From The Wellcome Trust Biocentre, University of
Dundee, Dundee, DD1 5EH and the ¶ Department of Biological
Sciences, University of Stirling, Stirling, FK9 4LA, United
Kingdom
Received for publication, January 31, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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The molybdate-dependent
transcriptional regulator ModE of Escherichia coli
functions as a sensor of intracellular molybdate concentration and a
regulator for the transcription of several operons that control the
uptake and utilization of molybdenum. We present two high-resolution
crystal structures of the C-terminal oxyanion-binding domain in complex
with molybdate and tungstate. The ligands bind between subunits at the
dimerization interface, and analysis reveals that oxyanion selectivity
is determined primarily by size. The relevance of the structures is
indicated by fluorescence measurements, which show that the oxyanion
binding properties of the C-terminal domain of ModE are similar to
those of the full-length protein. Comparisons with the apoprotein
structure have identified structural rearrangements that occur on
binding oxyanion. This molybdate-dependent conformational
switch promotes a change in shape and alterations to the surface of the
protein and may provide the signal for recruitment of other proteins to
construct the machinery for transcription. Sequence and structure-based
comparisons lead to a classification of molybdate-binding proteins.
Molybdenum is an essential trace element required for the
catalytic activity of several enzymes in animals, plants, and bacteria. In some cases the transition metal is complexed with a unique pterin
forming the molybdopterin cofactor, and in others it forms part of an
iron-molybdenum cluster cofactor (1, 2). Escherichia coli
acquires molybdenum in the form of
MoO E. coli ModE functions as a homodimer of subunits of 262 amino acids. The protein binds MoO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-helical (60%
-helix, 20%
-strand)
with a winged helix-turn-helix DNA binding motif. The C-terminal
molybdate-binding domains, which display a pronounced asymmetry,
comprise residues 122-262 and contain 60%
-strands, which form two
-barrels. The first barrel is constructed from a combination
of strands
6-
9 with
15 and the second from strands
10-
14. Each barrel constitutes a subdomain (Fig.
1) of similar folds, although the order
of secondary structure elements with respect to the sequence varies.
The subdomain fold is related to the oligomer binding fold (OB-fold
(17)), a five-stranded Greek-key
-barrel capped by an
-helix. The
subdomains share significant sequence and structural homology with each
other and also with the 7-kDa molybdenum-containing Mop proteins from Clostridium pasteurianum (18, 19) and Sporomusa
ovata (20). These proteins are implicated in the molybdenum
metabolism of a variety of microorganisms (3, 11, 21). We have
previously shown that each subdomain corresponds to the structure of a
single Mop unit and termed the C-terminal domain of ModE the DiMop
domain (16). The term molbindin has been introduced (19) to identify Mop-like proteins, and we made the further distinction that this refers
to proteins composed of only Mop domains.
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Fig. 1.
Ribbon diagram depicting the
architecture of intact apo-ModE (16). Trp18, shown as
a ball-and-stick representation, is labeled A186
and B186 to identify the subunit. The N-terminal DNA-binding
domains are red and orange for subunits A and B,
respectively, and the winged helix-turn-helix motifs are labeled
(wHTHA, wHTHB). The molybdate-binding domains are divided
into Mop subdomains labeled Mop1A, Mop2A,
Mop1B, and Mop2B according to subunit.
Although the structure of ModE has been determined, there are a paucity
of data concerning how the oxyanion ligands are bound, since we have
been unable to determine the structure of the complete ModE-molybdate
complex. Recent analysis of the Mop protein from S. ovata
provided the first structural details of oxyanion binding to a
molbindin, in this case tungstate (20), the results of which will be
described later. To study the protein-oxyanion interactions relevant to
the function of ModE we cloned and characterized the C-terminal (DiMop)
domain. We show that this domain retains the ability to bind oxyanions
selectively with properties similar to the intact protein, having
determined high-resolution crystal structures of both the molybdate and
tungstate complexes. These structures provide high-resolution
information on the protein-ligand interactions and allow us to compare
the apo-DiMop component of ModE with the oxyanion-loaded domain, a
comparison that identifies a conformational change induced by oxyanion
binding. Sequence and structural comparisons with other Mop proteins
led us to propose a classification of the proteins that utilize the Mop domain.
