Structural Studies of Molybdopterin Synthase Provide Insights into Its Catalytic Mechanism*

Michael J. RudolphDagger, Margot M. Wuebbens§, Oliver Turque, K. V. Rajagopalan§, and Hermann Schindelin

From the Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215 and the § Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, January 15, 2003, and in revised form, February 4, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molybdenum cofactor (Moco)1 biosynthesis is a phylogenetically conserved pathway present in all three kingdoms (1-3). This pathway has been studied most extensively in Escherichia coli, where five mo loci have been discovered encoding one or more enzymes involved in Moco biosynthesis (2). Studies using several mo-deficient mutants in E. coli have elucidated the major steps during Moco biosynthesis. Although not all of the details of this pathway have been established, it can be divided into three common steps, where the second step is accomplished by the enzyme molybdopterin (MPT) synthase (4). In humans, genetic defects in Moco biosynthesis result in Moco deficiency, a rare but severe disease accompanied by serious neurological symptoms including attenuated growth of the brain, untreatable seizures, dislocated ocular lenses, and mental retardation. Moco deficiency usually causes death in early infancy (5, 6).

MPT synthase catalyzes the incorporation of the dithiolene moiety into precursor Z (4) to form MPT (see Fig. 1 for structures). E. coli MPT synthase is composed of two subunits encoded by the moaD and moaE genes. In its active form, MoaD contains a thiocarboxylate at its C-terminal glycine. MPT synthase uses the thiocarboxylated C terminus as the sulfur donor during the synthesis of the dithiolene group. The formation of carbon-sulfur bonds is an integral process in the generation of both Moco and thiamin (7, 8). The lack of mechanistic information about sulfur transfer chemistry as well as the biological importance of sulfur put great emphasis on understanding the sulfur transfer chemistry in these systems. A common step during thiamin and molybdopterin biosynthesis is the ATP-dependent transfer of sulfur from L-cysteine to yield a C-terminal glycine thiocarboxylate on ThiS and MoaD, respectively (9-11). For Moco biosynthesis, MoeB, the MPT synthase sulfurylase, converts the C-terminal carboxylate of MoaD into the thiocarboxylate with the aid of an IscS-like protein (10). The C-terminal thiocarboxylate of MPT synthase and ThiS are the sulfur donors during either Moco or thiamin biosynthesis. In order for MoaD to interact with both MoeB and MoaE, MoaD must reversibly bind to either protein (Fig. 1a). This complex interaction is vital for the action of MPT synthase, where two MoaD subunits transfer the required sulfur atoms in two subsequent steps (12). The structural basis for the transfer of MoaD is important in clarifying the enzymatic function of MPT synthase and the sulfur transfer cycle between MPT synthase and MoeB.

The high resolution crystal structure of MPT synthase (Fig. 1b) revealed a heterotetrameric molecule in which the two MoaE subunits dimerize and the MoaD subunits are located on opposite ends of the molecule with their C termini deeply inserted into each MoaE subunit (13). MoaD adopts the same fold as ubiquitin despite the absence of detectable sequence homology, demonstrating a divergent evolutionary relationship between the MoaD-MoeB system and ubiquitin as well as the related protein modifiers SUMO and NEDD8 (Rub) and their activating (E1) enzymes. The MoaE subunit is characterized by an alpha /beta hammerhead fold with an additional subdomain containing a four-stranded anti-parallel beta -sheet. The active site of the enzyme is located in each MoaE subunit in close proximity to the MoaD C terminus (Fig. 1b), which is lined by conserved Lys, Arg, and His residues. While the majority of these residues originate from the proximal MoaE subunit, two residues (His-103' and Arg-104') are located in the distal MoaE subunit. Based on their phylogenetic conservation and their location in close proximity to the active site, these residues have been postulated to be important for the function of MPT synthase (13).


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Fig. 1.   MPT synthase function and structure. a, the roles of MoaD, MoaE, and MoeB in the conversion of precursor Z to MPT. The structures of precursor Z and MPT are indicated with the C-1'- and C-2'-positions labeled in precursor Z. b, ribbon diagram of MPT synthase in the monoclinic crystal form. The MoaD subunit is shown in yellow, the proximal MoaE subunit in cyan, and beta -strand 6 and alpha -helix 3 from the distal MoaE subunit in magenta. Figs. 1b, 2, 3, and 5-7 were generated with Molscript (34) and Raster3D (35).

