From the Department of Biochemistry and the Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219-1570
Received for publication, October 17, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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Serine hydroxymethyltransferase (SHMT; EC
2.1.2.1) catalyzes the reversible interconversion of serine and glycine
with transfer of the serine side chain one-carbon group to
tetrahydropteroylglutamate (H4PteGlu), and also the
conversion of 5,10-methenyl-H4PteGlu to
5-formyl-H4PteGlu. In the cell, H4PteGlu
carries a poly- The polyglutamate forms of tetrahydropteroylglutamate
(H4PteGlu)1 serve
as carriers of one-carbon groups that are required in the biosynthesis
of purines, thymidylate, choline, methionine, and other important
metabolites (1, 2). The circulating form of the coenzyme in blood is
the monoglutamate derivative (n = 1), which is readily
transported into cells. Once in the cell, additional glutamate residues
are added to form H4PteGlu3-7 in a reaction
catalyzed by folylpolyglutamate synthetase utilizing both ATP and
glutamate as substrates (3). Only polyglutamate forms are
physiologically active in the cell, although all but one enzyme
involved in one-carbon metabolism utilize the monoglutamate derivative
in vitro. Polyglutamylation blocks cellular export by
multidrug resistance proteins of H4PteGlun and
chemotherapeutic anti-folate analogs such as methotrexate and also
increases H4PteGlun affinity for most enzymes in
one-carbon metabolism (4).
The polyglutamate chain may also act as a tether in "channeling"
the H4PteGlun coenzyme between enzymes involved in
one-carbon metabolism (5, 6). The strongest evidence for channeling in
folate enzymes is between the dehydrogenase and cyclohydrolase
activities of the trifunctional enzyme C1-tetrahydrofolate synthase (7, 8) and between the dihydrofolate reductase and thymidylate
synthase sites on a bifunctional enzyme from Plasmodium falciparum (9-13). Kinetic evidence suggests that channeling of H4PteGlun may also be important for SHMT (14,
15).
There are now ~16 crystal structures of folate-requiring enzymes, but
few of these address the binding determinants of the polyglutamate
chain (16-21). Most of these structures have been determined without
any bound folate or folate analog or with folate analogs lacking a
polyglutamate chain or having only a single glutamyl residue. This
deficiency is due to several technical constraints and may also be
inherent in the nature of polyglutamylated H4PteGlun binding. Even in cases where
polyglutamylated folates might be used, the enzymes are often
crystallized in high salt, and the anions of these salts compete for
binding with the anionic polyglutamate part of the coenzyme, which may
cause weak or disordered binding of the polyglutamate chain. It is also
possible that the polyglutamate chain occurs as multiple conformers
whose occupancies are too low to be interpreted from an electron
density map. The only two published crystallographic structures with a bound polyglutamyl chain are those for tetraglutamylfolate analogs bound to thymidylate synthase (16, 17). In the first of these structures (folate analog CB3717-Glu4), there was
unambiguous electron density for triglutamate in one active site and
diglutamate in the other, but the group temperature factors for
Glu2 and Glu3 in the first site were high. The
failure to see the complete ligand in this structure may have been a
consequence of the high ionic strength (I SHMT catalyzes the conversion of serine and
H4PteGlun to glycine and
5,10-methylene-H4PteGlun, which is the major entry
point of one-carbon groups into one-carbon metabolism (1). An increase
in glutamate chain length from 1 to 6 increases the affinity of
H4PteGlun for several mammalian SHMTs by ~2
orders of magnitude, but results in only a 2-fold increase in affinity
for Escherichia coli SHMT (22, 23). Crystal structures have
been determined for the cytosolic SHMTs of human (24), rabbit (25), and
mouse (26) and for E. coli (27) and Bacillus stearothermophilus (28) SHMTs. The human and rabbit SHMT
structures have no bound folate ligand, whereas the E. coli
and mouse SHMT structures are ternary complexes of the monoglutamate of
5-CHO-H4PteGlu with glycine substrate. Crystal structures
for bsSHMT include the enzyme with no bound ligands, with either serine
or glycine bound, and the ternary complex of glycine and
5-CHO-H4PteGlu. However, none of these structures defines
the polyglutamate-binding site.
