A structural basis for lithium and substrate binding of an inositide phosphatase
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.014057
ISSN1083-351X
AutoresD.E. Dollins, Jian-Ping Xiong, Stuart Endo-Streeter, David E. Anderson, Vinay S. Bansal, Jay W. Ponder, Yi Ren, John D. York,
Tópico(s)Phytase and its Applications
ResumoInositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases. Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases. Inositol phosphate (IP) signaling is critically important to cellular communication networks. Among the numerous lipid and soluble IP messengers, inositol 1,4,5-trisphosphate (IP3) is crucial for mediating the agonist-induced release of intracellular calcium stores (1Michell R.H. Inositol lipids in cellular signalling mechanisms.Trends Biochem. Sci. 1992; 17: 274-276Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 2Berridge M.J. Irvine R.F. Inositol phosphates and cell signalling.Nature. 1989; 341: 197-205Crossref PubMed Scopus (3237) Google Scholar). Termination of IP3 signaling occurs, in part, through the action of IP kinases and phosphatases, the latter of which also function to replenish stores of inositol—metabolism reviewed in (3Irvine R.F. Schell M.J. Back in the water: the return of the inositol phosphates.Nat. Rev. Mol. Cell Biol. 2001; 2: 327-338Crossref PubMed Scopus (506) Google Scholar, 4Majerus P.W. Inositol phosphate biochemistry.Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (341) Google Scholar). Links of inositol signaling to the pharmacology of lithium emerged when it was first reported that treatment of rats with lithium resulted in decreased brain inositol levels and led to the accumulation of inositol monophosphates and polyphosphates (5Allison J.H. Stewart M.A. Reduced brain inositol in lithium-treated rats.Nat. New Biol. 1971; 233: 267-268Crossref PubMed Scopus (268) Google Scholar). Two IP phosphatase activities, inositol monophosphate phosphatase (IMPA1, IMPA2) and inositol polyphosphate 1-phosphatase (INPP1) were reported to be potently inhibited by lithium (6Inhorn R.C. Majerus P.W. Properties of inositol polyphosphate 1-phosphatase.J. Biol. Chem. 1988; 263: 14559-14565Abstract Full Text PDF PubMed Google Scholar, 7Inhorn R.C. Majerus P.W. Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+.J. Biol. Chem. 1987; 262: 15946-15952Abstract Full Text PDF PubMed Google Scholar, 8Hallcher L.M. Sherman W.R. The effects of lithium ion and other agents on the activity of myo-inositol-1-phosphatase from bovine brain.J. Biol. Chem. 1980; 255: 10896-10901Abstract Full Text PDF PubMed Google Scholar). Cloning and characterization of IMPA1 and INPP1 gene products confirmed they encoded metal-dependent lithium-inhibited enzymes with minimal sequence homology aside from a short six amino acid motif (9York J.D. Majerus P.W. Isolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate 1-phosphatase.Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9548-9552Crossref PubMed Scopus (43) Google Scholar, 10Diehl R.E. Whiting P. Potter J. Gee N. Ragan C.I. Linemeyer D. Schoepfer R. Bennett C. Dixon R.A. Cloning and expression of bovine brain inositol monophosphatase.J. Biol. Chem. 1990; 265: 5946-5949Abstract Full Text PDF PubMed Google Scholar). Structural studies of IMPA1 and INPP1 illuminated that the "DPIDxT" six-amino acid motif anchor metal binding sites likely involved catalysis (11York J.D. Ponder J.W. Chen Z.W. Mathews F.S. Majerus P.W. Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.Biochemistry. 1994; 33: 13164-13171Crossref PubMed Scopus (44) Google Scholar, 12Bone R. Springer J.P. Atack J.R. Structure of inositol monophosphatase, the putative target of lithium therapy.