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Membrane Association, Mechanism of Action, and Structure of Arabidopsis Embryonic Factor 1 (FAC1)

2006; Elsevier BV; Volume: 281; Issue: 21 Linguagem: Inglês

10.1074/jbc.m513009200

1083-351X

Byung Woo Han, C.A. Bingman, Donna K. Mahnke, Ryan M. Bannen, Sebastian Y. Bednarek, Richard L. Sabina, G.N. Phillips,

Plant Reproductive Biology

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-Å globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (α/β)8-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined Km (25-80-fold lower) and Vmax (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively. Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-Å globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (α/β)8-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined Km (25-80-fold lower) and Vmax (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively. Embryonic factor 1 (FAC1) 5The abbreviations used are: FAC1, embryonic factor 1; AMPD, AMP deaminase; ADA, adenosine deaminase; MIB, membrane isolation buffer; DTT, dithiothreitol; NMA, normal mode analysis; TIM, triose-phosphate isomerase; Hx, hypoxanthine. was recently identified as one of the earliest expressed plant genes and is essential for the zygote to embryo transition in Arabidopsis thaliana (1Xu J. Zhang H.Y. Xie C.H. Xue H.W. Dijkhuis P. Liu C.M. Plant J. 2005; 42: 743-758Crossref PubMed Scopus (52) Google Scholar). The zygote-lethal phenotype is characterized by developmental arrest at the 8-16-cell stage and mutant embryo shriveling 2-3 days after fertilization. The Arabidopsis FAC1 locus encodes an AMP deaminase (AMPD; EC 3.5.4.6), which is a eukaryotic enzyme that catalyzes the hydrolytic deamination of AMP to IMP. AMPD has also been identified as the intracellular target for a class of herbicides that are produced by fungal pathogens. Carbocyclic coformycin was initially discovered in Saccharothrix (2Bush B.D. Fitchett G.V. Gates D.A. Langley D. Phytochemistry. 1993; 32: 737-739Crossref Scopus (32) Google Scholar), and plant cells can take up this diffusible nucleoside and 5′-phosphorylate it to produce a potent transition state inhibitor of AMPD (3Dancer J.E. Hughes R.G. Lindell S.D. Plant Physiol. 1997; 114: 119-129Crossref PubMed Scopus (46) Google Scholar). Exposure to carbocyclic coformycin results in cessation of seedling growth, followed by paling and necrosis at the apical meristem (3Dancer J.E. Hughes R.G. Lindell S.D. Plant Physiol. 1997; 114: 119-129Crossref PubMed Scopus (46) Google Scholar). Coformycin, a structurally related compound produced by a number of microbes (4Nakamura H. Koyama G. Iitaka Y. Ohno M. Yagiawa N. Kondo S. Maeda K. Umezawa H. J. Am. Chem. Soc. 1974; 96: 4327-4328Crossref PubMed Scopus (116) Google Scholar, 5Isaac B.G. Ayer S.W. Letendre L.J. Stonard R.J. J. Antibiot. (Tokyo). 1991; 44: 729-732Crossref PubMed Scopus (30) Google Scholar), also has herbicidal properties (5Isaac B.G. Ayer S.W. Letendre L.J. Stonard R.J. J. Antibiot. (Tokyo). 1991; 44: 729-732Crossref PubMed Scopus (30) Google Scholar). Although the intracellular metabolism of this compound in plants has not been examined, its mode of action is presumably similar because coformycin 5′-phosphate is a transition state inhibitor of rabbit muscle AMPD (6Frieden C. Kurz L.C. Gilbert H.R. Biochemistry. 1980; 19: 5303-5309Crossref PubMed Scopus (123) Google Scholar). Both herbicides are inhibitors of mammalian adenosine deaminase (ADA) (3Dancer J.E. Hughes R.G. Lindell S.D. Plant Physiol. 1997; 114: 119-129Crossref PubMed Scopus (46) Google Scholar, 6Frieden C. Kurz L.C. Gilbert H.R. Biochemistry. 1980; 19: 5303-5309Crossref PubMed Scopus (123) Google Scholar), but the lack of this enzyme in plants (3Dancer J.E. Hughes R.G. Lindell S.D. Plant Physiol. 