In addition to the conversion of serine to glycine and the concomitant
transfer of the one-carbon group to H4PteGlun, SHMT
also catalyzes the physiologically important conversion of 5,10-methenyl-H4PteGlun to
5-CHO-H4PteGlun (29-31). The polyglutamate forms
of the product 5-CHO-H4PteGlu are slow tightly binding
inhibitors of the physiological reaction catalyzed by SHMT, but the
monoglutamate derivative binds rapidly (23). The polyglutamate forms of
H4PteGlu and 5-methyl-H4PteGlu have affinity
for the active site of rcSHMT comparable to
5-CHO-H4PteGlun, but are not slow binding. Of all
the reduced folate derivatives, only
5-CHO-H4PteGlun is stable to degradative oxidation, and this has permitted extensive spectroscopic binding and isothermal titration calorimetry studies of this compound with rcSHMT (23, 32).
In the work described here, we have measured solution equilibrium
binding and kinetic constants for wild-type and structure-based site
mutant tetrameric rcSHMTs with 5-CHO-H4PteGlun and H4PteGlun to define the binding site of the
polyglutamate tail. We have also determined the crystal structure at
2.7-Å resolution of a complex of rcSHMT with active site-bound
5-CHO-H4PteGlu3 obtained by soaking unliganded
crystals of rcSHMT in 5-CHO-H4PteGlu3. This is
the first crystal structure of a natural polyglutamylated folate bound
to an enzyme and is formed under conditions of low ionic strength. It
provides new information on the nature of the interaction of this
cofactor with proteins and, together with the solution data, provides a
consistent picture for the location and properties of the
H4PteGlun-binding site of SHMT.
Preparation of Site Mutants, Purification of
Expressed Enzymes, and Determination of Kinetic
Constants--
H4PteGlu1-5 were
purchased from Schircks Laboratory (Jona, Switzerland). These
compounds were reduced and converted to
(6S)-H4PteGlun as previously
described (33). (6S)-5-CHO-H4PteGlun was
also made by this method. Site-directed mutagenesis was performed using
a PCR-based method as previously described (34). Mutant enzymes were
expressed from a recA Crystallization and Diffraction Data--
Crystals of rcSHMT
were grown in 0.5-ml Eppendorf tubes at room temperature from 10-µl
drops of rcSHMT at ~50 mg/ml in 3% polyethylene glycol 4000 and 50 mM potassium MES (pH 7.0), sometimes with seeding (37).
Fresh crystals were stabilized in 4.8% polyethylene glycol 4000 in the
same buffer for 1 h and then soaked overnight in the same solution
with 0.4 mM
(6S)-5-CHO-H4PteGlu3. Crystals were
transiently (<30 s) placed in a cryoprotectant of 30% polyethylene glycol 400 and 6% polyethylene glycol 4000 in 50 mM
potassium MES (pH 7.0) and flash-frozen in liquid N2 for
data collection. Data for a 100° sector were collected on an RAxisII
with Osmics confocal optics at 60 kV and 150 mA. Oscillation
frames were integrated with Denzo and merged with Scalepack (38).
Merged intensity data were converted to structure factor amplitudes
using Truncate (39). Data collection statistics are summarized in Table
I. Although the unliganded rcSHMT structure was previously assigned to
space group P41212 with one dimer/asymmetric
unit, in this work, we indexed these crystals in the lower symmetry
P41 space group with one tetramer/asymmetric unit.