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MATERIALS AND METHODS |
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Expression of the C-terminal DiMop Domain of ModE-- A fragment of the modE gene coding for the C-terminal domain was amplified by polymerase chain reaction using pHW121 as the template plasmid, which carries a fragment covering the modE region in the vector pUC18 (8). The oligonucleotide primer used for the 5'-end of the C-terminal domain-encoding DNA was 5'-G CGC CAT ATG CAA ACC AGC-3', which incorporated an NdeI recognition site (underlined) and covered the first 3 codons of the C-terminal domain. The primer used for the 3'-end of the C-terminal domain was 5'-GCG CGG ATC CCA CGC TTA GCG CAG-3', which covered the last 6 codons of modE and contained a BamHI recognition site (underlined). The polymerase chain reaction-amplified fragment was cloned as an 0.45-kilobase NdeI-BamHI insert in the N-terminal hexa-His tag expression vector pET15b (Novagen) to create plasmid Plaa7. The expression of protein is under the control of the T7-Lac promoter, and the recombinant DiMop domain carries a thrombin cleavage site between the protein and the His tag.
Plasmid Plaa7 was heat shock-transformed into E. coli
BL21(DE3), and transformants were selected on Luria Bertani (LB) agar plates containing 100 µg ml1 ampicillin. A
transformant was grown at 37 °C in LB broth with ampicillin to
mid-log phase at which point 0.4 mM (final concentration) isopropyl-
-D-thiogalactopyranoside was added and growth
continued with vigorous aeration for a further 4 h. Cells were
harvested by centrifugation (2500 × g) at 4 °C and
were then resuspended in 50 mM Tris-HCl, pH 7.6, 250 mM NaCl, 5 mM benzamidine and stored at
20 °C. Bacterial cells were broken by passage through a French press, the insoluble cell debris pelleted by centrifugation at 4 °C
(18000 × g) for 15 min, and the cell extract passed
through a 0.2-µm filter and applied to a 5-ml metal chelate affinity
column (Hi-Trap, Amersham Pharmacia Biotech) charged using nickel
chloride. The column was washed with four column volumes of 50 mM Tris-HCl, pH 7.6, 250 mM NaCl, and 5 mM benzamidine. The His-tagged protein was eluted with a
0-500 mM imidazole gradient in the same buffer and then
incubated with thrombin (Amersham Pharmacia Biotech) for 12 h at
20 °C to remove the histidine tag. The DiMop domain was separated
from the thrombin, uncleaved fusion protein, and N-terminal peptide by
strong anion exchange chromatography using a RESOURCETM Q
(Amersham Pharmacia Biotech) column on a BioCAD 700E work station. Pooled fractions were concentrated to 20 mg
ml
1 in 50 mM Tris-HCl, pH 7.6, and sample purity was assessed by SDS-polyacrylamide gel
electrophoresis and MALDI-TOF (matrix-assisted laser desorption
time-of-flight) mass spectrometry (Voyager DE STR; PerSeptive
Biosystems). Protein concentration was estimated spectrophotometrically
at 280 nm using the calculated molar extinction coefficient of 12660 M
1 cm
1
(22). The yield of purified protein was ~40 mg
liter
1 of bacterial culture. Following
thrombin cleavage the DiMop domain consisted of residues 124-262 of
ModE with a Gly-Ser-His extension at the N terminus. For reasons of
consistency we maintained the amino acid numbering of the intact ModE.
Fluorescence Measurements--
Intrinsic protein fluorescence
spectra were recorded using a PerkinElmer LS50B spectrofluorimeter
maintained at 25 °C. Excitation was at 295 nm (5 nm bandpass for
both excitation and emission) at a scan rate of 100 nm
min1 with a protein concentration of 29 µM of monomer in 50 mM Tris-HCl, pH 7.6. Binding parameters were determined by changes in fluorescence at 347 nm
on successive additions of sodium molybdate to a solution of protein
(38 µM monomer). Corrections were made for dilution effects but not for the inner filter effect because molybdate does not
absorb significantly at the wavelengths used.