We present here the 1.9-Å resolution crystal structure of MPT synthase in a novel orthorhombic space group. This structure revealed a bound sulfate ion near the MoaD C terminus that probably mimics the binding site for the terminal phosphate group of MPT, the product of the reaction. In addition, the homodimeric structure of MoaE in the absence of MoaD was derived at 2.15-Å resolution from the crystal structure of a MoaE deletion variant (MoaE 141Delta ) in which the last 10 residues (positions 141-150) have been deleted. Including the earlier MPT synthase structure and the MoaD-MoeB complex, three structures each of MoaE and MoaD are now available, and these have been investigated to determine the conformational changes in MoaD and MoaE. From the available MoaE structures, the likely pterin-binding site was also deduced. Finally, the position of the thiocarboxylate sulfur of MoaD has been identified through anomalous scattering techniques. In an accompanying paper (12), site-directed mutagenesis has been employed to study the role of a number of highly conserved MoaE residues in the MPT synthase reaction. Taken together with these data, the structures presented here further define the mechanism of MPT synthase and the role of specific residues in its catalytic mechanism.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Crystallization-- Purification of partially activated MPT synthase was carried out as described (13). Fully activated MoaD, MoaE, and the E141Delta variant of MoaE were purified as described in the accompanying paper (12). Formation of fully activated MPT synthase was accomplished by mixing equimolar quantities of activated MoaD and MoaE. The resulting synthase was then passed through a Superdex 75 (Amersham Biosciences) size exclusion column, concentrated, and crystallized under the published conditions (13). A new crystal form of MPT synthase using partially activated MPT synthase was obtained with 1.1 M (NH4)2SO4 and 0.1 M Hepes, pH 7.5, as precipitant at a protein concentration of 15 mg/ml. The crystals grew extremely slowly and reached their final size of 0.2 × 0.04 × 0.04 mm3 within 4-8 months. The crystals belong to the orthorhombic space group I212121 with unit cell dimensions of a = 44.6 Å, b = 93.9 Å, and c = 137.0 Å and contain one MoaD-MoaE heterodimer in the asymmetric unit.

Initial attempts to structurally characterize full-length apo-MoaE resulted in crystals that diffracted to only 3.5-Å resolution. Despite this low resolution, a structure was determined by molecular replacement and partially refined. Since this structure revealed that the MoaE C terminus was disordered, crystallization of a truncated version of MoaE was attempted. The E141Delta C-terminally truncated version of MoaE was crystallized from a solution containing 600 mM sodium formate, 15% polyethylene glycol 4000, 10% isopropyl alcohol, and 100 mM Tris, pH 7.5, at a protein concentration of 20 mg/ml. The crystals grew to their final size of 0.3 × 0.3 × 1.0 mm3 in 2-3 days and belong to the C-centered orthorhombic space group C2221 with unit cell dimensions of a = 104.1 Å, b = 127.8 Å, and c = 138.1 Å. Three MoaE homodimers are present in the asymmetric unit.

Data Collection and Structure Determination-- Diffraction data for the I-centered orthorhombic form of MPT synthase were collected from cryocooled crystals at beamline X26C at the National Synchrotron Light Source at Brookhaven National Laboratory and at beamline X25 at the National Synchrotron Light Source for E141Delta MoaE at a wavelength of 1.1 Å. Crystals of both forms were soaked in mother liquor containing 25% (v/v) glycerol prior to cryocooling. A full sphere of diffraction data for fully activated MPT synthase crystals was collected at a wavelength of 1.54 Å on a rotating anode generator equipped with confocal optics and an R axis IV++ imaging plate detector. All data were indexed, integrated, and scaled using HKL (14).

The data set of the orthorhombic crystal has an overall completeness and Rsym of 98.4 and 8.1%, respectively (82.1 and 34.6% in the last shell). The data possess an overall I/sigma I of 16.1 and an I/sigma I in the highest resolution shell of 2.5 (Table I). The data set of the MoaE 141Delta deletion mutant contains 50,343 unique reflections resulting from a total of 167,567 recorded reflections in the resolution range from 50 to 2.15 Å. The overall completeness and Rsym are 92.7 and 6.3%, respectively (73.1 and 36.7% in the last shell), and the data possess an overall I/sigma I of 17.9 (1.7 in the highest resolution shell) (see Table I). Although the MoaE E141Delta crystals initially diffracted to 1.7 Å, radiation damage restricted the resolution limit to 2.15 Å, even with cryocooling.


                              
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Table I
Data collection statistics
Numbers in parentheses refer to the respective highest resolution data shell in each data set.