Model Refinement--
Initial phases were based on a
2.1-Å structure of unliganded
rcSHMT,2 which was nearly
isomorphous with the rcSHMT·5-CHO-H4PteGlu3 structure. Initially, the space group was assigned as
P41212 with one dimer/asymmetric unit. After 20 cycles of rigid body refinement in CNS Version 1.0 (40),
Rwork and Rfree were 35.8 and 35.6%, respectively. The resulting phases were used to calculate a
SIGMAA-weighted 2mFo Calculation of Surface Charge Distribution--
Electrostatic
field calculations were carried out by numerical solution of the
Poisson-Boltzmann equation using the program DELPHI as implemented in
the INSIGHT 2000 package (Accelrys, San Diego, CA). In this
calculation, the protein formal charge set, which corresponds to unit
charges on Lys, Arg, Glu, and Asp residues, was used. For the
triglutamate chain of 5-CHO-H4PteGlu3, unit negative charges were assigned to the Kinetic Properties and Ligand Affinity of rcSHMT and Its
Mutants--
The surface of rcSHMT has a net positive charge at
neutral pH (25), and we previously described a possible cationic
binding locus for the extended polyglutamate tail of
H4PteGlun emanating from the folate-binding site of
each of the subunits (27). Both Lys
Mutation of any one of 4 Lys residues to a neutral or negatively
charged side chain resulted in a 2-10-fold decrease in affinity for
H4PteGlu1/2/3/5. Mutation of
Lys1341, Lys1342, or
Lys346 3
affected the binding of H4PteGlu (1 glutamyl residue). None
of the mutations significantly affected the binding of
H4PteGlu2, but mutation of Lys3991
and Lys346 decreased the binding of
H4PteGlu3 and
H4PteGlu5. Removal of the insertion loop at
residue 244, which includes an Arg and a Lys residue, lowered the
affinity for the pentaglutamate by 2-fold and had no effect on the
shorter glutamate chain lengths. The crystal structure of the
soaked 5-CHO-H4PteGlu3 complex (see below) shows that the polyglutamate moiety of
5-CHO-H4PteGlu3 follows the channel
hypothesized from the earlier rcSHMT model to be the polyglutamate-binding site, on which the design of the site mutants was based.
Stability of Site Mutants--
Previous studies have shown that
saturation of rcSHMT with serine greatly increases the thermal
stability of the enzyme (36). Tm values increase from 65 °C
in the absence of serine to ~78 °C in the presence of serine.
Glycine also increases the thermal stability to a similar extent, but
only in the presence of 5-CHO-H4PteGlu. This increased
stability, as well as other changes in physical properties, was
interpreted to be the result of a closure of the active site upon
binding substrates and was inferred to require a group occupying the
substrate serine side chain site (36). We measured the thermal
stability of those mutant enzymes that showed altered folate affinity
to determine whether any of the residues mutated might be a trigger for
the closure of the active site (Table II). Such mutants might not show
the stabilizing effect of serine and glycine in the presence of
5-CHO-H4PteGlu. However, the mutations that resulted in the greatest decrease in affinity for H4PteGlun
(K1341Q, K1342Q, K346Q, and K3991Q/E) did not significantly
alter the thermal properties of rcSHMT. The K1342Q mutation did lower
the thermal stability of the enzyme by ~5 °C, but had little
effect on the thermal stabilization by substrates. Therefore, none of
the residues tested plays a role in the putative open-to-closed transformation.
Crystal Structure of rcSHMT Crystals Soaked in
5-CHO-H4PteGlu3--
Crystals of unliganded
rcSHMT in space group P41 with a single homotetramer of
54-kDa monomers/asymmetric unit were used in the soaking experiment.
The asymmetric unit of these crystals consists of two tight dimers,
A·B and C·D, with near-perfect 222 symmetry (see "Experimental
Procedures" for a discussion of the space group of unliganded
rcSHMT). Crystals were grown in 50 mM potassium MES (pH
7.0) and polyethylene glycol 4000, a low ionic strength buffer with a
large anion that has been shown not to inhibit substrate binding.
Numerous screenings of conditions did not yield any crystals of the
preformed mono- or polyglutamyl rcSHMT·5-CHO-H4PteGlu complex.
We previously described an inferred binding locus in rcSHMT for the
polyglutamate chain of H4PteGlun based on the structures of the eSHMT ternary complex and unliganded rcSHMT (25, 27).
