From the Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom
Received for publication, August 11, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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The active conformation of the dimeric
cofactor-dependent phosphoglycerate mutase (dPGM) from
Escherichia coli has been elucidated by crystallographic
methods to a resolution of 1.25 Å (R-factor 0.121;
R-free 0.168). The active site residue His10,
central in the catalytic mechanism of dPGM, is present as a phosphohistidine with occupancy of 0.28. The structural changes on histidine phosphorylation highlight various features that are significant in the catalytic mechanism. The C-terminal
10-residue tail, which is not observed in previous dPGM structures, is
well ordered and interacts with residues implicated in substrate
binding; the displacement of a loop adjacent to the active histidine
brings previously overlooked residues into positions where they may
directly influence catalysis. E. coli dPGM, like the
mammalian dPGMs, is a dimer, whereas previous structural work has
concentrated on monomeric and tetrameric yeast forms. We can now
analyze the sequence differences that cause this variation of
quaternary structure.
Phosphoglycerate mutases
(PGMs)1 are enzymes involved
in glycolysis and gluconeogenesis. They can be subdivided into two
types: cofactor-dependent PGM (dPGM) and
cofactor-independent PGM (iPGM). Whereas vertebrates, yeasts, and many
bacteria have only dPGM, and higher plants, nematodes, archaea, and
many other bacteria have only iPGM, a small number of bacteria
including Escherichia coli have both (1).
dPGMs have three catalytic activities. The main activity is that of a
mutase (EC 5.4.2.1), catalyzing the interconversion between
2-phosphoglycerate and 3-phosphoglycerate. A second activity is
as a phosphatase (EC 3.1.3.13), converting
2,3-bisphosphoglycerate and water to 3-phosphoglycerate or
2-phosphoglycerate and phosphate. The third activity is the
synthase activity (EC 5.4.2.4), where 1,3-bisphosphoglycerate is converted to 2,3-bisphosphoglycerate. The
label "cofactor-dependent" comes from the observation
in vitro that to be active, the native protein must be
phosphorylated by 2,3- bisphosphoglycerate.
The crystal structure of Saccharomyces cerevisiae
dPGM2 was first published in
1974 (Protein Data bank code 3PGM (2, 3)), and structures of different
crystal forms and inhibitor complexes at increasing resolution have
followed (4PGM, 5PGM, 1BQ3, 1BQ4, 1QHF (4-7)).
Schizosaccharomyces pombe dPGM has been studied by NMR, and
a backbone assignment has been published (8). In most organisms for
which a dPGM has been characterized, including E. coli and
mammals, the active enzyme exists as a dimer. S. cerevisiae
dPGM, however, is tetrameric, and S. pombe dPGM is
monomeric. Most recently, the crystal structure of the iPGM from
Bacillus stearothermophilus has been solved (9, 10), highlighting the absence of any similarity to dPGM in all aspects except its main mutase activity.
dPGM is the archetype of the "phosphoglycerate mutase-like" protein
fold superfamily (SCOP (11)), which also contains the phosphatase
domain of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family as well as prostatic acid phosphatase and phytase. The common
fold of these proteins is commensurate with their use of phosphohistidine as a catalytic intermediate.
Other examples of N-phosphorylation at histidine occur in
bacterial signaling proteins (12) and in enzymes such as fructose permease (13), nucleoside diphosphate kinase (14), and succinyl-CoA synthetase (15). Structures of intact phosphohistidine-containing proteins are particularly rare, those of HPr by NMR (16) and succinyl-CoA synthetase (15) and nucleoside diphosphate kinase (14) by
x-ray crystallography being the only examples to date. None of these
structures represent the phosphoglycerate mutase-like fold family.
We now report the structure of E. coli dPGM in its
phosphorylated, active conformation. The structure of a dimeric dPGM
provides a basis for examining the residues involved in interactions in the varying oligomerization states observed in dPGMs. The establishment of this structure as representative of the active conformation of the
enzyme and comparison with the available dephosphorylated structures
provide new information regarding the roles of specific residues in the
complex catalytic mechanism of this class of enzymes.