Crystal Growth and Data Collection--
Crystals were grown at
20 °C using the hanging drop method by mixing 3 µl of protein
solution (16 mg ml1 protein, 50 mM Tris-HCl, pH 7.6, 10 mM
Na2MoO4 or
Na2WO4) with 3 µl of a reservoir
solution. Similar crystals were obtained with reservoirs of 400 µl of
1.0 M trisodium citrate, 100 mM HEPES, pH 7.6, and 10% mM ethylene glycol or 1.4 M
ammonium sulfate in 100 mM Tris-HCl, pH 8.4. The crystals,
which attained a size of 0.3 mm in all dimensions over a period of 2 days, were cryoprotected with ethylene glycol and cooled to
173 °C, and the data were then measured using an RAXIS IV image
plate with a RU-200 rotating anode (Cu K
'3d
1.54Å). Two data sets were measured, one from a crystal grown
in the presence of WO
The structure of the DiMop-WO
Interactive computer graphics model building was carried out using the
program O (27). A Ni2+ ion was located in the same position
of the tungstate complex as seen in one of the subunits of the apo-ModE
structure and is considered an artifact of the purification procedure
(16). Refinement was completed using REFMAC (28) combined with
ARPP (29) to place the water molecules. The molybdate complex was
solved using the structure of the tungstate-bound form with the water
molecules removed and the tungstate replaced with molybdate as a
starting model for calculating phases for use in the auto model
building procedure in ARP/wARP. The model was refined in a procedure
identical to that used for the tungstate complex; relevant statistics
are presented in Table I. The molybdate
and tungstate complexes are essentially identical, with an r.m.s.
deviation of 0.3 Å for superposition of all C atoms. We
concentrated on the molybdate-bound structure, although for
completeness some details of the tungstate complex are provided. The
figures were produced using ALSCRIPT (30), MOLSCRIPT (31), and
RASTER-3D (32).
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Coordinates--
Atomic coordinates and structure factors have
been deposited with the Research Collaboratory for Structural
Bioinformatics Protein Data Bank (accession codes 1H9R/1H9S for the
tungstate and molybdate complexes, respectively).
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RESULTS AND DISCUSSION |
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Properties of the DiMop ModE Fragment--
The C-terminal DiMop
fragment of ModE was expressed and purified in high yield, and
analytical gel filtration analysis (data not shown) revealed it to be a
dimer in solution. The DiMop fragment retains two of the three
tryptophan residues of ModE (Trp131, Trp186),
and the intrinsic protein fluorescence emission spectrum is similar to
that reported for intact ModE with max at 347 nm. Upon
addition of 0.5 mM sodium molybdate to the DiMop fragment, the fluorescence emission spectrum was reduced by more than 60% of its
value in the absence of molybdate (data not shown). This molybdate-induced quench is slightly greater than that reported for
intact ModE (50% (11)), clearly demonstrating that the DiMop fragment
retains the ability to bind molybdate. Moreover, it suggests that the
third tryptophan (Trp49) of ModE, located in the
DNA-binding domain, makes a less important contribution to the
molybdate-induced fluorescence quench of ModE.
Titration of the DiMop fluorescence quench provided an estimate of the
stoichiometry of molybdate bound per monomer fragment of 1 with a
Kd of ~0.5 µM (Fig.
2). These values are in excellent
agreement with those reported for molybdate binding to intact ModE;
stoichiometry of 1 molybdate per monomer with a Kd
of 0.8 µM. The DiMop fragment effectively retains the
molybdate binding capacity and properties of intact ModE; this
observation validated our approach to the exploration of ModE molybdate
interactions by crystallographic analysis of the ligand-bound DiMop
fragment.