The I-centered orthorhombic MPT synthase structure was solved by molecular replacement using MPT synthase in the C2 monoclinic space group (13) as the search model (Protein Data Bank entry 1FM0) and the program AMORE (15). The rotational and translational searches were performed using data in the resolution range from 8 to 4 Å, and the correct space group (I212121) was identified during the translation search. The E141Delta MoaE structure was also solved by molecular replacement using a MoaE homodimer truncated after residue 140 as the search model. The rotational and translational searches were performed with data in the resolution range from 8 to 4 Å with the program MOLREP (16, 17). Two of the three MoaE dimers could be located by molecular replacement, but different molecular replacement programs (AMORE, MOLREP, COMO (18), and BEAST (19)) were unable to locate the third dimer. Therefore, the resulting phase information from the two correctly positioned homodimers was used to autobuild the polypeptide chain of the third homodimer with ARP (20) using structure factor amplitudes to 2.15-Å resolution. ARP was able to trace 23 separate chains containing 560 residues of 834 total residues with five of these chains belonging to the third homodimer. These five chains contained 98 of a total of 280 residues. After superposition of these fragments with an intact MoaE homodimer, the third dimer could be located in the asymmetric unit. Phases were subsequently improved by 6-fold noncrystallographic symmetry averaging, and the resulting electron density maps were used to fit and improve all six monomers of MoaE.

Refinement and Analysis-- The orthorhombic form of MPT synthase was refined at 1.9-Å resolution, the partially activated form of MPT synthase was refined at 2.5-Å resolution, and E141Delta MoaE was refined at 2.15-Å resolution, all with the program REFMAC (21). Water molecules for the orthorhombic form and E141Delta MoaE were added with the program ARP, and additional solvent molecules were included manually. Incorporation of the correct side chains and building of all additional residues was accomplished with O (22). For subsequent calculations, the CCP4 suite was utilized (23). The anomalous Fourier of partially activated MPT synthase was calculated at 2.5 Å. The active site cavity volume of MPT synthase was calculated with Voidoo (24) using a probe radius of 1.4 Å.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Localization of the Sulfur Atom in the MoaD Thiocarboxylate-- To locate the sulfur atom in the MoaD thiocarboxylate, activated MPT synthase was crystallized in the monoclinic crystal form, and diffraction data were collected to a resolution of 2.5 Å utilizing x-rays with a wavelength of 1.5418 Å (CuKalpha ) to improve the anomalous signal originating from sulfur atoms. The resulting anomalous Fourier map (Fig. 2) indicates the position of the thiocarboxylate sulfur at the C terminus of MoaD. The peak height corresponds to ~3.5 times the r.m.s. deviation of the map and is clearly weaker than the adjacent chloride ion and the sulfur of Met-E115. Following the earlier convention (13), residues in the MoaD and MoaE subunits will be identified with a D and E, respectively, preceding the residue number. This result was somewhat surprising, since the incorporation of the sulfur was assumed to be greater than 90% as estimated by mass spectrometry,2 taking into account the time required for crystallization. At the relatively low resolution of 2.5 Å, there is neither an indication for the formation of a covalent complex nor for disorder in the MoaD C terminus, which could potentially result in the weaker than expected anomalous signal. The model was refined with a carboxylate, and a positive peak in the difference density map (Fig. 2) at a somewhat larger distance than the position of the oxygen is additional proof of the presence of the sulfur atom. The oxygen of the thiocarboxylate forms a hydrogen bond with the amino group of the side chain of Lys-E126, whereas the sulfur atom forms a hydrogen bond with the side chain of Lys-E119. The latter interaction is presumably responsible for the formation of the covalent complex between the two subunits in which the side chain of Lys-E119 is covalently linked to the MoaD C terminus via an isopeptide bond (13). The formation of this covalent complex is an unwanted side reaction of MPT synthase that inactivates the enzyme and occurs on a time scale of months at 4 °C. Whereas this approach could identify the position of the thiocarboxylate sulfur, it is not inconceivable that the MoaD C terminus undergoes conformational changes upon substrate binding to yield the catalytically competent state.


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Fig. 2.   Structure of the MoaD thiocarboxylate. The MoaD C terminus and selected surrounding MoaE residues are displayed. The model has been refined with a carboxyl group at Gly-D81. The anomalous Fourier map (green) and difference electron density map (red) are contoured at 3 times the r.m.s. deviation. Atoms are color-coded with carbon in gray, oxygen in red, nitrogen in blue, sulfur in yellow, and chlorine in green.

Structure of Orthorhombic Form of MPT Synthase-- The structure of MPT synthase in the orthorhombic crystal form was refined at 1.9-Å resolution to an R-factor of 0.175 (Rfree = 0.206) (Table II). The final model comprises residues 1-81 of the MoaD subunit and residues 2-39 and 47-150 of the MoaE subunit, 198 water molecules, 3 sulfate molecules, and 2 glycerol molecules. Residues 40-46 of MoaE are also disordered in the original structure of MPT synthase (13). The overall quality of the resulting model was very good as corroborated by the crystallographic and free R-factors. In addition, the model has well defined stereochemistry, with 94.7% (180 residues) of all non-Pro and non-Gly residues (190 residues) in the most favored regions of the Ramachandran diagram (25), one residue (Ala-D8) in generously allowed regions, and two residues (Asp-D48 and Thr-E130) in disallowed regions. Both of these residues are located in weakly defined areas in the electron density maps.