Cloning, Expression, and Purification--
The E. coli (K12) pgm1 gene was amplified from genomic DNA by
a polymerase chain reaction using the 5' and 3' end-specific primers 5'
CCC-GCG-CAT-ATG-GCT-GTA-ACT-AAG 3' and 5'
CGC-GGA-TCC-TTA-CTT-CGC-TTT-ACC-CTG 3'. These
oligonucleotides (Amersham Pharmacia Biotech) introduced NdeI and BamHI restriction sites, respectively
(underlined). Taq polymerase, DNA ligase, and the relevant
restriction enzymes were obtained from Promega. The polymerase chain
reaction product (~0.75 kilobases) was gel-purified (Qiaex extraction
kit, Qiagen) and cloned into pUC18 (SureClone, Amersham Pharmacia
Biotech), and positive clones were identified by restriction
digest. The DNA fragment was ligated into the
BamHI/NdeI-cleaved plasmid pET3a (Novagen) to
give the pET3a-pgm construct that was amplified in E. coli
JM109 (Novagen), and the integrity of the gene was confirmed by
sequencing. E. coli strain BL21(DE3)pLysS (Novagen) was heat shock-transformed with pET3a-pgm and selected on Luria-Bertani agar
plates containing ampicillin and chloramphenicol. Single colonies were
cultured, and the expression of protein with
isopropyl- Crystallization--
Crystals were grown from hanging drops (2 µl of protein, 1-µl reservoir) from a protein solution containing
dPGM (15 mg ml Data Processing and Refinement--
Data (Table
I) were collected at SRS Daresbury
station 9.6 (
Refinement with SHELXL (20), addition of water molecules, manual
intervention using O (21), use of restrained anisotropic thermal
parameter refinement, and the inclusion of 17 dual side chain
conformers resulted in a structure containing 2069 nonhydrogen protein
atoms (residues 1-247), two sulfates, and a chloride; this structure
had an R-factor of 0.121 and R-free of
0.168. Multiple conformers were refined with total occupancy restrained
to 1.0. Occupancies were refined for the two sulfates in the active
site, whereas the chloride, located on a 2-fold axis at the dimer
interface, is modelled with 0.5 occupancy.
At an early stage in the refinement it was noted that three waters lie
adjacent to the N Quality of the Model--
The excellent quality, high resolution
data have led to a reliable, precise model with root mean square
deviations from Engh and Huber bond lengths and angle distances of
0.014 and 0.030 Å (26). No residues have disallowed Overall Fold--
The Quaternary Structure--
The active dimer is formed by the
antiparallel alignment of the C strands of two monomers.
Comparison of the dimeric structure with the S. cerevisiae
tetramer highlights the structural basis for the different
oligomerization states. The S. cerevisiae and E. coli monomers superpose in LSQMAN (28), with a root mean square deviation of 1.2 Å over 227 C
An explanation for the inability of S. pombe dPGM to
dimerize is less clear, particularly given the lack of a
three-dimensional structure. Sequence comparison suggests a number of
interactions present in the E. coli and S. cerevisiae proteins that are absent in S. pombe. These
dimer-forming interactions come from two stretches of sequence
including residues 58-77 and 136-139. The first stretch forms helix
These comparisons reveal that there are no major structural
rearrangements between dPGMs. Rather, the differences are restricted to
amino acid changes at the subunit interfaces. Given that all dPGMs,
whether monomeric, dimeric, or tetrameric, retain essentially the same
activity, a question remains as to the biological function of these
distinct quaternary assemblies.
Structural Consequences of Histidine Phosphorylation--
Although
no measurements have been made of the phosphohistidine half-life of
E. coli dPGM, there was no reason to expect it to be any
longer than the 35 min observed for S. cerevisiae dPGM; yet
the presence of phosphohistidine in the crystals indicates that a
certain level of phosphorylation must persist for considerably longer.
Whereas the native structure has only 0.28 occupancy phosphohistidine, the remainder of dephosphorylated protein also adopts the active conformation, with water molecules occupying the vacant phosphate oxygen positions. We cannot rule out the possible contribution of
crystal-packing forces in aiding the dephosphorylated protein to adopt
the active conformation.
The native structure presented here is representative of the enzyme in
its competent, phosphorylated form and is used as such in the following
discussion; the S. cerevisiae structures are typical of an
inactive or inhibited dephosphorylated form. Comparison of these two
forms indicates significant structural differences. The C-terminal tail
of the protein, with the exception of the final two residues, is
ordered in the phosphorylated form. This tail, the subject of
much speculation regarding its possible role in the catalytic cycle
(30), is not modelled in the S. cerevisiae structures
because of disorder. The conformation of His10 when
phosphorylated is distinct from that in the yeast structures, and the
adjacent residues in the loop from Arg9 to
Thr22 have moved up to 1.7 Å (C Active Site--
dPGM has a cup-shaped active site that is 16 Å deep and 10 by 8 Å wide, with a volume of ~1200 Å3
containing up to 36 ordered solvents and 2 sulfates. This extensive cavity is lined by atoms from 43 residues: 9-23, 36, 61, 88-91, 99, 111-116, 183-188, 203-209, and 239-247. The roles of these residues
can, to a large extent, be divided into three categories: the catalytic
machinery, the residues responsible for substrate binding, and the site
of access where substrates enter and products leave (Fig.