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The Molybdate Binding Site--
The DiMop dimer binds two
oxyanions, in sites designated I and II, at the dimerization interface
(Fig. 3) using residues from both
polypeptide chains. Each ligand binding site is created by the turn
between 5 and
6 from one DiMop and helix
6 and the C-terminal
region of strand
15 from the other (Fig. 3B). The oxyanion is held in position by accepting nine hydrogens bonds with
each oxygen participating in at least two such
interactions. Five of the hydrogen bonds
are donated from one polypeptide, three from the other, and one from a
water molecule (Table II, Fig. 4). The
MoO
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The charge compensation for the oxyanion is achieved by interactions with a single basic side chain (Lys183) and partial charges contributed from the three main chain NH groups in the binding site. A strong peak in the electron density near the molybdate was assigned as a water molecule, rather than a monovalent cation such as sodium, on the basis of a water-like hydrogen bonding pattern. This water forms hydrogen bonds with the side chain OD1 of Gln144 from one subunit and the carbonyl oxygen of Thr232 of the partner subunit. Of note is a particularly short contact, 3.0 and 3.1 Å, between the metals in each site and the carbonyl oxygens of Ser126 (Fig. 4). The carbonyl is approximately equidistant from three of the molybdate oxygens and may help to align the oxyanion for binding. In S. ovata Mop (20) we note that the same structural feature is observed in the corresponding tungstate binding sites.
Size Determines Oxyanion Selectivity in ModE--
ModE functions
by binding molybdate or tungstate but not other tetrahedral oxyanions
such as phosphate, sulfate, or vanadate (9). In this work, DiMop
retained bound MoO
ModA also exhibits a similar selectivity of tungstate and molybdate
over other oxyanions even though it has a fold and binding site similar
to SBP (4). ModA binds the oxyanion in a deep cleft formed at the
junction of two globular domains. There are seven direct hydrogen
bonds, all donated from main chain amides and side chain hydroxyls of
neutral protein ligands (4, 5), and a contribution to binding from
N-terminal -helix dipoles. Although the DiMop domain is a different
fold compared with ModA, both proteins use three OH groups from the Ser
or Thr residues in ligand binding and have residues that bind ligands
via a chelate-type interaction using both main chain nitrogen and side
chain OH groups.
Molybdate-specific proteins, irrespective of fold, have larger binding sites and use size in conjunction with charge compensation to determine oxyanion selectivity. The mode of dimerization imposes size restrictions because the binding of a smaller oxyanion would require readjustment of the protein backbone and dimerization interface to optimize the length and angles of the hydrogen bonds involved in ligand binding.
The Overall Structure of the Ligand-bound DiMop Domain and
Comparisons with the Apo Form--
Like intact ModE, the ligand-bound
DiMop forms a homodimer with a secondary structure similar to the
apoprotein (Figs. 1 and 3). An obvious difference between the two
structures is a decrease in the asymmetry between the partner subunits
that was observed in the apoprotein structure (16). A least-squares
superposition of the C atoms of chain A onto chain B gives an r.m.s.
deviation of 1.0 Å for the apoprotein, whereas for the DiMop-molybdate
complex the r.m.s. deviation is lowered to 0.6 Å. Residues 138-144
were excluded from this superposition because they are disordered in chain B of apo-ModE.
The binding of molybdate induces a change in the quaternary structure
that, as a first approximation, can be described as the rigid body
movement of the four Mop domains against each other; when aligned on
Mop1A (as described in the legend for Fig.
6), the center of mass of Mop1B moves
about 3.0 Å between the apo and
MoO movements up to 12.6 Å) is observed for residues 138-144 (loop I, Fig. 6). As
mentioned above, this loop is partially disordered in the apoprotein
with no density observed for one chain. However, it becomes ordered
upon molybdate binding, moving to cover another flexible loop formed by
residues 163-166 (loop II). This smaller loop is important
because its movement is crucial for the formation of the molybdate
binding site, one-half of which is constituted by residues 163 and 166 (see above) after the loop has moved
2 Å toward the anion. This movement closes the molybdate binding site and leads to the formation of new hydrogen bonds across the intersubunit interface,
e.g. between the backbone oxygen of Ser126 and
the hydroxyl of Ser166', as well as the NH2 of
Arg169' or between the guanidino moiety of
Arg128 and the Thr163' hydroxyl (prime is used
to denote a residue on the partner subunit). The latter two residues
are also involved in molybdate binding and are conserved in virtually
all known Mop domains (Fig. 5). Residues 152-155 form a third mobile
loop (loop III). In the apoprotein it contacts the
symmetry-related loop of the Mop2 of the same chain (residues
224-226), and its movement upon molybdate binding follows, to a
limited extent, that of the Mop2. We noted that all mobile loops are
located on the Mop1 domain and that Mop2 actually behaves like a rigid
body.