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10031-10035Crossref PubMed Scopus (139) Google Scholar). Remarkably, superimposition of alpha-carbons of these six residues of IMPA1 and INPP1 aligned 13 secondary structural elements of a 280 amino acid common core fold. Sequence comparison based on this three-dimensional alignment indicates they are prototype members of a small family of structurally conserved phosphatases, whose activities were not only limited to inositol signaling but also include regulation of gluconeogenesis and nucleotide metabolism (13York J.D. Ponder J.W. Majerus P.W. Definition of a metal-dependent/Li(+)-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5149-5153Crossref PubMed Scopus (152) Google Scholar). Upon completion of the human and mouse genome sequences, we now know that the family comprised of seven genes including IMPA1 and its ortholog IMPA2; INPP1; FBP1 and FBP2—fructose 1,6-bisphosphate phosphatases; BPNT1—adenosine 3',5' bisphosphate nucleotidase; and gPAPP—Golgi-resident 3'phosphoadenosine-5'phosphate phosphatase (14Frederick J.P. Tafari A.T. Wu S.M. Megosh L.C. Chiou S.T. Irving R.P. York J.D. A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 11605-11612Crossref PubMed Scopus (51) Google Scholar, 15Spiegelberg B.D. Dela Cruz J. Law T.H. York J.D. Alteration of lithium pharmacology through manipulation of phosphoadenosine phosphate metabolism.J. Biol. Chem. 2005; 280: 5400-5405Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Of interest, all seven gene products are potently inhibited in vitro at or below therapeutic lithium concentrations (9York J.D. Majerus P.W. Isolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate 1-phosphatase.Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9548-9552Crossref PubMed Scopus (43) Google Scholar, 10Diehl R.E. Whiting P. Potter J. Gee N. Ragan C.I. Linemeyer D. Schoepfer R. Bennett C. Dixon R.A. Cloning and expression of bovine brain inositol monophosphatase.J. Biol. Chem. 1990; 265: 5946-5949Abstract Full Text PDF PubMed Google Scholar, 14Frederick J.P. Tafari A.T. Wu S.M. Megosh L.C. Chiou S.T. Irving R.P. York J.D. A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 11605-11612Crossref PubMed Scopus (51) Google Scholar, 15Spiegelberg B.D. Dela Cruz J. Law T.H. York J.D. Alteration of lithium pharmacology through manipulation of phosphoadenosine phosphate metabolism.J. Biol. Chem. 2005; 280: 5400-5405Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16Hudson B.H. York J.D. Roles for nucleotide phosphatases in sulfate assimilation and skeletal disease.Adv. Biol. Regul. 2012; 52: 229-238Crossref PubMed Scopus (15) Google Scholar, 17Spiegelberg B.D. Xiong J.P. Smith J.J. Gu R.F. York J.D. Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate.J. Biol. Chem. 1999; 274: 13619-13628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Detailed comparison of the structures of four members of the family suggests a common metal-dependent catalytic mechanism (11York J.D. Ponder J.W. Chen Z.W. Mathews F.S. Majerus P.W. Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.Biochemistry. 1994; 33: 13164-13171Crossref PubMed Scopus (44) Google Scholar, 12Bone R. Springer J.P. Atack J.R. Structure of inositol monophosphatase, the putative target of lithium therapy.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10031-10035Crossref PubMed Scopus (139) Google Scholar, 18Albert A. Yenush L. Gil-Mascarell M.R. Rodriguez P.L. Patel S. Martinez-Ripoll M. Blundell T.L. Serrano R. X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity.J. Mol. Biol. 2000; 295: 927-938Crossref PubMed Scopus (57) Google Scholar, 19Ke H. Thorpe C.M. Seaton B.A. Marcus F. Lipscomb W.N. Molecular structure of fructose-1,6-bisphosphatase at 2.8-A resolution.Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1475-1479Crossref PubMed Scopus (54) Google Scholar). Conserved secondary structural elements comprise juxtaposed metal binding pockets and define a shared sequence pattern of D54…E79E80…D153PID156S157T158…D317 as shown for INPP1 (Fig. 1). Despite several reports that have defined the position of activating metal sites used for catalysis by this protein family (20Dutta A. Bhattacharyya S. Dutta D. Das A.K. Structural elucidation of the binding site and mode of inhibition of Li(+) and Mg(2+) in inositol monophosphatase.FEBS J. 2014; 281: 5309-5324Crossref PubMed Scopus (14) Google Scholar, 21Gill R. Mohammed F. Badyal R. Coates L. Erskine P. Thompson D. Cooper J. Gore M. Wood S. High-resolution structure of myo-inositol monophosphatase, the putative target of lithium therapy.Acta Crystallogr. 2005; 61: 545-555Crossref PubMed Scopus (51) Google Scholar, 22Johnson K.A. Chen L. Yang H. Roberts M.F. Stec B. Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities.Biochemistry. 2001; 40: 618-630Crossref PubMed Scopus (53) Google Scholar, 23Choe J.Y. Poland B.W. Fromm H.J. Honzatko R.B. Role of a dynamic loop in cation activation and allosteric regulation of recombinant porcine fructose-1,6-bisphosphatase.Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (58) Google Scholar), understanding the mechanism of uncompetitive lithium inhibition has been challenging. Nonetheless, studies for three family members, IMPA1, yeast 3'-nucleotidase (Hal2p), and FBP1, have provided insights into catalytic metal orientation and effects of lithium on these configurations (12Bone R. Springer J.P. Atack J.R. Structure of inositol monophosphatase, the putative target of lithium therapy.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10031-10035Crossref PubMed Scopus (139) Google Scholar, 18Albert A. Yenush L. Gil-Mascarell M.R. Rodriguez P.L. Patel S. Martinez-Ripoll M. Blundell T.L. Serrano R. X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity.J. Mol. Biol. 2000; 295: 927-938Crossref PubMed Scopus (57) Google Scholar, 20Dutta A. Bhattacharyya S. Dutta D. Das A.K. Structural elucidation of the binding site and mode of inhibition of Li(+) and Mg(2+) in inositol monophosphatase.FEBS J. 2014; 281: 5309-5324Crossref PubMed Scopus (14) Google Scholar, 24Villeret V. Huang S. Fromm H.J. Lipscomb W.N. Crystallographic evidence for the action of potassium, thallium, and lithium ions on fructose-1,6-bisphosphatase.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8916-8920Crossref PubMed Scopus (72) Google Scholar, 25Bone R. Frank L. Springer J.P. Pollack S.J. Osborne S.A. Atack J.R. Knowles M.R. McAllister G. Ragan C.I. Broughton H.B. Baker R. Fletcher S.R. Structural analysis of inositol monophosphatase complexes with substrates.Biochemistry. 1994; 33: 9460-9467Crossref PubMed Scopus (85) Google Scholar, 26Bone R. Frank L. Springer J.P. Atack J.R. Structural studies of metal binding by inositol monophosphatase: evidence for two-metal ion catalysis.Biochemistry. 1994; 33: 9468-9476Crossref PubMed Scopus (103) Google Scholar). Other data include fluorescence quenching experiments in IMPA1 (27Pollack S.J. Atack J.R. Knowles M.R. McAllister G. Ragan C.I. Baker R. Fletcher S.R. Iversen L.L. Broughton H.B. Mechanism of inositol monophosphatase, the putative target of lithium therapy.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5766-5770Crossref PubMed Scopus (118) Google Scholar) and inference from coordination geometries of Li+ modeled into the active site of IMPA1 (21Gill R. Mohammed F. Badyal R. Coates L. Erskine P. Thompson D. Cooper J. Gore M. Wood S. High-resolution structure of myo-inositol monophosphatase, the putative target of lithium therapy.Acta Crystallogr. 