4a).
The residues at the base of the active site that surround
His10 are strictly conserved among the E. coli,
S. cerevisiae, S. pombe, and human dPGM family
(black circles in Fig. 2a). Interactions between
the phosphohistidine and the rest of the protein are depicted in Fig. 4b, with interatomic distances given in Table
II. The His10 side chain is
held in position in both the phosphorylated and dephosphorylated forms
by a hydrogen bond between N
The binding and presentation of substrates for phosphorylation or
dephosphorylation is mediated by active site residues between 2 and 12 Å from the active histidine. In addition to the phosphohistidine, the
active site of the E. coli structure contains two sulfate ions derived from the crystallization medium. It is likely that these
binding sites are formed by residues that are involved in binding the
phosphate groups of the mono- and bisphosphoglycerate substrates. Two
of the S. cerevisiae crystal forms also have two sulfates in
the active site, and in the case of 1QHF a partially occupied
3-phosphoglycerate has been modelled overlapping one of these sulfates
(7). It is of particular interest that the two pairs of sulfate binding
sites in S. cerevisiae and E. coli are different
(their positions are displaced by 3.1 and 4.0 Å, respectively) and
thus in combination describe four sites where the phosphate moieties of
the substrates may bind, with implications for the enzyme mechanism.
For simplicity, the designations E1 and E2 are used to identify the two
sites observed in the E. coli structure, and Y1 and Y2 are
used to identify those sites identified in the S. cerevisiae
structures. When the protein structures are superposed, Y1 is 3.9 Å from the position of the phosphoryl group of the phosphohistidine
forming hydrogen bonds to the phosphohistidine-stabilizing residues
Arg61 and Asn16 and to Ser13. Site
E1 is located further from the phosphohistidine and also participates
in a hydrogen bond with Arg61, but its most important
interactions are with the amide nitrogens of Thr22 and
Gly23. Sites Y2 and E2 are formed, in the main part, by
interactions with Arg115 and Arg116, both of
which are strictly conserved residues. These arginines form hydrogen
bonds with residues in the access site (discussed below) and probably
contribute to linking catalytic events to structural change at the
access site.
Access Site--
The structure of the C-terminal tail of dPGMs has
remained a mystery throughout previous structural and biochemical
studies. Most S. cerevisiae structures do not include
residues past 236 (E. coli sequence numbering). 1QHF
contains residues up to 242, but those beyond 238 have temperature
factors that have been truncated at 100.12 Å2. In the
native structure presented here, the tail is well defined up to
Lys247 (Fig. 5). This
observation strongly implies that the ordering of the C terminus is
commensurate with enzyme phosphorylation and thus typical only of the
active enzyme.
The access site as a whole consists of the rim of the active site
cavity and the C-terminal tail, which forms a lid. The rim is formed by
residues Glu12, Lys17, Asn19,
Lys32, Glu36, Ala100,
Asp108, Lys112, Glu204,
Asn206, and Thr209, whereas the C-terminal tail
consists of residues 238-249
(Lys-Ala-Ala-Ala-Val-Ala-Asn-Gln-Gly-Lys-Ala-Lys) and is highlighted in
magenta in Figs. 2 and 4a.
The basic secondary structure of the tail is a
Ala239-O forms a hydrogen bond with
Asn206-N
Ala243-O and Gly246-N form the hydrogen bond
that makes the
It has been proposed that adoption of an ordered conformation by the
tail when the protein is phosphorylated prevents solvent access and
thus phosphoenzyme hydrolysis (31). The present work shows this to be
unlikely because up to 36 ordered water molecules are found in the
cavity, but there may be a less direct role in phosphohistidine
stabilization. S. pombe dPGM is typical of a group of
"short" dPGMs (also including Zymomonas mobilis and
Haemophilus influenzae) that have no C-terminal tail.