                              
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Table II
Refinement statistics

The structure of the orthorhombic form of MPT synthase exhibits surprising differences when compared with the monoclinic structure of MPT synthase. Comparison of the MoaD-MoaE heterodimers reveals an r.m.s. deviation of 1.1 Å after superposition of 223 equivalent C-alpha atoms. Substantial conformational changes are present for residues D12-D17, D21-D23, D40-D47, and E133-E150. This structural dissimilarity appears to be caused by crystal contact variations between the orthorhombic and monoclinic crystal forms. Despite this unexpected structural difference, the MoaD-MoaE interface remains essentially unchanged (Fig. 3a). The buried surface area in both complexes is very similar (around 1500 Å2 in both complexes with a slightly higher number in the orthorhombic crystal form), and there are 13 strong intersubunit hydrogen bonds in each form. Comparison of the MoaD-MoaE heterotetramers results in a slightly higher r.m.s. deviation of 1.33 Å, demonstrating that MoaE dimerization, mediated in both cases by a crystallographic 2-fold axis of symmetry, is slightly different between the two crystal forms. Accompanying the differences in MoaE dimerization, the buried surface area of the MoaE dimer is ~300 Å2 larger for the dimer in the orthorhombic crystal form than for the dimer in the monoclinic crystal form.


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Fig. 3.   Structural changes in MPT synthase. a, least squares superposition of the MoaD-MoaE heterodimer in the monoclinic (yellow and cyan) and orthorhombic (orange and blue) crystals. Aromatic residues in the interface are shown with all-bonds representation. b, sulfate-binding site in the orthorhombic form of MPT synthase. MoaD is shown in yellow, proximal MoaE subunit in cyan, and distal MoaE subunit in magenta. Active site residues are depicted in all-bonds representation, and the bound sulfate molecule in ball-and-stick representation. c, chloride binding site in the monoclinic form of MPT synthase. d, electron density maps of the unassigned density feature near the MoaD C terminus. The SIGMAA weighted 2Fo - Fc (blue) and Fo - Fc (red) electron density maps are drawn at 0.8 and 2.5 times the r.m.s. deviation, respectively. The MoaD C terminus (yellow) and the proximal MoaE subunit (cyan) are shown with their atoms as CPK models at 75% of their van der Waals radii. All protein side chains are also shown in split-colored bond representation. The residual density feature is mostly surrounded by main chain atoms originating from the residues indicated by the arrows. The bound sulfate molecule is shown for reference.

A key difference between the two MPT synthase crystal forms is the presence of a sulfate molecule (Fig. 3b) in the orthorhombic crystal form that essentially replaces a chloride (Fig. 3c) in the original monoclinic crystal form grown from very high concentrations of NaCl. Both anions are located in the same anion-binding pocket located in close proximity to the MoaD C terminus. The sulfate is stabilized by electrostatic interactions involving His-E'103 and Arg-E'104 and by three hydrogen bonds. Residues of the distal MoaE subunit are given the E' designation. Two of these hydrogen bonds involve the side chain N-delta 1 atom of His-E'103 and N-zeta 1 atom of Arg-E'104 (Fig. 3b), and the third hydrogen bond is formed by the main chain amide proton of Arg-E'104. Compared with the binding pocket in the monoclinic crystal form, Arg-E39 and His-E'103 undergo significant conformational changes in their side chains. In the orthorhombic crystals, Arg-E39 is oriented away from the anion and points toward the solvent, whereas His-E'103 rotates around the Calpha -Cbeta bond to make more room for the larger sulfate residue and to facilitate a hydrogen bond of this residue with its N-delta 1 nitrogen atom.

A total of three data sets for the orthorhombic crystal form of MPT synthase were collected, including an initial data set at 2.6 Å. Attempts to soak 6,7-dimethyltetrahydrobiopterin or guanine into these crystals produced data sets with resolutions of 2.3 and 1.9 Å, respectively. Careful analysis of the resulting electron density maps did not support binding of either added compound to the enzyme. However, in all three data sets, a consistent electron density feature was observed near the side chain of Arg-E140 in a pocket in close proximity to the bound sulfate (Fig. 3d). Since this residual density could not be modeled with any of the buffer components, it most likely represents substoichiometric amounts of bound substrate and/or product. If compound Z and/or MPT were indeed bound in a similar orientation, their pterin moieties would be inserted deeply into the binding pocket present near the MoaD C terminus (Fig. 4). The volume of this cavity is ~180 Å3, a size sufficient to accommodate the pterin moiety of either precursor Z or MPT. With the exception of the partially conserved Arg-E140 residue, none of the residues surrounding this pterin binding site are conserved. Instead, main chain atoms originating from three different regions of the polypeptide chains (residues E119-E120, E138-E139, and the MoaD C terminus) are the most likely partners for hydrogen-bonded interactions with the polar atoms of the pterin.