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We can now describe the probable order in which molybdate binding and the subsequent structural changes take place. We propose that molybdate initially binds to the half-site formed by residues 126, 128, 183, and 184 of one protein chain. The bound molybdate then effects the movement of loop II of the partner chain and with that consequently the rigid body movement of the partner subunit. This seems a reasonable assumption, as the binding site components of residues 126, 128, 183, and 184 are practically unchanged in the two structures and can be considered a preformed binding site, whereas loop II moves and is clearly influenced by molybdate binding. Further support for this half-site being responsible for initial ligand binding comes from the unequal distribution of protein-ligand interactions; as described above this is where three of the four oxygens of the molybdate are bound, whereas the loop II component provided by the other subunit binds only one.
The closing of the binding site by the movement of loop II then could allow the hitherto solvent-exposed and flexible loop I to rearrange itself and cover loop II, which leads to the formation of a number of new hydrogen bonds, e.g. Gln145' backbone oxygen and nitrogen with Ile162' backbone nitrogen and oxygen, respectively, or Val143' backbone oxygen with Ala164' backbone N, and also improves the hydrophobic packing. The now ordered loop I might then attract the loop formed by residues 210-216 of the other chain, thereby effecting the observed movement of that Mop2 domain and leading to the formation of further hydrophobic contacts. Finally the loop III of the other chain moves to "follow" the Mop2 domain movement.
The fluorescence perturbation experiments indicate that the
environments of one or both tryptophans in the DiMop domain change on
binding MoO6 and positioned in a cavity formed by the residues linking
7 and
8. Trp186, however, is at the dimerization
interface on a 310 helix. In apo-ModE the
Trp186 of subunit A is aligned parallel to
Trp186 from the B subunit and separated by about 6 Å (Fig.
1). The adjustment of the DiMop domains on binding
MoO
Implications for Other Mop Proteins--
Mop domains occur in a
variety of proteins other than ModE; the molbindins consist of either
one (e.g. Mop from Hemeophilus influenzae or Mop
I-III from C. pasteurianum) or two (e.g. ModG from A. vinelandii) Mop domains. The function of these small
proteins is unclear, but it has been suggested that they store
molybdate/tungstate and are thus involved in maintaining homeostasis of
these anions. As molybdate binding occurs at the interface between two
Mop units, it is obvious that functional molbindins must be at least
dimeric. The structure of a single Mop protein from S. ovata
(20) revealed a hexameric arrangement (Fig.
7A) that provides three
binding interfaces, similar to those described for DiMop, at which six WO
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A Mop-like sequence is also observed as a C-terminal extension in the ATP-binding subunit of the mod-encoded molybdate transporter (e.g. ModC from E. coli or ModD from A. vinelandii). Again the function of the Mop domain is unclear, but by analogy with similar extensions, e.g. of the maltose transporter ATP-binding protein MalK (34), we suggest that it could allow regulation of transporter activity depending on intracellular molybdate levels. Such rapid feedback regulation would complement the transcription level control exercised by ModE.
Fig. 5 shows a sequence alignment of Mop domains from a representative selection of Mop-carrying proteins. With the exception of some of the Mop2s from ModE proteins (see below), all sequences show conservation of the residues involved in molybdate binding. In a number of cases Ser166 (numbering for the Mop1 domain of E. coli ModE) is replaced with an alanine, but from the structural data it seems perfectly conceivable that in those cases a water could take the place of the serine hydroxyl group.
Based on the known structures, we propose that Mop-containing proteins
can be divided into at least four groups depending on the quaternary
arrangement of their Mop moieties. The first group comprises the
single-Mop molbindins. These Mops dimerize, and the dimers then
trimerize to form a hexameric assembly as shown in Fig. 7A.