1997; 114: 119-129Crossref PubMed Scopus (46) Google Scholar, 7Le Floc'h F. Lafleuriel J. Guillot A. Plant Sci. Lett. 1982; 27: 309-316Crossref Scopus (21) Google Scholar, 8Butters J.A. Burrell M.M. Hollomon D.W. Physiol. Plant Pathol. 1985; 27: 65-74Crossref Scopus (8) Google Scholar, 9Yabuki N. Ashihara H. Biochim. Biophys. Acta. 1991; 1073: 474-480Crossref PubMed Scopus (34) Google Scholar) supports the argument that AMPD is the intracellular target for their nucleotide derivatives. Taken together, these observations suggest that AMPD is essential throughout the plant life cycle. However, the underlying basis for lethality of a FAC1 null phenotype and for herbicidal toxicity related to the catalytic inhibition of this enzyme is not known. AMPD catalyzes the initial step in adenine to guanine ribonucleotide conversion and also in one of the four identified pathways of AMP catabolism that are illustrated in Fig. 1. Each catabolic route differs in the order by which the phosphate, ribose sugar, and 6-amino group are removed from the purine ring structure, then all routes rejoin at the level of hypoxanthine (Hx), which is then further catabolized. Most organisms contain the necessary enzymes for more than one of these pathways. For example, AMP catabolic flow in eukaryotes of the animal kingdom proceeds along pathways 1 and 2. Pathways 3 and 4 are unique to prokaryotes, but these organisms also use pathway 2. Conversely, plants use only pathway 1 because they lack ADA and adenase (ADEase) (7Le Floc'h F. Lafleuriel J. Guillot A. Plant Sci. Lett. 1982; 27: 309-316Crossref Scopus (21) Google Scholar). Consequently, in plants AMPD is absolutely required for catabolism of AMP to hypoxanthine, which is then oxidized and linearized to form the recyclable ureides, allantoin and allantoic acid. Although not as well characterized as orthologs in the animal kingdom, endogenous plant AMPD enzymes reportedly exhibit unique physical and regulatory behaviors. Sequence analysis of available cDNA for the single A. thaliana gene (At2g38280) identifies motifs that could play structural and regulatory roles in plant-specific properties of AMPD. For example, a putative N-terminal helical transmembrane domain (residues 6-31) may be responsible for the particulate distribution of plant enzymes, as reported in pea seeds (3Dancer J.E. Hughes R.G. Lindell S.D. Plant Physiol. 1997; 114: 119-129Crossref PubMed Scopus (46) Google Scholar, 10Turner D.H. Turner J.F. Biochem. J. 1961; 79: 143-147Crossref PubMed Scopus (16) Google Scholar), spinach leaves (11Yoshino M. Murakami K. Z. Pflanzenphysiol. 1980; 99: 331-338Crossref Google Scholar), and artichoke tubers (7Le Floc'h F. Lafleuriel J. Guillot A. Plant Sci. Lett. 1982; 27: 309-316Crossref Scopus (21) Google Scholar, 12Le Floc'h F. Lafleuriel J. Physiol. Veg. 1983; 21: 15-27Google Scholar). This behavior results in poor yields and impedes further purification efforts. In addition, a putative ATP/GTP binding motif (Walker A ((A,G)X4GK(S,T)), residues 289-296) may be involved in a dramatic Km and Vmax effect of ATP, as observed with the Catharanthus roseus enzyme (13Yabuki N. Ashihara H. Phytochemistry. 1992; 31: 1905-1909Crossref Scopus (17) Google Scholar). AMPD is one of more than 1000 documented enzymes in the amidohydrolase superfamily (14Seibert C.M. Raushel F.M. Biochemistry. 2005; 44: 6383-6391Crossref PubMed Scopus (318) Google Scholar). The most recognizable feature across the 16 unique members for which high-resolution x-ray crystal structures are available, including the functionally related ADA (15Wilson D.K. Rudolph F.B. Quiocho F.A. Science. 1991; 252: 1278-1284Crossref PubMed Scopus (443) Google Scholar, 16Wang Z. Quiocho F.A. Biochemistry. 1998; 37: 8314-8324Crossref PubMed Scopus (113) Google Scholar), is a mononuclear or binuclear metal center embedded within an (α/β)8-barrel structural fold. It has long been proposed that the functional conformation of AMPD would resemble that of ADA due to similarity of the catalyzed reactions and the potent inhibition of both enzymes by deoxycoformycin derivatives (6Frieden C. Kurz L.C. Gilbert H.R. Biochemistry. 