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Fig. 4.   Surface representations of MPT synthase active site. a, close-up view of the molecular surface in the vicinity of the active site in the orthorhombic crystal form with selected residues mapped to the surface: Gly-D81 (green), Arg-E39 (red), Lys-E119 (blue), Lys-E126 (yellow), Arg-E140 (orange), His-E'103 (cyan), and Arg-E104' (magenta). b, same as in a after rotation around the vertical axis. The bound sulfate molecule is shown in all-bonds representation. c, close-up view of the monoclinic crystal form. The opening seen in b is not present in this structure due to the conformational change of His-103'. The bound chloride is not shown. Fig. 4 was generated with SPOCK (36).

Structure of the C-terminally Truncated MoaE Homodimer-- The structure of the truncated MoaE homodimer (E141Delta MoaE) was solved by molecular replacement in combination with automated density interpretation with ARP. The six monomers (labeled A-F) in the asymmetric unit are organized into three dimers. The difficulties in locating the third dimer by molecular replacement techniques are presumably due to the fact that the atoms in this dimer have considerably higher average B-factors (by 13 Å2), and their contribution to the scattering is consequently reduced. Five of the six MoaE monomers show disordered C termini variably starting at residues 127-129, but one monomer (D-chain) shows interpretable density up to Arg-140, the last residue present in the protein. In contrast to both structures of intact MPT synthase, the residues in the disordered loop region from positions 40-47 are somewhat less flexible in the deletion mutant. In the B-subunit, all residues in this region could be tentatively modeled, and these residues form a loop structure that partially conceals the active site. In two of the other subunits, additional residues (compared with the two MPT synthase structures) are visible, but a gap still exists. The structure of E141Delta MoaE was refined to an R-factor of 0.177 (Rfree of 0.227) including all working data to 2.15-Å resolution (Table II). The overall quality of the model is very good, as corroborated by the crystallographic and free R-factors. In addition, the model has 95.4% (585 of 613 non-Pro and non-Gly residues) in the most favored regions, 0.3% (His-41 in the B-chain and Ala-122 in the E-chain) in generously allowed regions, and 0.2% (Asp-134 in the D-chain) in disallowed regions of the Ramachandran diagram (25).

The E141Delta MoaE structure shows clear conformational changes when compared with the MoaE subunits in the monoclinic and orthorhombic MPT synthase structures. The overall r.m.s. deviation in C-alpha -positions is 1.9 Å upon superposition of residues 2-140 of the E141Delta MoaE B-chain with the corresponding residues of MoaE in monoclinic MPT synthase (Fig. 5a). The region with the largest deviations is the beta -hairpin formed by beta -strands 7 and 8, where r.m.s. deviations as high as 7.4 Å for Glu-E132 are observed. If residues 128-140 are omitted from the superposition, the r.m.s. deviation drops to 0.7 Å. Besides the hairpin, the majority of the structural changes are confined to the region that forms the interface with MoaD in the intact MPT synthase (Fig. 5a). In this region, conformational changes of the side chains of several residues (Glu-E53, Tyr-E55, Trp-E125, and Trp-E136) involved in interactions with MoaD are observed. The absence of MoaD produces this structural deviation between the two MoaE homodimers and influences the mobility of the corresponding atoms as measured by their atomic displacement factors (Fig. 5b). The most striking differences in mobility are observed for residues E117-E123, which do not undergo major main chain conformational changes, but become highly mobile upon removal of the MoaD C terminus. Additional increases in mobility in the E141Delta MoaE structure were observed along the entirety of alpha -helix 2 with residues E55-E58 at the N terminus of this helix exhibiting significantly increased motion. The conformational changes accompanying MoaD binding also bring the side chains of the two lysine residues at positions E119 and E126 closer together (from 10.3 to 4.5 Å as measured by the separation of their side chain nitrogen atoms), allowing them to interact with the MoaD C terminus. A shift in the position of Lys-E126 accounts for most of this decrease in separation between the two residues.


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Fig. 5.   Conformational changes in E141Delta MoaE. a, least squares superposition of MoaE from the monoclinic crystal form (cyan, with the MoaD C terminus in yellow) and monomer D of the E141Delta MoaE variant (red). Residues contributing their side chains to the MoaE-MoaD interface in MPT synthase are shown, and those that undergo substantial conformational changes are labeled. The beta -hairpin formed by beta -strands 7 and 8 undergoes the most significant conformational changes (top). b, side by side comparison of the temperature factor distribution in MoaE from the monoclinic crystal form (left, with the MoaD C terminus in yellow) and monomer D of the MoaE141Delta variant (right). Both proteins are shown as coils with residues exhibiting low thermal mobility in blue and high thermal mobility in red. Residues in the MoaE-MoaD interface are shown as gray spheres.

