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Passive Acquisition of Ligand by the MopII Molbindin fromClostridium pasteurianum

2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês

10.1074/jbc.m201005200

1083-351X

Alexander W. Schüttelkopf, J.A. Harrison, David H. Boxer, William N. Hunter,

Biochemical and Molecular Research

MopII from Clostridium pasteurianumis a molbindin family member. These proteins may serve as intracellular storage facilities for molybdate, which they bind with high specificity. High resolution structures of MopII in a number of states, including the first structure of an apo-molbindin, together with calorimetric data, allow us to describe ligand binding and provide support for the proposed storage function of the protein. MopII assembles as a trimer of dimers and binds eight oxyanions at two types of binding sites located at intersubunit interfaces. Two type 1 sites are on the molecular 3-fold axis and three pairs of type 2 sites occur on the molecular 2-fold axes. The hexamer is largely unaffected by the binding of ligand. Molybdate is admitted to the otherwise inaccessible type 2 binding sites by the movement of the N-terminal residues of each protein chain. This contrasts with the structurally related molybdate-dependent transcriptional regulator ModE, which undergoes extensive conformational rearrangements on ligand binding. Despite similarities between the binding sites of ModE and the type 2 sites of MopII the molbindin has a significantly reduced ligand affinity, due, in part, to the high density of negative charges at the center of the hexamer. In the absence of ligand this effects the movement of an important lysine side chain, thereby partially inactivating the binding sites. The differences are consistent with a biological role in molybdate storage/buffering. MopII from Clostridium pasteurianumis a molbindin family member. These proteins may serve as intracellular storage facilities for molybdate, which they bind with high specificity. High resolution structures of MopII in a number of states, including the first structure of an apo-molbindin, together with calorimetric data, allow us to describe ligand binding and provide support for the proposed storage function of the protein. MopII assembles as a trimer of dimers and binds eight oxyanions at two types of binding sites located at intersubunit interfaces. Two type 1 sites are on the molecular 3-fold axis and three pairs of type 2 sites occur on the molecular 2-fold axes. The hexamer is largely unaffected by the binding of ligand. Molybdate is admitted to the otherwise inaccessible type 2 binding sites by the movement of the N-terminal residues of each protein chain. This contrasts with the structurally related molybdate-dependent transcriptional regulator ModE, which undergoes extensive conformational rearrangements on ligand binding. Despite similarities between the binding sites of ModE and the type 2 sites of MopII the molbindin has a significantly reduced ligand affinity, due, in part, to the high density of negative charges at the center of the hexamer. In the absence of ligand this effects the movement of an important lysine side chain, thereby partially inactivating the binding sites. The differences are consistent with a biological role in molybdate storage/buffering. Mop 1Mono-mop molbindins are generally known as Mop proteins; we shall use “Mop” (uppercase) to refer to Mop proteins and “mop” (lowercase) for mop domains.domains, which occur in a variety of bacterial and archeal proteins, specifically bind molybdate. The simplest mop-containing proteins are the so-called molbindins (1.Lawson D.M. Williams C.E. White D.J. Choay A.P. Mitchenall L.A. Pau R.N. J. Chem. Soc. Dalton Trans. 1997; 21: 3981-3984Crossref Scopus (29) Google Scholar, 2.Gourley D.G. Schüttelkopf A.W. Anderson L.A. Price N.C. Boxer D.H. Hunter W.N. J. Biol. Chem. 2001; 276: 20641-20647Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), consisting entirely of either one or two mop domains. The physiological role of these proteins is unclear, although they have been implicated in molybdate storage and homeostasis (3.Grunden A.M. Shanmugam K.T. Arch. Microbiol. 1997; 168: 345-354Crossref PubMed Scopus (131) Google Scholar). Other mop-containing proteins are ModC, a component of the high affinity ABC-type molybdate transporter (4.Pau R.N. Klipp W. Leimkuhler S. Winkelmann G. Carrano C.J. Iron and Related Transition Metals in Microbial Metabolism. Harwood Academic Publishers, London, UK1997: 217-234Google Scholar) and ModE, the molybdate-dependent transcriptional regulator (5.Anderson L.A. Palmer T. Price N.C. Bornemann S. Boxer D.H. Pau R.N. Eur. J. Biochem. 