1980; 19: 5303-5309Crossref PubMed Scopus (123) Google Scholar). Although there is very little overall sequence homology between these two enzymes, many of the essential catalytic residues in the active site of ADA (15Wilson D.K. Rudolph F.B. Quiocho F.A. Science. 1991; 252: 1278-1284Crossref PubMed Scopus (443) Google Scholar, 16Wang Z. Quiocho F.A. Biochemistry. 1998; 37: 8314-8324Crossref PubMed Scopus (113) Google Scholar) are conserved in AMPD, including a signature motif, SL(S/N)TDDP (17Chang Z.Y. Nygaard P. Chinault A.C. Kellems R.E. Biochemistry. 1991; 30: 2273-2280Crossref PubMed Scopus (109) Google Scholar). This study explores the relationship between a putative N-terminal transmembrane domain in the A. thaliana FAC1 polypeptide and the particulate behavior of this plant enzyme. Physical and catalytic properties of FAC1 are also measured using purified soluble N-truncated enzymes. Polyclonal antiserum raised against one of these purified enzymes is used to examine the subcellular fractionation and the membrane extraction of FAC1 in Arabidopsis T87 protoplasts. Finally, because of its central role in nucleotide metabolism and the lack of sequence homology to any structure in the Protein Data Bank, the Center for Eukaryotic Structural Genomics undertook a structure determination. The x-ray crystal structure of the globular catalytic domain of FAC1 is presented with bound herbicide-based transition state inhibitors to reveal connections of function with structure. Expression and Purification of FAC1 Recombinant Enzymes—Recombinant plasmids containing wild type and N-truncated (ΔL31M and ΔI139M) FAC1 cDNA sequence (see supplementary Materials and Methods) were co-transfected into Spodoptera frugiperda (Sf9) cells together with a modified baculoviral genome (BaculoGold; BD Pharmingen) and the recombinant viral plaques were purified and amplified. Recombinant virus was used to infect 12 confluent T-185 flasks of Sf9 cells and recombinant enzymes were purified by phosphocellulose chromatography using a previously described protocol (18Mahnke-Zizelman D.K. Sabina R.L. Biochem. Biophys. Res. Commun. 2001; 285: 489-495Crossref PubMed Scopus (20) Google Scholar). Unless otherwise stated, leupeptin was included in all extraction and storage buffers to minimize N-terminal proteolysis, as previously described (19Haas A.L. Sabina R.L. Protein Expr. Purif. 2003; 27: 293-303Crossref PubMed Scopus (15) Google Scholar). Subcellular Fractionation Analysis of FAC1 Distribution in Arabidopsis T87 Protoplasts—Subcellular fractionation of Arabidopsis suspension cultured cells was performed as described (20Rancour D.M. Dickey C.E. Park S. Bednarek S.Y. Plant Physiol. 2002; 130: 1241-1253Crossref PubMed Scopus (91) Google Scholar) in membrane isolation buffer (MIB: 20 mm HEPES-KOH, pH 7.0, 50 mm KOAc, 1 mm Mg(OAc)2, 250 mm sorbitol) supplemented with 1 mm dithiothreitol (DTT), and a protease inhibitor mixture containing 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 1 μg/ml chymostatin, 1 mm p-aminobenzamidine, 1 mm ϵ-aminocaproic acid, 5 μg/ml aprotinin, 1 μg/ml leupeptin, 10 μg/ml E64. The subcellular distribution and activity of FAC1 were analyzed by immunoblotting and enzyme assay, which included 1 mm ATP to achieve maximal enzyme activity. To characterize the membrane association of FAC1, 223 μg of protein in the P150 fraction was diluted (10-fold) into MIB containing 1 mm DTT (MIBDTT), or MIBDTT supplemented with either 2.5 m NaCl, 100 mm Na2CO3, pH 11.5, or Triton X-100 (1% (v/v)). Samples were incubated for 30 min on ice followed by centrifugation (Beckman TLA100.1, 90,000 rpm, 30 min, 4 °C). Pellet fractions were washed with MIBDTT and 20 μg of protein equivalents of supernatant and pellet fractions were analyzed by immunoblotting using antisera specific for FAC1 (see supplementary Materials and Methods), cytosolic (PUX1) (21Rancour D.M. Park S. Knight S.D. Bednarek S.Y. J. Biol. Chem. 2004; 279: 54264-54274Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), integral membrane (AtSEC12) (22Bar-Peled M. Raikhel N.V. Plant Physiol. 