycoside phosphotransferase type IIIa (37Hon W.C. McKay G.A. Thompson P.R. Sweet R.M. Yang D.S. Wright G.D. Berghuis A.M. Cell. 1997; 89: 887-895Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) (APH(3′)-IIIa) (1J7I) (Fig. 2B) despite limited sequence identities of 15 and 12%, respectively. FATCAT calculates that MTR kinase has Cα root mean square deviations of 3.4 and 3.0 Å when compared with choline kinase and APH(3′)-IIIa, respectively. MTR kinase is also structurally similar to many eukaryotic protein kinases including casein kinase (38Xu R.M. Carmel G. Sweet R.M. Kuret J. Cheng X. EMBO J. 1995; 14: 1015-1023Crossref PubMed Scopus (181) Google Scholar), cAMP-dependent protein kinase (PKA) (39Zheng J. Trafny E.A. Knighton D.R. Xuong N.H. Taylor S.S. Ten Eyck L.F. Sowadski J.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 362-365Crossref PubMed Google Scholar), and G protein-coupled receptor kinase (40Lodowski D.T. Tesmer V.M. Benovic J.L. Tesmer J.J. J. Biol. Chem. 2006; 281: 16785-16793Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In the structural alignment, the anti-parallel β sheet found in the N-lobe of MTR kinase, choline kinase, and APH(3′)-IIIa superimpose readily (Fig. 2C), whereas the C-lobes reveal larger structural variations that account for the diversity of the substrates catalyzed. Nevertheless, the alignment reveals “core helices” corresponding to helices α4, α6, α8, α9, α10, and α11 of MTR kinase that are common to all three proteins. Examination of the quaternary structure of MTK kinase, choline kinase, and APH(3′)-IIIa reveals that although they share a common fold, their quaternary structures are unique. MTR kinase dimerizes mainly via interactions between helices α8 (Fig. 1B), whereas choline kinase dimerizes via interactions between helices that are structurally equivalent to helix α2 of MTR kinase (Fig. 2A). APH(3′)-IIIa is held together as a dimer in the crystal structure via disulfide bonds (37Hon W.C. McKay G.A. Thompson P.R. Sweet R.M. Yang D.S. Wright G.D. Berghuis A.M. Cell. 1997; 89: 887-895Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) (Fig. 2B) between strand β1 and the poorly conserved region between helices α6 and α7. The quaternary structure of MTK kinase, choline kinase, and APH(3′)-IIIa differs from the catalytic domains of many protein kinase that appear to function as monomers in the absence of their regulatory domains (38Xu R.M. Carmel G. Sweet R.M. Kuret J. Cheng X. EMBO J. 1995; 14: 1015-1023Crossref PubMed Scopus (181) Google Scholar, 41Zhao B. Bower M.J. McDevitt P.J. Zhao H. Davis S.T. Johanson K.O. Green S.M. Concha N.O. Zhou B.B. J. Biol. Chem. 2002; 277: 46609-46615Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Nucleotide-binding Site—Overall the nucleotide-binding site is relatively unperturbed between the apo, ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR bound MTR kinase structures (Fig. 3A) and the residues in the substrate binding pocket can be readily superimposed (Fig. 3B). The nucleotide binds in the cleft region between the two lobes of the MTR kinase, making extensive interactions with residues from both lobes as well as with residues from the linker region (residue 115-120). The adenine ring of the nucleotide binds near the linker region in a hydrophobic pocket consisting of residues Val46, Ile59, Met114, Leu117, Phe240, and Ile249 (Fig. 3B). Only the hydrophobicity of these residues is conserved across species. Besides these hydrophobic interactions, the adenine ring is also held by two hydrogen bonds: one from the backbone carbonyl oxygen of Glu115 to the N-6 amino nitrogen of the adenine, and the other from the amide nitrogen of Leu117 to the N-1 nitrogen (Fig. 4). A structurally conserved water molecule donates two hydrogen bonds to the nucleotide: one to the N-7 nitrogen of the adenine ring and the second to the O-1 oxygen of the α phosphoryl group of the nucleotide (Fig. 4). This water molecule is observed in all but the lowest resolution MTRK-ADP-PO4 structure. In all nucleotide complexes, the O-1 oxygen atoms of the α phosphoryl group of the nucleotide also interact via ionic interactions with the Nζ nitrogen of the strictly conserved Lys61 (Fig. 4), which in turn forms a salt bridge with the Oϵ carboxyl oxygen of Glu84 from helix α2.FIGURE 4Active site of B. subtilis MTR kinase. A and B, schematic diagram showing the non-hydrophobic interactions between the ligands and the protein (in A) the MTRK-ADP and MTRK-ADP-PO4 complexes and the MTRK-AMPPCP and MTRK-AMPPCP-MTR complexes (in B). In A, the nucleotide and protein are presented as sticks with C, N, O, and P atoms colored green, blue, red, and magenta, respectively. In B, C atoms are colored in cyan. Magnesium ions and water molecules are shown as yellow and blue spheres, respectively. The distances, in Å, are the average of four monomers with the standard deviations given in square brackets. The water molecule in A is not clearly observed in the MTRK-ADP-PO4 complex thus the distances given refer to those in chain A of the MTRK-ADP complex; distances in chain B are given in