2005; 61: 545-555Crossref PubMed Scopus (51) Google Scholar). In opposition to those data, support for Li+ inhibition at metal site 3 (MG3) include structural studies of the archael IMPA1/FBP1s implicating conformational changes associated in a loop region responsible for forming metal site 3 (28Stec B. Yang H. Johnson K.A. Chen L. Roberts M.F. MJ0109 is an enzyme that is both an inositol monophosphatase and the 'missing' archaeal fructose-1,6-bisphosphatase.Nat. Struct. Biol. 2000; 7: 1046-1050Crossref PubMed Scopus (75) Google Scholar, 29Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. Crystal structure of a dual activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus. The story of a mobile loop.J. Biol. Chem. 2002; 277: 22863-22874Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). These results agree with earlier mutagenesis studies of human IMPA1 that showed reduced lithium sensitivity and the loss of inhibition by high concentrations of Mg2+ after mutation of a lysine residue found in the loop region that helps form metal site 3 (30Ganzhorn A.J. Lepage P. Pelton P.D. Strasser F. Vincendon P. Rondeau J.M. The contribution of lysine-36 to catalysis by human myo-inositol monophosphatase.Biochemistry. 1996; 35: 10957-10966Crossref PubMed Scopus (22) Google Scholar). To further our understanding of lithium's mechanism of action, here were report a systematic series of crystallographic studies of INPP1 that reveal the effect of lithium on the binding to the metal-enzyme complex when substrate or product is present. From these data, we infer where and how lithium binds in the INPP active site illuminating a structural basis for its uncompetitive pattern of inhibition. Additionally, we used this information to determine the structure of the INPP/substrate complex which provides additional mechanistic understanding of active site determinants and reaction mechanism. We initiated a series of crystallographic studies building upon the original structure determination of INPP1 by multiple isomorphous replacement (11York J.D. Ponder J.W. Chen Z.W. Mathews F.S. Majerus P.W. Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.Biochemistry. 1994; 33: 13164-13171Crossref PubMed Scopus (44) Google Scholar). In these studies, the lanthanide metal gadolinium (Gd3+), a potent competitive inhibitor with respect to magnesium, was used in the presence of Li+ as one of the heavy atom derivatives and provides two key observations. The gadolinium ions found in the INPP1/Gd3+/Li+ structure bound predominantly to two sites in the INPP1 active site. Compared with the native INPP1/Mg2+/Li+ structure, the first gadolinium site (GD1) was well correlated with the position of the magnesium metal site 1 (MG1). The second gadolinium, however, was found 3.7 Å from the magnesium in site 2 (MG2) and was coordinated by a different set of protein and (presumably) solvent interactions. Given the anomalous differences of Gd3+, the identity of the metal in sites GD1 and the previously unseen metal site GD3 is unequivocal. Second, based upon difference Fourier analysis, the gadolinium metal density peaks were unequally occupied. Magnesium in the native structure had densities peaks of 4.4σ and 3.3σ in metal sites MG1 and MG2, respectively, giving a relative occupancy of 1.3. The gadolinium derivative showed a strong density peak (34.9σ) at GD1 and a relatively much weaker density peak (7.9σ) at GD3, giving a relative occupancy of the sites of 4.4 (Fig. 2A). The crystals of INPP1 used for the initial structure determinations were grown in the presence of very high Li+ concentrations (11York J.D. Ponder J.W. Chen Z.W. Mathews F.S. Majerus P.W. Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.Biochemistry. 