The crystals of the MoaE deletion mutant were grown in the absence of sulfate and in the presence of significantly lower chloride concentrations than in the original monoclinic crystal form of MPT synthase. Nevertheless, the anion-binding pocket is not empty but instead contains a formate ion in five of the six MoaE monomers. In four of the monomers, the formate is in an identical position, where it interacts with the side chains of Arg-39 and Arg-104' (Fig. 6). Relative to the sulfate molecule in the orthorhombic crystal form, these formate ions are displaced by ~4 Å, primarily toward the position where the MoaD C terminus would be located in MPT synthase. Compared with the two MPT synthase structures, the conformations of Arg-39 and Arg-104' in truncated MoaE are very similar to the chloride-containing structure, whereas His-103' adopts the same conformation as in the sulfate-bound MPT synthase. In the fifth E141Delta MoaE subunit, the formate is present at roughly the same position but does not closely interact with either arginine residue. The binding pocket of the sixth subunit is empty.


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Fig. 6.   Formate binding site in the E141Delta MoaE variant. The proximal MoaE subunit (cyan), and distal MoaE subunit (magenta) are shown together with key side chains.

Structural Variability of MoaD-- Together with the structure of the MoaD-MoeB complex (26), there are now three MoaD crystal structures available. Residues 1-77 of these structures can be superimposed with pairwise r.m.s. deviations of the Calpha atoms ranging from 0.68 Å (for MoaD in the two MPT synthase complexes) to 0.93 Å (for the MoaD-MoeB complex and the orthorhombic MPT synthase structure) (Fig. 7). Not surprisingly, the largest deviations are observed between MoaD in the complex with MoeB and MoaD in the two MPT synthase structures. However, these differences are rather small if the four MoaD C-terminal residues are omitted. An analysis of these residues reveals large deviations in the MoaD C termini (close to 12 Å in the positions of the Gly-D81 C-alpha atoms), which reflect the completely different conformation of the MoaD C termini in both complexes. The C terminus of MoaD in the absence of a partner protein is predicted to be highly flexible, as has been observed for the C termini of ubiquitin (27, 28), SUMO (29), Rub/NEDD8 (30, 31), and ThiS (32). Additional conformational changes in MoaD are located in solvent-exposed regions of the protein, where differences between the two MPT synthase-derived MoaD structures also exist. All three MoaD structures are very similar at their interfaces with either MoaE or MoeB. An almost identical subset of MoaD residues is contacted by both MoaE and MoeB (Fig. 7), and a similar number of interchain hydrogen bonds (13 in the two MPT synthase structures and 14 in the MoaD-MoeB complex) is present in the two complexes. However, a larger surface area (1950 Å2) is buried in the MoaD-MoeB complex compared with the MPT synthase structures (~1500 Å2).


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Fig. 7.   Conformational changes in MoaD. Least squares superposition of MoaD from the monoclinic MPT synthase (yellow), orthorhombic MPT synthase (green), and the MoaD-MoeB complex (red). Residues involved in complex formation (either with MoaE or MoeB) are highlighted by transparent spheres at their C-alpha atoms.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MPT synthase catalyzes the conversion of precursor Z into MPT through sulfur transfer from the MoaD thiocarboxylate. The mechanism of MPT synthase relies heavily on the precise interplay between the active form of MoaD carrying the thiocarboxylate and the MoaE protein. In fact, both proteins have to go through two cycles of association and dissociation to transfer the two sulfur atoms required for the conversion of precursor Z to MPT (Fig. 8). The thermodynamic and kinetic aspects of the MoaD interactions with either MoaE or MoeB are crucial for the overall reaction in which precursor Z is converted to MPT, and the structural data on these complexes suggest that at least the enthalpic component of the MoaD interactions is of comparable magnitude in both protein complexes. As far as the interaction in MPT synthase is concerned, complex formation is accompanied by a decrease in MoaE flexibility as demonstrated by the atomic displacement factors (Fig. 5b) and presumably also for MoaD itself, which is predicted to have an unstructured C terminus in its free form. The exposure of hydrophobic residues located in the interface regions of MoaD and MoaE will partially compensate for the decrease in entropy upon MPT synthase formation through the hydrophobic effect. Nevertheless, complex formation presumably results in an unfavorable decrease in entropy, which has to be clearly outweighed by the enthalpic contribution to give rise to the observed stability of the MPT synthase complex.