brackets. In B, the distances between MTR and the protein refer to those in chain A of the MTRK-AMPPCP-MTR complex; distances in chain B are given in brackets. The water labeled in gray in B does not have a conserved position in all structures although its distance to Mg(1) is comparable. For consistency between the two panels, some distances >3 Å are also depicted but are shown in light gray. Residues that are part of the G-loop are labeled green. The side chain of Asp40 is only visible in monomers A of MTRK-AMPPCP and MTRK-AMPPCP-MTR complexes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Apparent Lack of Lobe Movement—The apparent rigidity of the nucleotide-binding site between the two lobes of MTR kinase is supported by the lack of interactions between the ribosyl moiety of the nucleotide and residues from C-lobe. In PKA, nucleotide binding induces lobe closure. Interactions between residues in the C-lobe of PKA and the bound nucleotide have been defined as “closure-inducing residues” as these interactions produce a torque about the hinge axis and drive lobe closure (42Hayward S. J. Mol. Biol. 2004; 339: 1001-1021Crossref PubMed Scopus (71) Google Scholar). The most important closure-inducing residue in PKA is Glu127, which is located on the C-lobe and interacts with both ribosyl hydroxyl oxygens of ATP (39Zheng J. Trafny E.A. Knighton D.R. Xuong N.H. Taylor S.S. Ten Eyck L.F. Sowadski J.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 362-365Crossref PubMed Google Scholar). No residue in MTR kinase plays an equivalent role. A general problem associated with every crystal structure is that crystal-packing forces can occasionally lock the protein into a single conformation, open or closed. Whereas the presence of lobe or domain movement is perhaps easier to identify given two structures with different conformations, proving that no lobe movement occurs is more challenging. Additional evidence that supports our hypothesis that the lack of lobe closure observed between apo-MTRK and the various complexes is not the result of crystal packing, but relevant to the function of the protein, is as follows. First, B. subtilis MTR kinase in the absence of detergent crystallizes in a different space group (12Ku S.Y. Yip P. Cornell K.A. Riscoe M.K. Howell P.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 116-119Crossref PubMed Scopus (11) Google Scholar) with different surfaces involved in the packing interactions. Despite the differences in packing environments no major structural differences occur as a result of the different crystal contacts. Second, we have recently determined the structure of Arabidopsis thaliana MTR kinase complexed with ADP and MTR. 4S.-Y. Ku, K. A. Cornell, and P. L. Howell, manuscript in preparation. This co-crystal structure packs in yet another space group and reveals that the plant enzyme has the same conformation as the apo and complexed forms of the bacterial enzyme. Third, soaking (data not shown) and co-crystallization of B. subtilis MTRK with ATP produced in both cases ADP rather than ATP in the active site. For the soaking experiment this suggests that MTR kinase is active in the crystalline state and that ATP hydrolysis can occur without disrupting the crystal lattice. If significant inter-lobal movement was required for ATP binding or hydrolysis, and this movement was prevented by crystal contacts, one would have expected the crystals to shatter during the soaking experiment. The similarity of the ADP-PO4 (co-crystallized) and ADP (produced by soaking ADP) complexes also supports our hypothesis that no lobe movement occurs because, regardless of when hydrolysis of the ATP occurred, crystal-packing forces could not have influenced the conformation of the protein complex prior to its incorporation into the lattice. As the nucleotide-binding site of MTR kinase comprises residues from both lobes as well as the linker region, and the entire active site is readily superimposable in all MTRK structures determined to date (Fig. 3), current structural data suggests that unlike protein kinases, but comparable with APH(3′)-IIIa, MTRK does not undergo large lobe movement upon ATP binding, ATP hydrolysis, and ADP release. The Gly-rich Loop—A Gly-rich motif or G-loop is found between β-strands β1 and β2 in the N-lobe of MTR kinase. The G-loop of MTR kinase has a highly conserved GXGNXN motif (residue 39-44), although the last residue, Asn44, is not structurally in the loop but located on strand β2 according to DSSP (see supplemental Fig. S1). The G-loop is structurally analogous to the “nucleotide positioning loop” in APH(3′)-IIIa (44Thompson P.R. Boehr D.D. Berghuis A.M. Wright G.D. Bioch" @default.
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• W1978558133 date "2007-07-01" @default.
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• W1978558133 title "Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding" @default.
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• W1978558133 doi "https://doi.org/10.1074/jbc.m611045200" @default.
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