The position of the 5-CHO-H4PteGlu inhibitor in the eSHMT
structure, subsequently confirmed in mocSHMT and bsSHMT crystal
structures (26, 28), defines the binding site of the pteridine ring,
the p-aminobenzoic acid (PABA) group, and the first
glutamate of the polyglutamate tail. The computed surface charge
distribution of the rcSHMT tetramer shows a positively charged channel
emanating from the folate-binding site of each of the subunits (27),
and with this as a guide, we constructed a hypothetical model for the
binding of the extended polyglutamate tail of
H4PteGlu3. This hypothetical model was the
basis for identifying residues that could interact with the extended
polyglutamate chain.
The SIGMAA-weighted 3mFo
Subunits A and C (but not B and D) had electron density in the amino
acid substrate-binding site contiguous with the PLP ring and consistent
with the presence of a glycine bound as a gem-diamine. No
exogenous glycine was added either to the enzyme solution prior to
crystallization or to the crystals soaked in
5-CHO-H4PteGlu3, and we surmise that
adventitious glycine was present in some component of the sample. In
folate-bound subunits B and D, there was a single peak of electron
density in the amino acid-binding site, but it was not connected to
C-4' of the PLP ring and was assigned as a solvent molecule.
Interactions of Bound 5-CHO-H4PteGlu with
rcSHMT--
The binding of 5-CHO-H4PteGlu3 did
not cause any large-scale changes in the structure of the enzyme. The
molecular interactions between the pteridine and PABA portions of
5-CHO-H4PteGlu3 in the soaked rcSHMT crystals
were similar (but not identical) to those observed in the mocSHMT and
eSHMT structures that were grown in the presence of
5-CHO-H4PteGlu. Electron density for the pteridine ring in
subunit B is broken, with C-7 missing; in subunit D, the tetrahydropyrazine N-5, C-6, and C-7 positions have weak or missing density, and the edges of the PABA ring are shaved. The C-6-C-9 bond
is axial to the formyltetrahydropyrazine of the pteridine so that the
PABA ring is nearly perpendicular to the pteridine ring. The
formyltetrahydropyrazine ring of the pteridine is in the half-chair
form, as is observed in the ternary complexes of eSHMT and mocSHMT with
5-CHO-H4PteGlu; but the broken density in this ring
indicates conformational flexibility not seen in the structures of the
other complexes. The formyl group in the complex described here is
oriented roughly perpendicular to the pseudoplane of the
formyltetrapyrazine ring, resembling that of the mocSHMT ternary
complex and differing from that of the eSHMT ternary complex, in which
the formyl oxygen is oriented diametrically away from O-4 of the
pteridine ring. The formyl oxygen of
5-CHO-H4PteGlu3 in subunits B and D makes
hydrogen bonds with the solvent in the amino acid-binding site. In
subunit B, the carboxylate of GluB57 also makes a long
hydrogen bond with this formyl oxygen, but this distance is too long in
subunit D for a hydrogen bond.
The side chain amide of AsnB347 is not visible in subunit B
and is broken in subunit D, but is well defined in subunits A and C. The hydrogen bond contacts of Asn347 with the pteridine
ring vary in three of the four complexes for which structures are
available. In the eSHMT and bsSHMT ternary complexes, the
Asn347 side chain makes a double hydrogen bond with N-1 and
N-8 of the pteridine, whereas in the mocSHMT ternary complex, this side
chain makes two different sets of contacts, one with N-1 and the
exocyclic amino group of the pteridine and the other with the exocyclic amino group alone. The disorder in the buried Asn347
residue in subunits B and D of the
rcSHMT·5-CHO-H4PteGlu3 complex, but not in
unliganded subunits A and C, may be transmitted from the adjacent
flexible loop around position 355, which, in subunits B and D,
interacts with the polyglutamate chain.