Each pair of dimers provides a molybdate binding interface like that
observed in ModE, altogether supplying six binding sites. Two
additional MoO
E. coli ModE is a member of the third group. Here only two DiMop chains dimerize to form an assembly that can be superimposed almost perfectly onto two "`dimers" from the S. ovata Mop structure (Fig. 7C). As the "missing" dimer participated in the formation of not only the two "3-fold" sites but also two of the binding interfaces, it is clear that ModE-like proteins can bind only 2 mol of molybdate/mol of (dimeric) protein. It is furthermore clear that two of the four Mop domains (one from each dimer, dark green and purple in Fig. 7C) are no longer involved in oxyanion binding. Accordingly it is not surprising that in some ModE-like proteins (e.g. the ones from E. coli or H. influenzae, Fig. 5) the Mop2 domain has undergone amino acid changes that render molybdate binding impossible. Consider the Mop2 domain of E. coli ModE; the residue corresponding to Mop1 Ser126 is Asn198, which, in the observed conformation, would for steric reasons be unable to bind molybdate. Arg128 is substituted by Asp200, its side chain facing toward the hypothetical binding site, preventing the approach of an oxyanion. Asn252 from the Mop2 domain is the equivalent residue to Lys183 of Mop1, and again a residue important for molybdate binding is lost. The other two binding site residues, Thr163 and Ser166, are replaced by Pro234 and Glu237, respectively, which abolishes all hydrogen bonding and introduces another negative charge incompatible with oxyanion binding.
We note that Mop proteins with a proposed storage function maximize the number of molybdates bound per Mop unit (=1.33), whereas the oligomerization state of other Mop proteins with a function in signaling/feedback provide only two binding sites, thus ensuring that no molybdate is "wasted" (e.g. E. coli ModE binds 0.5 molybdates/Mop).
The fourth group of Mop proteins are the ATP-binding components of the molybdate-transporting ABC transporter system, for example E. coli ModC and A. vinelandii ModD. On the basis of sequence alignments (Fig. 5), these proteins carry a Mop domain that should be able to bind molybdate. To do so it must at least dimerize with a Mop domain-containing protein, and it is possible that this involves another subunit of ModC itself.
Conclusions--
Our analysis shows that the molybdate binding
properties of the C-terminal DiMop fragment of E. coli ModE
are the same as for the intact protein; it has provided details of
oxyanion binding together with the conformational and quaternary
changes that result. Structural data have revealed that E. coli ModE discriminates between oxyanions based on size and
charge. This molecular recognition is vital for the biological role of
ModE in sensing and then controlling the internal molybdate
concentration in E. coli by regulation of the transcription
of the modABCD operon. The DiMop domain undergoes a
ligand-induced conformational change that produces an alteration to the
surface of the DiMop dimer and by implication to the surface of ModE.
We previously suggested a role for the C-terminal domain of ModE in the
recruitment of partner proteins to form the complexes necessary for the
regulation of transcription (16). The conformational alteration that
the C-terminal domain undergoes when oxyanion binds may represent a
molecular switch that regulates the recruitment of such partner
proteins necessary for the positive regulation of transcription. The
possibility exists that this conformational switch in the C-terminal
domain may extend to the N-terminal domain and influence the
interactions with DNA, although further work will be required to
investigate this hypothesis.
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ACKNOWLEDGEMENTS |
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We thank C. Bond, D. Hall, and J. Harrison for their help and useful discussions.
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FOOTNOTES |
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* This work was funded by the Biotechnology and Biological Sciences Research Council and The Wellcome Trust.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 1H9R/1H9S) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ These authors made equal contributions to this paper.
Present address: IBLS, Div. of Biochemistry and Molecular
Biology, Joseph Black Bldg., University of Glasgow, Glasgow G12 8QQ, UK.
** To whom correspondence should be addressed. Tel.: 1382-345745; Fax: 1382-345764; E-mail: w.n.hunter@dundee.ac.uk.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M100919200
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ABBREVIATIONS |
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The abbreviations used are: SPB, sulfate-binding protein of Salmonella typhimurium; r.m.s., root-mean-square; Mop, molybdate-binding protein.
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