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Fig. 8.   Catalytic cycle of MPT synthase. The MoaD subunits are colored yellow and blue, and the MoaE subunits are cyan and magenta. The MoaE dimer (lower left) binds precursor Z (Pr. Z) and activated MoaD (with the thiocarboxylate sulfur in red) and converts precursor Z to the hemisulfurated intermediate (Int.) (see "Discussion"). This intermediate molecule is proposed to stay bound to the MoaE dimer after dissociation of the discharged MoaD subunits. The MoaE dimer has been modeled based on the D-subunit present in the E141Delta MoaE crystals. Following binding of two new activated MoaD subunits, MPT synthase transforms the bound intermediate into MPT, which dissociates from MoaE together with the second set of discharged MoaD subunits to complete the cycle.

A comparison of the MPT synthase active sites in the monoclinic form, the orthorhombic form, and the MoaE homodimeric structure reveals conformational changes within the anion-binding pocket that appear to be of relevance for the catalytic mechanism (Figs. 3, b and c, and 6). The anion-binding pocket interchangeably consists of the strictly conserved residues Arg-E39 from the proximal MoaE subunit and His-E'103 and Arg-E'104 from the distal MoaE subunit. Among those three residues, Arg-E39 and His-E'103 adopt two different conformations, whereas Arg-E104' has an essentially unchanged conformation. The static behavior of Arg-E104' is influenced by its involvement in ionic interactions with the side chain of Asp-E116 present in all three structures. In addition, Arg-E104' also forms a salt bridge with Glu-E143 in the two MPT synthase structures.

The pterin moiety of the substrate and product presumably binds into the deep binding pocket (Fig. 4) that harbors the residual electron density feature observed in the orthorhombic crystal form (Fig. 3d). A structural comparison of the different conformations of the Moco bound to various enzymes suggests that one of the pterins (Q-pterin) of the bis-MGD cofactor present in dimethyl-sulfoxide reductase (33) could be easily fit into the MPT synthase structure such that its pterin moiety is in the binding pocket and its terminal phosphate group is in the sulfate binding site. Only minor conformational changes involving rotations around the two single bonds connecting the phosphate group to the pyran ring would be necessary to achieve an optimal fit. This conformation would then correspond to the MPT synthase-product complex after formation of the dithiolene moiety. The pterin ring system of the substrate (precursor Z) is predicted to be arranged in a similar fashion, but due to the presence of the cyclic phosphate forming a fourth ring system in precursor Z, different interactions between MPT synthase and the phosphate group of precursor Z are required. The flexibility of the anion-binding site observed in the three MoaE structures suggests that MPT synthase can accommodate precursor Z through conformational changes involving the side chains of Arg-E39 and His-E'103. The observed ~4-Å displacement of the formate relative to the sulfate is similar in magnitude to the predicted shift in the position of the phosphorous atom between precursor Z and MPT. Therefore, the conformation observed in the E141Delta MoaE variant might mimic the one required for precursor Z.

Multiple sequence alignments of 90 MoaE subunits from different organisms reveal that three residues, Lys-E119, Trp-E125, and Lys-E126 are invariant. Additional highly conserved residues include Arg-E39, His-E103, Arg-E104, and Trp-E136. Both Trp residues (E125 and E136) are located in the MoaE-MoaD interface, and their primary role appears to be the stabilization of the MoaD-MoaE interface. The role of the positively charged residues Arg-E39, His-E103, and Arg-E104 would be to variably interact with the cyclic phosphate present in the substrate precursor Z or the terminal phosphate present in the product molybdopterin as described above. This leaves two invariant residues, Lys-E119 and Lys-E126, as putative catalytic residues. Whereas Lys-E119 and Lys-E126 form hydrogen bonds and ionic interaction with the MoaD C terminus, the nature of these interactions does not require strict conservation of these residues, since other residues such as arginine would also be able to form similar interactions. Furthermore, positively charged residues are not required to stabilize the MoaD C terminus as can be seen from the MoaD-MoeB complex, where the MoaD C terminus forms hydrogen bonds to uncharged atoms of MoeB (26). The strict sequence conservation of Lys-E119 and Lys-E126 therefore suggests that both of these residues directly participate in the catalytic mechanism of MPT synthase. The sequence conservation analysis reveals no additional highly conserved residues that could be involved in binding the pterin moiety of the substrate. In line with this observation is the putative assignment of the pterin binding site to the region shown in Figs. 3d and 4, where mostly main chain atoms are present as hydrogen binding partners for the polar atoms in the pterin moiety. Although residue Arg-E140 is partially conserved, a large group of MoaE orthologs contain C-terminal truncations that in many cases eliminate the position of this residue along with the C-terminal helix (alpha 4) present in E. coli MoaE. Evidence that mutation of this residue to an alanine only moderately decreases its activity (12) also indicates that this residue is not likely to play a crucial role in substrate binding.