Other hydrogen bonds between the pteridine and the protein observed in
the ternary complex structures of mouse and E. coli structures are similar in the soaked
rcSHMT·5-CHO-H4PteGlu3 complex, although
there are variations in angle and bond length. The PABA ring is
approximately parallel with Tyr64 of the opposing subunit
of the tight dimer (subunits A and C), as is observed in the structures
of the eSHMT and mocSHMT ternary complexes. Although the location of
5-CHO-H4PteGlu3 in this soaked crystal
structure is the same as that in the eSHMT, bsSHMT, and mocSHMT ternary
complexes with glycine, the small differences noted above and the
incomplete electron density for the pteridine indicate that the
cofactor is not optimally bound and is present in more than one conformation.
Structure of the Polyglutamate Chain in the
rcSHMT·5-CHO-H4PteGlu3 Complex--
Omit map
electron density (3mFo
In subunit B, the
These differences in interactions of the polyglutamyl chain in subunits
B and D are the result of differences in the conformation of the
triglutamyl group and also of small differences in the loop
conformations around residues 134 and 356 in these two subunits. The
electron density in both of these loops in subunits B and D is broken
and apparently less ordered than their counterparts in subunits A and
C. In the unpublished 2.1-Å resolution electron density map of
unliganded rcSHMT, these loops are closely similar and well ordered in
all four subunits, in contrast to the four loops in the
5-CHO-H4PteGlu3 complex structure, two of which
(subunits A and C) are well ordered and two of which (subunits B and D) are disordered.
The half-site binding of 5-CHO-H4PteGlu3 in the
crystal structure of the soaked complex is consistent with the
equilibrium binding studies showing half-site occupancy of
H4PteGlun-bound rcSHMT for all mutants and all
congeners of the folate. This strengthens the inference that the form
of the enzyme in the crystal is similar (if not identical) to that on
which the solution measurements were made. The lattice packing presents
no obvious obstruction of access to ligands diffusing into the active
sites on all four subunits. When both glycine and
5-CHO-H4PteGlu3 were included in the soak, the
crystals cracked, most likely due to the enzyme going from an
open-to-closed structure.
Comparison of the SHMT·5-CHO-H4PteGlu3
Complex with Related Structures--
In the mocSHMT ternary complex
structure with 5-CHO-H4PteGlu and glycine, all subunits
have a glycine conjugated to PLP. One tight dimer binds
5-CHO-H4PteGlu in both subunits (A and B), whereas the
other dimer binds 5-CHO-H4PteGlu in only one subunit and in a slightly different orientation and at a lower occupancy than the
other tight dimer (26). Although the resolution of this structure may
not be sufficient to draw detailed conclusions about local
stereochemistry, the Protein Data Bank entry shows the linkage of the glycine to the PLP group in each subunit to be variable, suggesting the coexistence of different intermediates in the tetramer. There also appears to be some variation between the 2 inferred glycine
residues bound to PLP of subunits A and C in the soaked rcSHMT·5-CHO-H4PteGlu3 complex. Although both
are gem-diamines, that in subunit A is almost exactly
between gem-diamines I and II, and that in subunit C is
closer to gem-diamine II (51).
There is breakdown of the 222 symmetry of rcSHMT in crystals soaked in
5-CHO-H4PteGlu3, although it differs from that
observed in the mocSHMT ternary complex. The soaked
rcSHMT·5-CHO-H4PteGlu3 complex shows degraded
pseudosymmetry about two of the three local dyads due to the
differential ligand binding to subunits B and D versus
subunits A and C. The third local dyad relating the two tight dimers
retains the symmetry of ligand binding, but because the ligands and the
loops around residues 134 and 356 have different conformations, this
operation also deviates from the near-perfect symmetry of the
unliganded structure.