Limited proteolysis of S. cerevisiae dPGM, removing the
C-terminal seven residues, produces a protein of similar character to
S. pombe dPGM with markedly reduced mutase activity and
enhanced phosphatase activity (30). Most of the residues involved in
interactions that hold the tail in place are conserved among all
"full-length" dPGMs and link, via one or two residues, directly to
the substrate binding region. This hydrogen-bonding network is shown in
Fig. 4a.
We propose that Arg115 and Arg116 provide the
switch with which the substrate in the active site induces a change
between active and inactive forms by making interactions with the tail
residues more or less favorable. When the active form is selected, the interactions of the tail with Asp108 provide further
stability to the conformation. The real key to preserving the
phosphohistidine is the conformation of Asn16. This residue
forms two hydrogen bonds with the phosphohistidine and lies on the loop
from residues 9-21, which may well be constrained in the active form
by the interaction of Asn19 with the C-terminal tail.
The crystal structure of a dPGM in its competent, phosphorylated form
advances our understanding of the contributions from various parts of
the dPGM structure in determining and regulating catalysis. Of
particular importance is the ordered structure of the C-terminal
portion of the polypeptide, which has been an enigma for many years.
The structure of this tail differs from predictions and redefines the
proposed roles of a number of residues. The dimeric structure has
allowed us to identify the determinants of the distinct oligomerization
states of dPGMs, although it remains unclear why such variety exists.
The analysis of the structural changes on phosphorylation suggests
important roles for previously overlooked residues, such as
Asn16 and Asn19, which can now be investigated
through site-directed mutagenesis. The existence of a crystal form of
dPGM that diffracts to atomic resolution provides an excellent basis
for such studies and also allows further investigation of substrate and
inhibitor binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside was tested under a
range of conditions. E. coli dPGM was then overexpressed and
purified as described previously (1).
1) in 20 mM
Tris-HCl buffer (pH 8.0) with 100 mM NaCl and a reservoir comprising 100 mM Tris-HCl, pH 8.75, 200 mM
Li2SO4, and 20% polyethylene glycol 4000. The
crystals are orthorhombic (P 21212
with a
62 Å, b
113 Å, and
c
40 Å) with a solvent content of 50%,
corresponding to one monomer of protein per asymmetric unit.
= 0.87 Å) on an ADSC Quantum-4 CCD detector,
processed with DENZO, and scaled with SCALEPACK (17). Molecular
replacement was performed with AMORE (18) using data to 2.0 Å and a search model derived from S. cerevisiae dPGM (the
highest resolution structure available at 1.7 Å, Protein Data Bank
code 1QHF (7)), using one monomer and truncating all side chains to
C
. The suitability of the best solution was confirmed by the
presence of the correct dimer interface provided by the
crystallographic 2-fold. Subsequent phase improvement and automated
building were achieved using wARP (19), resulting in a model
with an R-factor of 0.229.
Crystallographic data and refinement statistics
2 of His10 in a tetrahedral
arrangement. In the center of these atoms was a prominent peak of
residual electron density corresponding to ~0.5 that of an omitted
ordered solvent molecule. His10 is the nucleophilic
histidine that becomes phosphorylated during the catalytic cycle.
Although there are two histidines that have roles in catalysis, the
term "active site histidine" will be used exclusively for
His10. The geometry of the imidazole, three water
molecules, and peak (N-P distance, 1.74 Å; O-P distances,
1.50 ± 0.02 Å) was in agreement with the structure of
phosphorylimidazole found in the Cambridge Structural Data base ((22,
23); CADPIM (24)). The outstanding quality of the diffraction
data allowed us to successfully refine His10 as a partially
occupied phosphohistidine with its occupancy coupled to a
histidine and three solvent water molecules (Fig.
1). The resulting model has 0.28 occupancy phosphohistidine and 0.72 occupancy histidine plus three
water molecules. We had no reason to expect histidine
phosphorylation prior to crystallization, because the half-life of the
phosphohistidine is expected to be of the order of 35 min, as observed
with the S. cerevisiae enzyme (25).
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Fig. 1.
Electron density
(blue) at the active site histidine.
a, 6 Fo
Fc
calc electron density omitting the phosphorus.
b, 2
2Fo
Fc
calc electron density calculated omitting the atoms
shown. The phosphohistidine, at an occupancy of 0.28, is shown as
semitransparent ball and stick. The solid ball and stick shows the
remaining 0.72 occupancy histidine (blue) with three water
molecules (red). Figs. 1, 2b, 3, 4b,
and 5 were prepared using Molscript (32) and Raster3D (33).