1997; 114: 315-324Crossref PubMed Scopus (113) Google Scholar), and peripheral membrane (AtCDC48) (20Rancour D.M. Dickey C.E. Park S. Bednarek S.Y. Plant Physiol. 2002; 130: 1241-1253Crossref PubMed Scopus (91) Google Scholar) proteins. Prior to immunoblotting, the nitrocellulose membrane was analyzed by Ponceau S staining to confirm protein recovery and equal loading. Crystallization and Structure Determination of ΔI139M FAC1 Recombinant Enzyme in Complex with a Transition State Inhibitor— Crystals of ΔI139M FAC1 recombinant enzyme in complex with a transition state analogue inhibitor, coformycin 5′-phosphate (Fig. 6C), were obtained by hanging-drop vapor diffusion at 22 °C. The precipitant solution contained 0.4 m monoammonium dihydrogen phosphate, 0.1 m trisodium citrate, pH 5.6, and 10% (v/v) ethanol, as previously described (23Han B.W. Bingman C.A. Mahnke D.K. Sabina R.L. Phillips Jr., G.N. Acta Crystallogr. Sect. F. 2005; 61: 740-742Crossref PubMed Scopus (2) Google Scholar). Native and a mercury derivative data set (thimerosal), respectively, were collected at synchrotron beamlines 22-ID and 19-ID at the Advanced Photon Source of the Argonne National Laboratory as previously described (23Han B.W. Bingman C.A. Mahnke D.K. Sabina R.L. Phillips Jr., G.N. Acta Crystallogr. Sect. F. 2005; 61: 740-742Crossref PubMed Scopus (2) Google Scholar). The data sets of diffraction images were integrated and scaled using the HKL2000 suite (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). ΔI139M FAC1 structure was phased using the molecular replacement method with the rabbit AMPD1 structure (25Bazin, R. J., McDonald, G. A., and Phillips, C. (September 23, 2003) UK Patent GB2373504.Google Scholar) as a phasing model by MolRep (26Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) in the CCP4i suite (27Number Collaborative Computational Project Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The model was improved using alternate cycles of manual building in Xfit (28McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) and refinement in REFMAC5 (29Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar), and the final structure was refined with the Crystallography & NMR System (CNS) (30Brunger 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. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Normal Mode Analysis (NMA) of ΔI139M FAC1 Dimer—The dynamical domains in the ΔI139M FAC1 dimer were determined using NMA (31Hinsen K. Thomas A. Field M.J. Proteins. 1999; 34: 369-382Crossref PubMed Scopus (181) Google Scholar) in the Molecular Modeling ToolKit (32Hinsen K. J. Comp. Chem. 2000; 21: 79-85Crossref Scopus (272) Google Scholar). A deformation measure was calculated for the carbon α (Cα) atom in each residue followed by normal mode calculations. The two lowest frequency non-trivial normal modes were combined and the results were scaled with a heuristic scaling factor. AMPD Enzyme Assay—AMPD activity was measured in 100-μl reactions containing 25 mm imidazole, pH 7.0, 150 mm ammonium sulfate, 20 μg of bovine serum albumin, and 20 mm AMP. Substrate and product were resolved and quantified by anion-exchange high pressure liquid chromatography as previously described (33Swain J.L. Sabina R.L. McHale P.A. Greenfield Jr., J.C. Holmes E.W. Am. J. Physiol. 