1997; 246: 119-126Crossref PubMed Scopus (75) Google Scholar, 6.McNicholas P.M. Mazzotta M.M. Rech S.A. Gunsalus R.P. J. Bacteriol. 1998; 180: 4638-4643Crossref PubMed Google Scholar). ModE-like proteins occur in a number of organisms such asEscherichia coli, where this protein mediates both the molybdate-dependent transcriptional repression of themodABCD operon (MoO42−transport (7.Grunden A.M. Self W.T. Villain M. Blalock J.E. Shanmugam K.T. J. Biol. Chem. 1999; 274: 24308-24315Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 8.Rech S. Deppenmeier U. Gunsalus R.P. J. Bacteriol. 1995; 177: 1023-1029Crossref PubMed Google Scholar, 9.Walkenhorst H.M. Hemschemeier S.K. Eichenlaub R. Microbiol. Res. 1995; 150: 347-361Crossref PubMed Scopus (42) Google Scholar)) and the molybdate-dependent activation of the moaABCDE operon (molybdopterin biosynthesis (10.Anderson L.A. McNairn E. Leubke T. Pau R.N. Boxer D.H. J. Bacteriol. 2000; 182: 7035-7043Crossref PubMed Scopus (58) Google Scholar)). ModE also influences the expression of molybdoenzymes such as dimethyl sulfoxide reductase (6.McNicholas P.M. Mazzotta M.M. Rech S.A. Gunsalus R.P. J. Bacteriol. 1998; 180: 4638-4643Crossref PubMed Google Scholar) and nitrate reductase A (11.Self W.T. Grunden A.M. Hasona A. Shanmugam K.T. Microbiology. 1999; 145: 41-55Crossref PubMed Scopus (56) Google Scholar). Functional ModE is a homodimeric protein, which folds into two distinct domains, an N-terminal DNA-binding domain and a C-terminal molybdate-binding domain. The latter consists of four mop domains (two from each chain). The structure of the mono-mop molbindin from Sporomusa ovatain complex with tungstate (12.Wagner U.G. Stupperich E. Kratky C. Structure. 2000; 8: 1127-1136Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) shows Mop subunits assembled into a trimer of dimers forming eight oxyanion binding sites. A similar arrangement was found for ModG from Azotobacter vinelandii, a di-mop molbindin. The crystal structure of this protein shows a trimeric arrangement where one ModG molecule takes the place of each Mop dimer (13.Delarbre L. Stevenson C.E.M. White D.J. Mitchenall L.A. Pau R.N. Lawson D.M. J. Mol. Biol. 2001; 308: 1063-1079Crossref PubMed Scopus (37) Google Scholar). The structures of complete Escherichia coliModE in its apo form (14.Hall D.R. Gourley D.G. Leonard G.A. Duke E.M.H. Anderson L.A. Boxer D.H. Hunter W.N. EMBO J. 1999; 18: 1435-1446Crossref PubMed Scopus (71) Google Scholar) as well as its ligand-bound C-terminal domain (2.Gourley D.G. Schüttelkopf A.W. Anderson L.A. Price N.C. Boxer D.H. Hunter W.N. J. Biol. Chem. 2001; 276: 20641-20647Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) have been determined. The protein binds two molybdate anions per dimer. The quaternary structure adopted by the four mop domains of the dimer superimposes on two Mop dimers or two di-mops from the molbindins. The nitrogen-fixing bacterium Clostridium pasteurianumpossesses three distinct genes encoding molbindins (15.Hinton S.M. Freyer G. Nucleic Acids Res. 1986; 14: 9371-9380Crossref PubMed Scopus (16) Google Scholar); the three proteins, Mop I through III, are ≈7 kDa in size and show a high degree of amino acid sequence conservation (>86% identity). They consist of a single mop domain and thus belong to the mono-mop molbindin family. We report a number of high resolution crystal structures for C. pasteurianum MopII: two different structures of the apoprotein (Apo1 and Apo2), one structure of the tungstate complex, and two structures of Mop-molybdate complexes (Moo1 and Moo2, named after the Protein Data Bank name for molybdate). The apoprotein and the ligand-bound protein crystallized in different monoclinic crystal forms, both with six Mop molecules per asymmetric unit. In the case of the apoprotein they form a complete hexamer, whereas the ligand-bound structures contain two separate Mop trimers, which are complemented to hexamers by crystallographic symmetry. The structures provide the first view of an apo-molbindin and allow us to characterize the mechanism by which Mop acquires ligands and to compare this mechanism to that described for ModE (2.Gourley D.G. Schüttelkopf A.W. Anderson L.A. Price N.C. Boxer D.H. Hunter W.N. J. Biol. Chem. 2001; 276: 20641-20647Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Calorimetric studies complement the crystallographic analysis, further characterizing molybdate binding to Mop and enabling us to compare ligand affinities with those of ModE. The cloning, expression, and purification of MopII as well as crystallization conditions and data collection procedures for the tungstate, Moo1, and Apo1 complexes are as previously described (16.Harrison J.A. Schüttelkopf A.W. Boxer D.H. Hunter W.