/
angles, and only Ala182 has generously allowed values, as
defined by PROCHECK (27).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
fold of the monomer of E. coli dPGM is the same as S. cerevisiae dPGM, as
expected from their 54% sequence identity (Fig.
2a). In summary, the protein
core consists of a six-stranded
-sheet, C-B-D-A-E-F, with all but E
being parallel, flanked by six
-helices (Fig. 2b). The
active site is located at the C-terminal edge of the
-sheet and is
constructed from stretches of sequence dispersed throughout the amino
acid sequence.
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Fig. 2.
A, alignment of four dPGM
sequences from E. coli (PMG1_ECOLI), human brain
(PMGB_HUMAN), S. cerevisiae
(PMG1_YEAST), and S. pombe
(PMGY_SCHPO) from the Swiss Protein Database (34),
numbered according to the E. coli sequence. Secondary
structure elements assigned by PROMOTIF (35) to the E. coli structure are marked on, colored, and labeled as in
b. Dark blue boxes signify identity, and
cyan boxes signify similarity. Where the S. pombe
sequence has deletions, identity and similarity are calculated for the
other three sequences. Black circles highlight
phosphohistidine binding residues; red boxes underline
residues involved in dimerization; green boxes underline
residues involved in tetramerization; magenta boxes
indicate the C-terminal tail. Prepared using CLUSTALW (36) and
ALSCRIPT (37). B, a ribbon diagram of E. coli dPGM. The -strands labeled A-F are shown as
yellow arrows, most of the
- and 310
(
)-helices are colored cyan. Helix
10 and the
C-terminal tail are magenta, and helix
8 is
gray. Blue spheres represent the active
phosphohistidine. The two active site sulfates (red and
yellow) and a chloride ion (green) are also
depicted as spheres.
atoms. Despite having a largely similar backbone structure, the region of lowest
sequence similarity (residues 124-145) provides a number of important
differences at the S. cerevisiae tetramerization interface.
Significant substitutions include Asp141(S.
cerevisiae)-Ser143(E. coli), which
abolishes a hydrogen bond to Trp162* (* signifies a residue
of another subunit); Pro142-Glu144, which
alters the local backbone conformation significantly for a stretch of
four residues; Val144-Glu146, which produces a
steric clash with Gln163*;
Asp164-Glu166, which breaks a salt bridge to
Arg83*; and Lys168-Pro170, which
causes a steric clash with the main chain of Tyr139*. The
S. cerevisiae K168P mutant has indeed been shown to
reduce the Km of tetramerization (29); but whereas
the introduction of this proline into a helix does disrupt the hydrogen
bond network, the change in backbone conformation is insignificant, and
it is the position of the proline side chain that disrupts the
interface interaction (Fig. 3).
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Fig. 3.
Stereo view highlighting differences
between E. coli and S. cerevisiae
dPGMs at the tetramerization interface of the latter.
Subunits of S. cerevisiae are shown in magenta
and black, and subunits of E. coli are shown in
green. Side chains are shown for residues that may be
responsible for the inability of E. coli dPGM to
tetramerize.
3 and the adjacent strand
C, whereas the second stretch is part
of the region discussed above that promotes tetramerization in S. cerevisiae. The positions where the S. pombe sequence
differs from both E. coli and S. cerevisiae
include Val58(E.
coli)-Ala63(S. pombe), which causes the
loss of a hydrophobic contact, and Ala76-Pro81, which is likely to cause some
distortion of the backbone. A significant hydrophobic packing
interaction of Trp77 with Arg138* and
Tyr139* is also lost on substitution of Trp to
Asn82 and deletion of the loop from 124-147.
-C
distance) relative
to the equivalent residues in the S. cerevisiae structure.
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Fig. 4.
a, the surface of dPGM
(semitransparent gray) is cut away to reveal the network of
hydrogen bonds linking the catalytic machinery and substrate
binding residues to the active site entrance. The
sulfate binding sites E1 and E2 are labeled. GRASP (38),
Molscript (32), GL_render (L. Esser and J. Deisenhofer,
personal communication), and POVRAY were used to prepare this figure.
b, the chemical environment of the phosphohistidine.
Red spheres represent oxygens, blue spheres
represent nitrogens, and yellow spheres represent sulfurs.