1982; 242: H818-H826PubMed Google Scholar, 34Bausch-Jurken M.T. Mahnke-Zizelman D.K. Morisaki T. Sabina R.L. J. Biol. Chem. 1992; 267: 22407-22413Abstract Full Text PDF PubMed Google Scholar). Kinetic studies were conducted under the assay conditions described above using ∼40 milliunits of enzyme per assay, variable substrate concentrations (0.017-106 mm AMP), and initial velocity conditions (product not exceeding 15% of substrate). Kinetic parameters were calculated by fitting data to the following equation: log v = log[VA/(K + A)], where A was AMP concentration and V was the rate. A log fit was called for because data measurement errors were proportional to the velocities. Residual (difference between experimental and calculated V) did not exceed 2.6 σ (the 99 percentile criterion for throwing out a point and refitting) for any fit. Coordinates—The coordinates and diffraction data of the ΔI139M FAC1 structure in complex with coformycin 5′-phosphate have been deposited in the Protein Data Bank (accession code 2A3L). Expression and Purification of FAC1 Recombinant Enzymes—FAC1 recombinant enzymes were expressed in Sf9 insect cells using baculoviral technology. Expression of the full-sized FAC1 cDNA produces a robust activity that partitions predominantly into the particulate fraction of insect cells, as evidenced by the low recovery from a freshly prepared sonicate following a 10,000 × g spin (see supplementary Table 1). However, there is a time-dependent increase in recovery when the sonicate is stored at 4 °C in the absence of protease inhibitors prior to centrifugation. SDS-PAGE analysis of the peak fraction eluting from a phosphocellulose column following adsorption of crude extract derived from a sonicate stored at 4 °C for 93 h reveals a series of bands between 70 and 95 kDa (see supplementary Fig. 1). These combined data suggest that proteolytic events occur in the sonicate over time at 4 °C and remove a region of the 95-kDa FAC1 polypeptide that is responsible for the particulate distribution of this enzyme. Furthermore, this occurs without any substantial loss of catalytic activity, as evidenced by similar total sonicate activities over this period of time (see supplementary Table 1). To test this hypothesis, two N-truncated enzymes, ΔL31M and ΔI139M, were constructed and expressed. Both enzymes are soluble and have robust activities (see supplementary Table 1), and phosphocellulose chromatography purification in the presence of protease inhibitor (leupeptin) followed by SDS-PAGE fractionation yields single bands of approximate 92 and 80 kDa (see supplementary Fig. 1), the predicted masses of the ΔL31M and ΔI139M polypeptides, respectively. Notably, the specific activities of these purified preparations are 3 orders of magnitude higher than those previously reported for endogenous plant AMPD enzymes (120-458 milliunits/mg of protein) (3Dancer J.E. Hughes R.G. Lindell S.D. Plant Physiol. 1997; 114: 119-129Crossref PubMed Scopus (46) Google Scholar, 11Yoshino M. Murakami K. Z. Pflanzenphysiol. 1980; 99: 331-338Crossref Google Scholar, 12Le Floc'h F. Lafleuriel J. Physiol. Veg. 1983; 21: 15-27Google Scholar, 13Yabuki N. Ashihara H. Phytochemistry. 1992; 31: 1905-1909Crossref Scopus (17) Google Scholar). The combined results demonstrate that up to 31 N-terminal amino acids in the FAC1 polypeptide are responsible for partitioning of the wild type enzyme into the particulate fraction. Subcellular Fractionation Analysis of FAC1 Distribution in Arabidopsis T87 Cells— Rabbit polyclonal antiserum was raised against purified ΔI139M antigen and this reagent was used to examine FAC1 distribution and membrane extraction in T87 cells. AMPD enzyme activity and immunoreactive polypeptide are located exclusively in the microsomal membrane fraction (P150) of these cells (Fig. 2). Furthermore, high salt (2.5 m NaCl), high pH (Na2CO3, pH 11.5), and anionic detergent (Triton X-100) all remove a portion of FAC1 from the P150 fraction, but the majority of immunoreactive polypeptide is not extracted into the soluble fraction under any of these conditions. Conversely, an integral membrane protein with a single transmembrane domain, AtSEC12, is extracted by anionic detergent, but not by either high salt or pH. In addition, a peripheral membrane protein, AtCDC48, is