1994; 33: 13164-13171Crossref PubMed Scopus (44) Google Scholar, 31York J.D. Chen Z.W. Ponder J.W. Chauhan A.K. Mathews F.S. Majerus P.W. Crystallization and initial X-ray crystallographic characterization of recombinant bovine inositol polyphosphate 1-phosphatase produced in Spodoptera frugiperda cells.J. Mol. Biol. 1994; 236: 584-589Crossref PubMed Scopus (10) Google Scholar). Thus, an explanation for the finding of metal mainly at site GD1 may be that Li+, for which the electron density is essentially invisible, may interfere with Gd3+ binding, either by directly competing for metal site GD3 or by inducing a conformational change thereby preventing binding. We first sought to rule out that the unequal occupancy of GD1 and GD3 was because of competition among the lesser electron dense metal. New crystals were grown under similar conditions with Gd3+ replacing Mg2+ but in the absence of Li+. Under these conditions, the electron densities for metal sites GD1 and GD3 gave a similar but less pronounced pattern of unequal occupancy (Fig. 2B). The Fo-Fc difference density gave peaks of 18.0σ and 10.5σ in metal sites GD1 and GD3, respectively, with a relative occupancy of 1.7. Similarly, the anomalous difference density showed peaks of 13.7σ and 5.7σ in metal sites GD1 and GD3, respectively, giving a relative occupancy of 2.4 (Fig. 2C). In the absence of substrate, both the Gd3+ and Gd3+/Li+ structures had gadolinium atoms that were poorly ordered in metal site GD3, refined to only half occupancy and had B-factors more than two-fold higher than the global average B-factor. This indicates in the absence of substrate or product, the addition of Li+ did not significantly affect the ratio of Gd3+ at sites GD1 and GD3, with metal binding strongly preferred at site GD1. The activity of INPP1 is cooperative with Mg2+ having a Hill co-efficient of 1.9 (7Inhorn R.C. Majerus P.W. Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+.J. Biol. Chem. 1987; 262: 15946-15952Abstract Full Text PDF PubMed Google Scholar), supporting the idea that substrate binding alters Mg2+ affinity at additional metal sites. We tested the effects of substrate addition by co-crystallization of INPP1 with a 10X molar excess of Ins(1,3,4)P3 substrate and 2 mM inhibitory Gd3+. Although we could not confidently model the substrate in the resultant structure, metal sites GD1 and GD3 became equivalently occupied (Fig. 2D). Difference Fourier analysis showed densities peaks of 20.5σ and 20.0σ in metal sites GD1 and GD3, respectively, giving a relative occupancy of 1.0. Consistent with the Fo-Fc differences, anomalous differences peaks of 15.4σ and 14.0σ were calculated, giving a relative occupancy 1.1 (Fig. 2E). Whereas in the absence of substrate, the gadolinium in metal site GD3 were found at half occupancy and had B-factors greater than twice the average, and in the presence of substrate, the gadolinium in site metal site GD3 was well ordered, refined to full occupancy and had B-factors well below the global average. In the absence of substrate, the addition of lithium did not eliminate Gd3+ metal binding in INPP1. These data are consistent with an uncompetitive pattern of Li+ inhibition that predicts that substrate or product must be bound in order for Li+ inhibition to occur. We therefore tested the effect lithium addition in the presence of substrate. INPP1 was co-crystallized in the presence of 10X molar excess of Ins(1,3,4)P3 and 2 mM Gd3+, but also 200 mM Li+. As seen in Figure 2F, in the presence of substrate, no electron density peak was observed for Gd3+ at metal site GD3 with little change in density observed at metal site GD1. A 15.4σ difference density peak was found for metal site GD1, with no detectable peak for metal site GD3 indicating lithium displaces metal at site GD3 and that binding requires the presence of substrate. Interestingly, a 4.9σ difference density peak was found for gadolinium binding at GD2, the equivalent of MG2, indicating poorly ordered Gd3+ in that site (Fig. 2F). This provides further evidence Li+ does not displace metals at either sites GD1 or GD2. The native INPP1 structure was re-refined and agrees well with the original structure with an all-atom root mean square deviation of 0.25 Å. Importantly, the structural core and active