The phosphohistidine is represented by green bonds, and
sulfate is represented by yellow. Residues are represented
as follows: blue, basic; red, acidic;
gray, apolar; magenta, polar. Hydrogen bonds are
shown as cyan dashed lines.
1 and the amide oxygen of the adjacent
Gly11. The length of this hydrogen bond decreases on
phosphorylation, accompanied by the movement of residues 9-22. One of
these residues, Asn16, alters its side chain conformation
to allow N
2 to form a hydrogen bond with a phosphate oxygen (3.18 Å) and the O
1 to participate in a CH···O hydrogen bond with
C
1 of His10 (3.14 Å). A second phosphate oxygen accepts
a hydrogen bond from His183 N
1 (2.72 Å), which is
itself hydrogen-bonded via its N
2 atom to Ser57 (data
not shown). His183 may also serve as a proton source during
catalysis, because it is spatially adjacent to the active site acid
Glu88. The other basic residues that bind the phosphate
group are Arg61, which forms a hydrogen bond to the same
oxygen as His183 via its N
atom (2.96 Å), and
Arg9, which forms a hydrogen bond to the third phosphate
oxygen via N
(2.74 Å). This oxygen is also hydrogen-bonded to the
amide nitrogen of Gly184, which is the N-terminal residue
of a 12-residue
-helix (
8). This helix (gray in Fig.
2b) is conspicuous on a ribbon diagram because it lies more
perpendicular to the
-sheet than do the other flanking helices and
is oriented such that the N-terminal helix dipole contributes to
stabilization of the phosphohistidine, rather than to the stabilization
of another substrate phosphate group, as previously proposed (31). In
addition to the polar interactions with the phosphoryl group, the
aliphatic segments of the side chains of Arg9 and
Arg61 also provide a series of hydrophobic contacts that
contribute to the orientation of the imidazole ring of
His10. Whereas the interactions with Arg61
are conserved on dephosphorylation, those with Arg9
are completely abolished.
Phosphohistidine hydrogen bonding interactions
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Fig. 5.
The C-terminal tail of E. coli dPGM with 1
2Fo
Fc
calc electron density
(blue). Hydrogen bonding interactions with
residues elsewhere on the structure are labeled.
-hairpin based around
a
-turn at Ala243-Gly246. This motif
extends away from helix
10 across the active site opening, forming a
number of hydrogen bonds with residues of the rim and substrate binding
region (Fig. 5).
2, whereas Asn206-O
1 accepts a
hydrogen bond from the substrate binding residue Arg116.
The side chain of Val242 interacts with Lys247,
whereas its amide oxygen forms a hydrogen bond with the side chain of
another substrate binding residue, Arg115.
Asn244-O
1 also forms a hydrogen bond to
Arg115. In the S. cerevisiae structures these
two arginine side chains are oriented differently to bind sulfate
rather than to form the interactions listed above, which may promote
the disorder of the tail. The hydrogen bond between
Asn244-O and the side chain of Asn19 is the
only direct link between the C-terminal tail and the stretch of
residues from 9-22.
-turn. This places Asn244 and
Gln245 in the correct orientation to hydrogen-bond to the
active site rim residue, Asp108, a residue that is
conserved throughout the full-length dPGMs and was previously proposed
to bind the C-terminal lysine residues. The observed interactions serve
as a clasp to pin the tail over the active site.
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ACKNOWLEDGEMENTS |
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We thank E. Tetaud and staff at Daresbury Laboratory for their help. This work made use of the Engineering and Physical Sciences Research Council Chemical Data Base Service at Daresbury Laboratory.
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FOOTNOTES |
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* This work was funded by 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 1E58) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
A Royal Society University Research Fellow. Current Address:
Centre for Biomolecular Science, University of St. Andrews, Fife KY16
9ST, UK.
§ To whom correspondence should be addressed. Tel.: 44-1382-345745; Fax: 44-1382-345764; E-mail: W.N.Hunter@dundee.ac.uk.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M007318200
2
Sequence numbering used throughout is that based
on the gene sequence of the E. coli protein. The S. cerevisiae sequence has extensions at the N and C termini and one
insertion and one deletion compared with the E. coli
sequence, occurring at positions 226 and 229, respectively. Hence for
the majority of the sequence residue n in the E. coli sequence corresponds to residue n 2 in the
S. cerevisiae sequence.
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ABBREVIATIONS |
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The abbreviations used are: PGM, phosphoglycerate mutase; dPGM, cofactor-dependent PGM; iPGM, cofactor-independent PGM.
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REFERENCES |
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