extracted by high pH and partially extracted by high salt. Effect of ATP on the Kinetic Properties of FAC1 Recombinant Enzymes— As reported for isolated pea seed AMPD (10Turner D.H. Turner J.F. Biochem. J. 1961; 79: 143-147Crossref PubMed Scopus (16) Google Scholar), the ΔL31M and ΔI139M FAC1 recombinant enzymes prefer sulfate anion for maximal activity (data not shown), rather than chloride, which is typically used to study the catalytic behaviors of animal orthologs. Therefore, sulfate anions were included in the assay buffer for the kinetic analysis of these two recombinant enzymes. In the absence of ATP, Km(app) and Vmax values for the ΔL31M enzyme are 6.7 ± 0.8 mm and 68 ± 6 units/mg of protein, respectively, and 12 ± 3 mm and 17 ± 3 units/mg of protein, respectively, for the ΔI139M enzyme. Vo versus [S] plots presented in Fig. 3 show that 1 mm ATP exerts a combined Km and Vmax effect on both enzymes. Km and Vmax values for the ΔL31M enzyme are 0.26 ± 0.03 mm and 375 ± 25 units/mg of protein, respectively, and 0.15 ± 0.02 mm and 113 ± 7 units/mg of protein, respectively, for the ΔI139M enzyme. Overall Structure of ΔI139M FAC1 in Complex with Coformycin 5′-Phosphate—The ΔI139M FAC1 recombinant enzyme was crystallized with bound coformycin 5′-phosphate. The crystals belong to space group P6222, with unit cell parameters a = b = 131.3, c = 208.3 Å. The final model, one per asymmetric unit, was refined using data to a resolution of 3.3 Å, and comprises 616 residues (212-273 and 286-839), one coformycin 5′-phosphate, and one phosphate ion. Statistics for data collection and refinement are summarized in Table 1. An anomalous difference Fourier map generated with the mercury data set revealed six mercury atoms positioned adjacent to the sulfur atoms of six cysteine residues, Cys254, Cys299, Cys348, Cys397, Cys470, and Cys764, confirming the amino acid sequence alignment with the electron density map.TABLE 1Data collection and refinement statisticsData collectionΔI139M FAC1 in complex with coformycin 5′-phosphateMercury derivative (thimerosal)Space groupP6222P6222Cell dimensions a, b, c (Å)131.3, 131.3, 208.3132.6, 132.6, 207.6 α, β, γ (°)90, 90, 12090, 90, 120 Resolution (Å)a50.00–3.34 (3.42–3.34)50.00–4.05 (4.12–4.05) RmergeaValues in parentheses are for the highest resolution shell,bRmerge = ΣhΣi|Ii(h) – I(h)ñ|/ΣhΣiIi(h), where Ii(h) is the intensity of an individual measurement of the reflection and 〈I(h) 〉 is the mean intensity of the reflection0.06 (0.49)0.12 (0.36) I/σIaValues in parentheses are for the highest resolution shell30.6 (4.1)26.5 (10.3) Completeness (%)aValues in parentheses are for the highest resolution shell99.5 (99.6)99.9 (100) RedundancyaValues in parentheses are for the highest resolution shell6.1 (5.7)17.0 (17.8)Refinement Resolution (Å)50.00–3.34 No. reflections15,038 RcrystcRcryst = Σh||Fobs| –|Fcalc||/Σh|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively/RfreedRfree was calculated as Rcryst using 5.0% of the randomly selected unique reflections that were omitted from structure refinement0.237/0.323No. atoms Protein5050 Ligand/ion30 Water24B-factors Protein73.47 Ligand/ion77.27 Water75.90Root mean square deviations Bond lengths (Å)0.016 Bond angles (°)2.40a Values in parentheses are for the highest resolution shellb Rmerge = ΣhΣi|Ii(h) – I(h)ñ|/ΣhΣiIi(h), where Ii(h) is the intensity of an individual measurement of the reflection and 〈I(h) 〉 is the mean intensity of the reflectionc Rcryst = Σh||Fobs| –|Fcalc||/Σh|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectivelyd Rfree was calculated as Rcryst using 5.0% of the randomly selected unique reflections that were omitted from structure refinement Open table in a new tab The structure shows an incomplete triose-phosphate isomerase (TIM) (α/β)8 barrel fold characterized by irregular loops in place of the 5th and 6th strands (Fig. 4). The space group has several 2-fold rotation axes, one of