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1715-1717Crossref PubMed Scopus (3) Google Scholar). After it was determined that the Moo1 structure contained only partially loaded binding sites, more crystals were grown, but this time with the addition of 1 μl of 8 mmNa2MoO4 directly to the drop. Data from these crystals were collected in-house using a Rigaku RU-200 rotating anode x-ray source (Cu Kα, λ = 1.5418 Å) and an R-Axis IV image plate detector. The resulting data set (Moo2) was processed and scaled to 2.4 Å using DENZO/SCALEPACK (17.Otwinowski Z. Minor V. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). In the hope of improving the resolution limit of the apo-structure (Apo1: 1.8 Å) a second data set was collected in-house from a crystal grown under the same conditions as described before (16.Harrison J.A. Schüttelkopf A.W. Boxer D.H. Hunter W.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1715-1717Crossref PubMed Scopus (3) Google Scholar). The experimental setup, scaling, and processing were as for the Moo2 data; the high resolution limit for the new data set (Apo2) is 1.5 Å. Phases were obtained by single isomorphous replacement with anomalous scattering (SIRAS) using the tungsten anomalous signal from the tungstate data and the isomorphous differences between the tungstate and the Moo1 data. We note that the partial occupancy of the MoO42− sites was fortuitous for the phasing calculations. SOLVE (18.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) found and refined all eight oxyanion sites, producing phases with a figure of merit (FOM) 2The abbreviations used are: FOMfigure of meritr.m.s.d.root-mean-square deviationITCisothermal titration calorimetryMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight of 0.47–2.0 Å. Density modification using RESOLVE (19.Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1634) Google Scholar) improved the FOM to 0.72. The resulting map was used with ARP/wARP in warpNtrace mode (20.Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar, 21.Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2564) Google Scholar) to obtain an initial model containing 371 of the 408 expected amino acid residues. Cycles of model building using O (22.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), water placement with ARP/wARP, and refinement using CNS (23.Brünger 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 (16967) Google Scholar) and/or REFMAC (20.Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar, 24.Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13869) Google Scholar) led to the final refined structure. The other data sets were solved by molecular replacement with the refined tungstate structure using AMoRe (25.Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) and refined in a similar fashion. For a summary of data collection, processing, and refinement statistics see Table I.Table IData collection, processing, refinement, and model statisticsTungstateMoo1Moo2Apo1Apo2Unit cell, a, b, c (Å); β (°)56.37, 78.51, 94.84; 90.0156.81, 78.38, 95.24; 90.0155.14, 77.76, 93.49; 89.9979.08, 82.40, 56.82; 93.2378.96, 82.33, 57.03; 93.44Resolution range (Å)20.00–1.6020.00–1.8330.00–2.4025.00–1.8030.00–1.50No. of measurements405,754206,409173,881124,4321,132,889No. of unique reflections53,57836,04310,09733,11255,394Redundancy1.9 (1.7)2.8 (2.8)4.0 (2.9)1.9 (1.7)4.0 (2.9)Completeness (%)93.6 (90.4)97.4 (98.8)98.3 (89.2)97.6 (93.9)95.5 (85.4)I/ς(I)14.7 (4.3)16.7 (3.7)27.7 (4.4)16.8 (2.6)18.9 (4.2)R sym(%)6.8 (26.4)5.5 (33.6)5.0 (25.9)4.1 (28.0)6.7 (24.8)Protein residues402402400402402Solvent atoms24816613316407Oxyanions88800R andR free(%)15.4/18.321.5/24.821.0/25.717.9/21.717.8/22.2Wilson B(Å2)10.021.859.119.517.6Average isotropic B (Å2) Overall19.327.652.021.919.1 Protein18.627.352.220.717.8 Protein backbone17.525.651.318.816.3 Protein side chains20.029.353.423.019.5 Solvent28.733.644.333.829.2 Ligand11.125.340.8n/an/aCruickshank's DPI (Å)1-aDPI, Data Precision Indicator (20).0.090.180.380.150.09r.m.s.d. bond lengths (Å)0.0130.0140.0110.0190.011r.m.s.d. bond angles (°)1.31.91.52.01.5r.m.s.d. dihedral angles (°)8.29.29.78.46.3Ramachandran plot: Residues in most favored region (%)93.793.791.396.896.6 in additionally allowed region (%)6.36.38.73.23.4PDB accession codes1GUG1GUN1GUO1GUS1GUT1-a DPI, Data Precision Indicator (20.Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar). Open table in a new tab figure of merit root-mean-square deviation isothermal titration calorimetry matrix-assisted laser desorption ionization time-of-flight Figs. 1 A and 2 through 5 were generated with MOLSCRIPT (26.Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (27.Merrit E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3875) Google Scholar). Fig. 1 B was produced using ALSCRIPT (28.Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1111) Google Scholar). Cavity volumes were calculated with the program VOIDOO (29.Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (983) Google Scholar) using default parameters.Figure 2A, stereo view of the complete hexameric structure. Tungstate ions are shown as CPK models, and the dotted gray line represents the molecular 3-fold symmetry axis.B, the hexamer seen down the 3-fold axis. C, subunit arrangement: The hexameric molecule can be abstracted as a (distorted) trigonal antiprism with the six subunits (represented asspheres and labeled by chain name) occupying its corners. The edges of this antiprism represent the intersubunit interfaces; they are colored by interface type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Comparison of type 2 sites from the DEF half of the tungstate complex (top) and Apo2 (bottom). The colors used are similar to those in the previous figures. Protein residues are shown as CPK representations and are labeled with the one-letter amino acid code, the residue number, and the chain name. In the apo-structure the position of the oxyanion from the superimposed tungstate structure is shown as semi-transparent spheres.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Experiments were carried out on a Voyager MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometer (Amersham Biosciences, Inc.) using a sinapinic acid matrix. MopII was used as a 1 mg/ml solution in 50 mm Tris/HCl, pH 8.0, with and without addition of a 5-fold molar excess of Na2MoO4. Calorimetric measurements were performed using a VP-ITC instrument (Microcal, Inc.). All experiments were carried out at 25 °C. The concentration of the MopII solutions was determined by the Bradford method (30.Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar), a necessity due to the absence of a (UV) chromophore in the protein. A 100 μm solution of MopII in 50 mm Tris/HCl, pH 8.0, in the calorimetric cell was titrated with sodium molybdate (concentrations between 0.5 and 1.5 mm in the injection syringe) dissolved in the same buffer. The syringe was refilled with titrate during the measurements. As a dilution correction a titration of buffer with sodium molybdate solution in buffer was carried out. After integration, the determined heat of dilution was subtracted from the heat of reaction. The data were integrated and analyzed using Origin 5 (Microcal, Inc.). The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (www.rcsb.org). Accession codes are given in Table I. The 68-amino acid Mop polypeptide consists predominantly of β-strands (52% of the residues in six strands), most of which (β2–β5) form a twisted antiparallel β-sheet. There is also a short α-helix (10% of residues) between β4 and β5 and two 310-helical segments (η1 and η2; 8% of residues) between β3 and β4 and between β5 and β6, respectively (Fig. 1, A and B). In all crystal structures six Mop subunits form a compact hexamer with 32-point group symmetry and a diameter of about 60 Å (Fig. 2, A and B). An area of approximately 17,400 Å2 (≈55% of the accessible monomer surface) is buried upon oligomerization. The previous structures of S. ovata Mop and ModG show a similar quaternary structure. The Mop hexamer is observed by mass spectrometry and by native polyacrylamide gel electrophoresis (data not shown). Moreover, considering that all ligand binding sites are formed through the interaction of two or more subunits, it is likely that the hexamer represents the functional unit. We are furthermore confident that the reported hexameric apo-structures are physiologically meaningful, because the mass spectra show the hexamer peak even in the absence of ligand. For the following discussion the six chains have been assigned chain identifiers A through F as shown in Fig. 2 C. We use these chain names to differentiate between the two trimers and more generally the two poles of the molecule (ABC and DEF). In the case of the two apo-structures, the chain names used are identical to those in the deposited coordinate sets. The crystallographic symmetry in the ligand-bound structures means that the chain identifiers A–F as used here are actually chains A, B, C, A*, B*, C* or D*, E*, F*, D, E, F (where X* is symmetry-related to X). The hexamer possesses three unique intersubunit interfaces (Fig. 2 C), one between subunits from the same trimer (yellow edges in Fig. 2 C) and two interfaces between subunits from opposite trimers (both with internal 2-fold symmetry;red and green edges). The most extensive interaction occurs at the “red” interface, which contributes ∼3 × 2950 Å2 of the buried accessible surface and involves 29 residues on each side. The main feature of this interface is the interaction between β5 from either subunit and β6 from the other subunit. This results in the extension of the central β-sheets to two Greek-key β-barrels, which display structural similarity to the oligonucleotide/oligosaccharide binding fold (31.Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (774) Google Scholar). The interaction between β5 and β6′ (primes are used to denote a different chain) accounts for eight of the sixteen direct hydrogen bonds formed across the red interface, the remaining hydrogen bonds are formed between strand β4 and side chains from β2′ as well as backbone atoms from η2′. In addition to these direct interactions, this interface also includes 16 water-bridged hydrogen-bonding contacts. Contacts across the green interface (about 3 × 1180 Å2 of buried surface) are formed by 13 residues from helix α1, strands β1 and β4, and the 310-helix η2 on either side; interactions include a salt bridge between Arg6 and Glu46′ and 10 direct hydrogen bonds. Six of the eight oxyanion binding sites are formed at this interface (see below). The yellow interface buries 6 × 1040 Å2of solvent-accessible surface area with about 70% of this surface being hydrophobic in nature. Its principal feature is the antiparallel alignment of β6 with β1′, which is stabilized by three direct hydrogen bonds and two bridging waters and further extends the central β-sheet. Strands β3 and β4 as well as η2 from both sides participate in further contacts, providing three more direct hydrogen bonds. With the exception of Apo2 (see below) the backbone conformations are remarkably similar between subunits both within any structure (root mean square deviation (r.m.s.d.) values for Cα atoms after pairwise alignment less than 0.4 Å) and between different structures (r.m.s.d. values for Cα atoms less than 0.5 Å). In the case of the two apo-structures, an even higher degree of similarity is observed between subunits from the same trimer (r.m.s.d. less than 0.08 Å). The observed similarity is not an artifact of refinement, because only weak restraints on non-crystallographic symmetry were applied or, in the case of Apo2, none at all. C. pasteurianum MopII possesses eight oxyanion binding sites per hexamer, all of which make use of the oligomeric assembly: two sites lie on the molecular 3-fold axis at the centers of the two trimers, whereas the remaining six are located at the green interfaces (Fig. 2, A andB). In accordance with Wagner et al. (12.Wagner U.G. Stupperich E. Kratky C. Structure. 2000; 8: 1127-1136Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) these anion binding positions are referred to as type 1 and type 2 sites, respectively. Type 1 sites are formed by the bend between β2 and β3 from three Mop chains related by the molecular 3-fold axis (Figs. 1 A,2, and 3 A). The symmetry of the binding site is mirrored by the ligand binding mode: The central metal atom and one of the oxygen atoms lie on the 3-fold axis. This axial oxygen points into the hexamer, whereas the other three oxygens face outwards and each one toward a different subunit (cf. Fig. 2 B). The bound oxyanion forms twelve hydrogen bonds with the protein, three of them at the axial oxygen, which is contacted by the side-chain hydroxyl of Thr22 from all three Mop chains. The other oxygen atoms each accept three hydrogen bonds from the backbone amides of Val20, Val21, and Thr22 of one protein molecule, although the latter is relatively weak at an average distance of 3.3 Å. It is noteworthy that no positively charged groups are involved in binding of the oxyanion at this site, in fact no such groups exist within a radius of 9 Å from the oxyanion. With the exception of a hydrogen bond from the Thr22 side chain, interactions with the ligand are limited to the protein backbone. The lack of sequence conservation for the residues involved (Fig. 1 B) is consistent with this observation. Type 2 sites occur in pairs at the three green interfaces and are created by β1 as well as η2 from one subunit and the α1′ helix from the other subunit. The protein donates eight hydrogen bonds to the ligand, five from β1/η2, and three from α1′. Two of the ligand oxygens accept only one hydrogen bond (from the Ser4hydroxyl and the Lys60 amine, respectively), the third oxygen forms the remaining three hydrogen bonds with the same subunit (with the Arg6 backbone amide and both the amide and the side chain hydroxyl from Ser61). The fourth oxygen accepts all three hydrogen bonds from the other subunit (amide and hydroxyl of Ser40′ and hydroxyl of Ser43′ from consecutive turns of the helix, Fig. 3 B). In this case partial compensation for the negative charge of the ligand is afforded by the side chain