site, including Mg2+ in metals sites MG1 and MG2, are essentially identical. In conjunction with the four INPP1-Gd3+ complexes presented here, at least three metal binding sites are seen in the active site of INPP1. Metal site MG1/GD1 has been observed in all structures, metal site MG2/GD2 has been observed in the Mg2+/Li+ and Gd3+/Li+/Ins(1,3,4)P3 structures, and the inhibitory metal site GD3 has been observed in the Gd3+, Gd3+/Li+, Gd3+/Ins(1,3,4)P3 structures. The INPP1 GD3 metal site is mediated by two protein–metal contacts to residues D54 and E80. In other family members, there appears to be only one protein-mediated contact to the activating metal site MG3. The other five coordinating ligands of MG3 are water molecules giving this site a unique plasticity that could accommodate different coordination geometries (Fig. 3A) (21Gill R. Mohammed F. Badyal R. Coates L. Erskine P. Thompson D. Cooper J. Gore M. Wood S. High-resolution structure of myo-inositol monophosphatase, the putative target of lithium therapy.Acta Crystallogr. 2005; 61: 545-555Crossref PubMed Scopus (51) Google Scholar). The lithium sensitive GD3 binding site corresponds to one of the five waters that coordinate the MG3 metal site (Fig. 3A). The GD3/coordinating water site is also in close proximity to a lysine residue in IMPA1 that when mutated dramatically reduces lithium sensitivity and renders the enzyme resistant to Mg2+ inhibition at high concentration (30Ganzhorn A.J. Lepage P. Pelton P.D. Strasser F. Vincendon P. Rondeau J.M. The contribution of lysine-36 to catalysis by human myo-inositol monophosphatase.Biochemistry. 1996; 35: 10957-10966Crossref PubMed Scopus (22) Google Scholar), further suggesting a role for this site in lithium inhibition. An overlay of the INPP-metal complexes with structurally related lithium-inhibited phosphomonoesterase proteins with one or more active site metals reveals that their active site metals cluster around defined sites (Fig. 3B) (11York J.D. Ponder J.W. Chen Z.W. Mathews F.S. Majerus P.W. Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.Biochemistry. 1994; 33: 13164-13171Crossref PubMed Scopus (44) Google Scholar, 12Bone R. Springer J.P. Atack J.R. Structure of inositol monophosphatase, the putative target of lithium therapy.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10031-10035Crossref PubMed Scopus (139) Google Scholar, 18Albert A. Yenush L. Gil-Mascarell M.R. Rodriguez P.L. Patel S. Martinez-Ripoll M. Blundell T.L. Serrano R. X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity.J. Mol. Biol. 2000; 295: 927-938Crossref PubMed Scopus (57) Google Scholar, 21Gill R. Mohammed F. Badyal R. Coates L. Erskine P. Thompson D. Cooper J. Gore M. Wood S. High-resolution structure of myo-inositol monophosphatase, the putative target of lithium therapy.Acta Crystallogr. 2005; 61: 545-555Crossref PubMed Scopus (51) Google Scholar, 22Johnson K.A. Chen L. Yang H. Roberts M.F. Stec B. Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities.Biochemistry. 2001; 40: 618-630Crossref PubMed Scopus (53) Google Scholar, 23Choe J.Y. Poland B.W. Fromm H.J. Honzatko R.B. Role of a dynamic loop in cation activation and allosteric regulation of recombinant porcine fructose-1,6-bisphosphatase.Biochemistry. 1998; 37: 11441-11450Crossref PubMed Scopus (58) Google Scholar, 24Villeret V. Huang S. Fromm H.J. Lipscomb W.N. Crystallographic evidence for the action of potassium, thallium, and lithium ions on fructose-1,6-bisphosphatase.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8916-8920Crossref PubMed Scopus (72) Google Scholar, 25Bone R. Frank L. Springer J.P. Pollack S.J. Osborne S.A. Atack J.R. Knowles M.R. McAllister G. Ragan C.I. Broughton H.B. Baker R. Fletcher S.R. Structural analysis of inositol monophosphatase complexes with substrates.Biochemistry. 