which leads to an obvious dimer interface. This interface buries 2,514 A2 or 9.5% of the total monomeric surface and 62% of the atoms in the interface are non-polar, supporting the hypothesis that FAC1 is a dimeric protein (35Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2289) Google Scholar). This conclusion is further supported by gel filtration chromatography data for the ΔI139M enzyme (see supplementary Results and supplementary Fig. 2A). A possible crystallographic tetramer contact interface comprises 727 Å2, which is only 2.7% of the total monomeric surface, and 69% of the atoms in the interface are non-polar (see supplementary Fig. 3). This weak dimer-dimer contact likely results from the high protein concentration in the crystal and may also explain why chemical cross-linking captures tetrameric species of this enzyme (see supplementary Fig. 2B). The apparent physiological dimer exhibits 2-fold symmetry, at the edge of which is an unusually flat and wide surface that is predominantly positively charged (Fig. 5, A and C).FIGURE 5Electrostatic surface view of FAC1 dimer. A, positively charged flat surface: bottom view with the 10 basic residues labeled; B, vector representation of normal mode analysis showing the putative direction of motion in the two-lowest frequency normal mode; C, side view before (dimer orientation identical to that in Fig. 4B); and D, side view after normal modes applied. With the normal mode perturbation, the basic surfaces of each monomer can become quite flat in the region of positive charge.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The active site of FAC1 with bound coformycin 5′-phosphate is positioned on the C-terminal side of the imperfect (α/β)8 barrel, surrounded by multiple helices and loops. The catalytic zinc ion is coordinated to the coformycin 5′-phosphate, an aspartic acid (Asp736) and three histidine (His391, His393, and His659) residues (Fig. 6A). There are 7 identified variations of the metal ligand centers in the amidohydrolase superfamily (14Seibert C.M. Raushel F.M. Biochemistry. 2005; 44: 6383-6391Crossref PubMed Scopus (318) Google Scholar), and this configuration is referred to as subtype III. A fourth histidine (His681) not ligated to the zinc resides on loop 6 and is positioned to function in catalysis as a general base during proton abstraction from a water molecule that is complexed with this ion. Alternatively, a glutamate residue (Glu662) is similarly positioned and could perform this catalytic role. Residues Phe463 and Tyr467 are important in positioning the inhibitor in the FAC1 active site by causing the ribose ring to assume a different orientation compared with the coformycin base (Fig. 6B). The phosphate group of the inhibitor is located in a polar environment consisting of residues Lys462, Lys466, Arg476, Asp737, and Gln740 (Fig. 6B). All are within 6 Å of the phosphate, and even closer to the associated oxygen atoms. Other notable structural features of FAC1 are the putative Walker A motif and a bound phosphate ion (Fig. 4). The ATP/GTP binding motif is located 28 Å from the catalytic zinc and spans residues 289-296 (AHYPQGKS) that comprise part of a loop structure exposed to solvent in both subunits of the physiological dimer. The phosphate ion is located at the N-terminal side of the imperfect TIM barrel 30 Å from the catalytic zinc and is surrounded by several basic residues (Arg380, Arg386, and Lys506; not shown). NMA of ΔI139M FAC1 Dimer—NMA of the ΔI139M FAC1 dimer shows that it likely moves with an opening and twisting hinge motion (Fig. 5B) that would make the basic surface of each monomer flat with respect to each other (compare Fig. 5, C and D; see supplementary Movie 1). The distance between the α-carbon atoms of residue Arg351,a basic residue located in the middle of helix 3, would increase from 50 Å in the crystal structure to 71 Å in the flattened form. In addition, the distance between