A critical aspect of catalysis by MPT synthase is the stoichiometry of the reaction. Whereas two sulfurs are needed for the conversion of each precursor Z molecule to MPT, a single MoaD thiocarboxylate can only donate one sulfur atom. The possibility that the two MoaD C-terminal thiocarboxylates in heterotetrameric MPT synthase act together on a single precursor Z molecule is ruled out by the structure of MPT synthase, since the two active sites are separated by ~31 Å. The model building studies described above suggest that the amino groups of Lys-E119 and Lys-E126 would be located in close proximity (within ~4 Å) of the C-1' or C-2' carbon atoms in precursor Z onto which the dithiolene moiety of MPT will be incorporated and to within 3.5 Å of the MoaD C-terminal thiocarboxylate, the sulfur donor during the reaction. Assuming that Lys-E119 and Lys-E126 indeed are catalytic residues, one could envision that they could be either involved in acid-base catalysis or covalent catalysis, two general catalytic strategies in which lysine residues participate. Whatever the exact role of these residues, formation of the dithiolene group would be a two-step process in which two sulfur atoms are sequentially incorporated into precursor Z (Fig. 8). The accompanying paper (12) provides experimental evidence for the formation of a reaction intermediate that contains a single sulfur atom. After insertion of the first sulfur atom into precursor Z via a nucleophilic attack of the thiocarboxylate sulfur atom, an intermediate would be formed, which putatively contains a thiol at either the C-1'- or C-2'-position. Whereas two scenarios for the initial attack are possible (see Fig. 7 of the accompanying paper (12)), the enzyme, through its specific interactions with the substrate, will select either the C-1'- or C-2'-position. Incorporation of the first sulfur atom into the C-2'-position with concomitant hydrolysis of the cyclic phosphodiester present in precursor Z appears more straightforward as described below.

As demonstrated in the accompanying paper (12), Lys-E119 is essential for the first step during MPT biosynthesis from precursor Z, since its replacement with alanine prevents the formation of the intermediate containing a single sulfur atom. Precursor Z must therefore bind in an orientation that allows Lys-E119 to assist in the incorporation of the first sulfur atom. Incorporation of the first sulfur atom also leads to hydrolysis of the cyclic phosphodiester of precursor Z, generating the terminal phosphate group also present in MPT. If the first sulfur is incorporated at the C-2'-position, these two steps could be directly coupled, whereas initial incorporation at the C-1'-position would require an independent hydrolytic event to cleave the cyclic phosphodiester of precursor Z. The structural change resulting from the formation of the terminal phosphomonoester group could then trigger 1) the dissociation of the discharged MoaD subunit through alterations in direct interactions presumably present between the MoaD C terminus and the intermediate and 2) a somewhat altered binding of the intermediate compared with precursor Z. The altered binding of the intermediate would ensure incorporation of the second sulfur atom at the C-1'-position presumably assisted by Lys-E126 after binding of a recharged MoaD subunit in the subsequent step. Evidence of tight binding of the intermediate to MPT synthase suggests that it remains bound at the active site after the discharged MoaD subunits have dissociated from the MPT synthase holoenzyme. The MoaE homodimer would thus represent a functional state during the MPT synthase-catalyzed reaction, and the observed decrease in flexibility of residues 40-46 in the loop region near the active site could indicate that these residues stabilize the intermediate and prevent it from dissociation. After incorporation of the second sulfur atom, the product would be released by MPT synthase to become available to the enzymes catalyzing the metal incorporation step during Moco biosynthesis.

The structural investigations of MPT synthase in different functional states has identified residues involved in binding of the phosphate group present in precursor Z and MPT and revealed conformational changes in two of these residues that would accommodate binding of the substrate, the intermediate, and the product. The pterin-binding region of MPT synthase has been inferred from the location of the anion-binding pocket and the MoaD C terminus and a surface analysis of MPT synthase. Taken together, these results suggest that the strictly conserved residues Lys-E119 and Lys-E126 are directly involved in the catalytic mechanism of MPT synthase as demonstrated in the accompanying paper (12).

    ACKNOWLEDGEMENTS

We thank the staff at beam lines X25 and X26C of the National Synchrotron Light Source for excellent technical support.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK54835 (to H. S.) and GM00091 (to K. V. R.). The National Synchrotron Light Source in Brookhaven is supported by the Department of Energy and National Institutes of Health, and beam line X26C is supported in part by the State University New York at Stony Brook and its Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1NVI (I-centered orthorhombic crystal form of MPT synthase) and 1NVJ (E141|gD MoaE deletion variant)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger Present address: Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215.

To whom correspondence should be addressed. Tel.: 631-632-1022; Fax: 631-632-1555; E-mail: hermann.schindelin@sunysb.edu.

Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M300449200

2 M. Wuebbens, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; r.m.s., root mean square; E1, ubiquitin-activating enzyme.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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