1994; 33: 9460-9467Crossref PubMed Scopus (85) Google Scholar, 26Bone R. Frank L. Springer J.P. Atack J.R. Structural studies of metal binding by inositol monophosphatase: evidence for two-metal ion catalysis.Biochemistry. 1994; 33: 9468-9476Crossref PubMed Scopus (103) Google Scholar, 28Stec B. Yang H. Johnson K.A. Chen L. Roberts M.F. MJ0109 is an enzyme that is both an inositol monophosphatase and the 'missing' archaeal fructose-1,6-bisphosphatase.Nat. Struct. Biol. 2000; 7: 1046-1050Crossref PubMed Scopus (75) Google Scholar, 29Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. Crystal structure of a dual activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus. The story of a mobile loop.J. Biol. Chem. 2002; 277: 22863-22874Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 32Choe J.Y. Fromm H.J. Honzatko R.B. Crystal structures of fructose 1,6-bisphosphatase: mechanism of catalysis and allosteric inhibition revealed in product complexes.Biochemistry. 2000; 39: 8565-8574Crossref PubMed Scopus (61) Google Scholar, 33Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. Structural enzymology of Li(+)-sensitive/Mg(2+)-dependent phosphatases.J. Mol. Biol. 2002; 320: 1087-1094Crossref PubMed Scopus (30) Google Scholar, 34Patel S. Yenush L. Rodriguez P.L. Serrano R. Blundell T.L. Crystal structure of an enzyme displaying both inositol-polyphosphate-1-phosphatase and 3'-phosphoadenosine-5'-phosphate phosphatase activities: a novel target of lithium therapy.J. Mol. Biol. 2002; 315: 677-685Crossref PubMed Scopus (36) Google Scholar, 35Arai R. Ito K. Ohnishi T. Ohba H. Akasaka R. Bessho Y. Hanawa-Suetsugu K. Yoshikawa T. Shirouzu M. Yokoyama S. Crystal structure of human myo-inositol monophosphatase 2, the product of the putative susceptibility gene for bipolar disorder, schizophrenia, and febrile seizures.Proteins. 2007; 67: 732-742Crossref PubMed Scopus (16) Google Scholar, 36Stieglitz K.A. Roberts M.F. Li W. Stec B. Crystal structure of the tetrameric inositol 1-phosphate phosphatase (TM1415) from the hyperthermophile, Thermotoga maritima.FEBS J. 2007; 274: 2461-2469Crossref PubMed Scopus (16) Google Scholar). The positions of metal sites corresponding to INPP1 MG1/GD1 and MG2/GD2 are well conserved with an average pairwise distance for 26 and 27 metal atoms of 0.8 Å and 0.7 Å, respectively, and represent two of three activating metal sites. In contrast, the lithium-sensitive and inhibitory gadolinium metal binding site has only yet been observed in INPP1. Members of this family conserve a third activating metal site (MG3) (Fig. 3B). Notably, this site is distinct from the Li/GD3 binding site observed in the INPP1 structures, and its position away from putative water nucleophile may explain the inhibitory effect these metals exert. Based on our crystallographic studies, we found that the lithium-sensitive, inhibitory metal binding site was coordinated by the carboxylates of two conserved residues, D54 and E80. Residue D54 was mutated to alanine, and we kinetically examined the effect of Li+ inhibition upon the wild-type and mutant enzymes. Mutant INPP1D54A exhibited a 4300-fold inactivation relative to the wild-type enzyme with a Vmax of 13.9 nmol/min/mg compared with 59.7 μmol/min/mg for the wild-type enzyme (Fig. 4A) (31York J.D. Chen Z.W. Ponder J.W. Chauhan A.K. Mathews F.S. Majerus P.W. Crystallization and initial X-ray crystallographic characterization of recombinant bovine inositol polyphosphate 1-phosphatase produced in Spodoptera frugiperda cells.J. Mol. Biol. 1994; 236: 584-589Crossref PubMed Scopus (10) Google Scholar). While a large change was observed for Vmax values, the substrate affinity appeared to be relatively unaltered with Km values of 11 μM to 31 μM for the D54A mutant and wild-type enzymes (Fig. 4A) (31York J.D. Chen Z.W. Ponder J.W. Chauhan A.K. Mathews
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