of Lys60. Most of the residues participating in the formation of type 2 sites are at least functionally conserved, as should be expected given that most of them contact the oxyanion with their side chains. Only Ser43 and Ser61 are replaced by alanine or glycine in some molbindins (Fig. 1 B). Arg6 is conserved in almost all mop proteins, despite the fact that its side chain does not contribute to molybdate binding, instead forming an important salt bridge with Glu46′ as described above. Aside from a few exceptions that will be described, the similarity in backbone conformation between the various MopII structures can be extended to the side-chain conformations, with subunit-subunit alignments typically resulting in r.m.s.d. values below 0.7 Å for ≈480 atoms. Most of the ligand binding sites in the apo-structures are also similar to the corresponding ligand-filled sites. In the absence of ligand, the positions of its oxygen atoms are populated by solvent atoms. The best correspondence between apo- and ligand-bound structures is observed for type 1 sites (Fig. 4 A), alignment of Apo1 with the tungstate complex puts solvent molecules within 0.7 Å of all tungstate oxygens. However, a noticeable difference lies in the position of Val20: Its side chains, which limit access to the site from the bulk solvent, move slightly outwards in the apo forms compared with ligand-bound structures, thereby widening the access bore from an average diameter of 5.6 Å (ligand-bound) to ∼7.5 Å (apo) and thus facilitating ligand access. The slightly increased space requirements of the solvent structure compared with the oxyanion may account for this. The three type 2 sites located closer toward the DEF pole of Apo1 are also structurally similar to their liganded counterparts (Fig. 4 B). One minor difference concerns the side chain of Ser61, which appears in two alternate conformations in Apo1. Again ligand oxygen equivalent positions are occupied by solvent molecules. Only the solvent molecule hydrogen-bonding to the Lys60 side chain is about 1.1 Å from the ideal position, which allows it to form an additional hydrogen bond to the hydroxyl of Ser61 in its alternate conformation (asterisk in Fig. 4 B). A more prominent difference is observed for the type 2 sites closer to the ABC poles of both apo-structures (Fig. 4 C): The side chain of Lys60 adopts a completely different conformation pointing away from the binding site. Subsequently, the solvent molecule that accepted a hydrogen bond from Lys60 in the other sites has moved even further (up to 2.0 Å from the closest aligned oxyanion oxo position, not shown). The largest difference from the ligand-bound structures can be observed in the DEF half of Apo2 (Fig. 5): The N-terminal six residues of chains A, B, and C (chain B in Fig. 5) move up to 6 Å outwards, thereby partly exposing the type 2 site underneath. Concomitantly minor movements of surrounding residues, including the C terminus of a neighboring chain (chain A in Fig. 5) and residues 46–48 from another chain (chain D in Fig. 5), can be observed. This more open conformation demonstrates how the otherwise solvent-inaccessible type 2 sites may acquire ligand molecules. Initial refinement of the Moo1 complex resulted in the eight molybdenum atoms having unexpectedly high temperature factors compared with their oxo ligands. Given the solvent arrangement in the apo-structures, coupled with the absence of lower-mass oxyanions from the crystallization conditions, we interpret the observed electron density as resulting from partially molybdate-occupied sites. The superposition of actual oxyanion oxygens and solvent molecules (both with partial occupancy) mimics fully occupied oxo ligands. The occupancies for the molybdenum atoms were reduced to 0.5 and 0.25 for type 1 and type 2 sites, respectively, which brought the temperature factors more in line with those of the surrounding protein atoms. A further indication of the partial ligand occupancy in this structure is the observation that the side chain of Lys60 exhibits a dual conformation in two of the protein chains, pointing both toward the nearby type 2 site (as in the fully loaded structures) and toward the center of the oligomer as in the apo-subunits. The partially occupied binding sites in Moo1 also underline the lack of structural differences between apo- and ligand-bound Mop. In addition, the oxyanions in the two type 1 sites of Moo1 adopt a different orientation from the one seen in the other loaded structures: The ligand is again oriented along the 3-fold axis, only now the axial oxygen points outwards. The three equatorial oxygens are in approximately the same position as in the other structures (Fig. 4 D). The hydrogen bond donors and the number