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
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ABSTRACT |
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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.
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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
hammerhead fold with an additional subdomain containing a
four-stranded anti-parallel
-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 -strand 6 and
-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 141) 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.
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EXPERIMENTAL PROCEDURES |
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Crystallization--
Purification of partially activated MPT
synthase was carried out as described (13). Fully activated MoaD, MoaE,
and the E141 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 E141 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 E141
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/I of 16.1 and an
I/
I in the highest resolution shell of 2.5 (Table I). The data set of the MoaE 141
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/
I of 17.9 (1.7 in the highest
resolution shell) (see Table I). Although
the MoaE E141
crystals initially diffracted to 1.7 Å, radiation
damage restricted the resolution limit to 2.15 Å, even with
cryocooling.
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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 E141 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 E141 MoaE was
refined at 2.15-Å resolution, all with the program REFMAC (21). Water
molecules for the orthorhombic form and E141
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.
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RESULTS |
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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 (CuK) 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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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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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- 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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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-1 atom of His-E'103 and N-
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 C
-C
bond to make more room for the larger sulfate residue
and to facilitate a hydrogen bond of this residue with its N-
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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Structure of the C-terminally Truncated MoaE Homodimer--
The
structure of the truncated MoaE homodimer (E141 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 E141
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 E141 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-
-positions is 1.9 Å upon superposition of residues 2-140 of the
E141
MoaE B-chain with the corresponding residues of MoaE in
monoclinic MPT synthase (Fig.
5a). The region with the
largest deviations is the
-hairpin formed by
-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 E141
MoaE structure were observed along the entirety of
-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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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 E141 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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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 C 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-
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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DISCUSSION |
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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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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
E141 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 (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).
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ACKNOWLEDGEMENTS |
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We thank the staff at beam lines X25 and X26C of the National Synchrotron Light Source for excellent technical support.
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FOOTNOTES |
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* 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/).
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.
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ABBREVIATIONS |
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The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; r.m.s., root mean square; E1, ubiquitin-activating enzyme.
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