Correlation of Crystal Structure and Solution Studies of rcSHMT
Binding of 5-CHO-H4PteGlu3--
In this work,
we used the formyl derivative of H4PteGlu3
because of its high stability and comparable affinity to
H4PteGlu3 in binding SHMT. In solution, the
pteridine ring of 5-CHO-H4PteGlu is reported to be in a
half-chair conformation with the C-6-C-9 bond axial to the
formyltetrahydropyrazine ring (52, 53), whereas H4PteGlu
exists as a roughly equal mixture of two half-chair conformations with
C-6-C-9 either axial or equatorial (54). However, the data reported in
two NMR studies (52, 53) on the conformation of
5-CHO-H4PteGlu in solution are not in complete agreement
and are consistent with more than one conformation of the pteridine
ring. Such polymorphism is also suggested by the pattern of broken
density in the formyltetrahydropyrazine ring in both subunits B and D
of the soaked rcSHMT·5-CHO-H4PteGlu3 complex
structure described here. As found in other enzymes that bind reduced
folates, the C-6-C-9 bond is axial in eSHMT, bsSHMT, rcSHMT, and
mocSHMT.
The binding of the pteridine and PABA portions of
5-CHO-H4PteGlu3 to preformed rcSHMT crystals
occurs at the same site as that observed in the crystal structures of
the eSHMT, bsSHMT, and mocSHMT ternary complexes with glycine (26, 27).
However, the enzyme ligands to NH2, C-2, N-3, O-4, and the
N-5 formyl group of the pteridine ring are similar (but not identical)
in the three structures. The electron density of the complex described
here shows suboptimal hydrogen bonding for some of the pteridine donors and acceptors and weak electron density for N-3 and the
tetrahydropyrazine ring of the pteridine and for the side chains of
AsnB347 and AsnD347 in the folate-occupied
subunits. The mocSHMT ternary complex also shows variable and, in some
cases, suboptimal hydrogen bonding to the pteridine ring in the three
liganded subunits. In the eSHMT and bsSHMT complexes with
5-CHO-H4PteGlu, the side chain amide of AsnB347
makes dual hydrogen bonds with N-1 and N-8 of the pteridine ring, and
all hydrogen-bonding groups have good stereochemistry. This suggests
that, in the current structure, 5-CHO-H4PteGlu3
is not optimally aligned at the active site of rcSHMT and that the
disorder in the side chain of Asn347 in subunits B and D is
due to the presence of 5-CHO-H4PteGlu3, since
these side chains have clear density in unliganded subunits A and C.
Solution studies suggest that the slow step in binding
5-CHO-H4PteGlu3 with glycine to SHMT requires a
conformational change that occurs late in the binding process (23).
Because the crystals of the soaked complex of
5-CHO-H4PteGlu3 with rcSHMT are isomorphous with the unliganded native crystals, lattice forces or the absence of
glycine is likely to have blocked this putative conformational change
in the soaked complex described here, and some of the observed poorly
formed enzyme-ligand interactions of this complex may become optimal upon transition through this conformational change. This conformational change may account for the crystals cracking when glycine was included in the soak. It should be noted that, in the
mocSHMT ternary complex, Asn347 makes variable and
generally poorer hydrogen bonds with the pteridine ring than is
observed in the "closed" eSHMT and bsSHMT ternary complexes,
suggesting that, in the mocSHMT structure, 5-CHO-H4PteGlu may also not be optimally bound and may not, as thought, be in the
closed conformation (26). The observations in the
rcSHMT·5-CHO-H4PteGlu3 complex that the
formyl group of 5-CHO-H4PteGlu3 is oriented
away from O-4 of the pteridine and that the PABA ring is stacked and parallel to Tyr64, as found in the other structures,
support the current structure as being on the pathway to the final
structure characterized in our solution studies. Substitution of
Asn347 with Ala in eSHMT essentially abolished the affinity
for
H4PteGlu3,4
demonstrating that the interactions of this side chain with the pteridine are essential for the optimal folate binding assumed in the
productive reaction. Ligand-induced flexibility in loop 355 in an early
complex of the SHMT reaction may be coupled to limited disorder of the
pteridine-binding groups of the active site and to multiple
conformations of the tetrahydropyrazine ring that are necessary to
realize productive binding of a single conformer in a subsequent
reaction step. A pre-binding folate site has also been proposed for a
folate-soaked complex of thymidylate synthase (55).
Structural and Site Mutant Studies of the Binding of
the Polyglutamate Chain--
Two studies have
previously used chemical modification to locate the folate-binding site
on SHMT. In one study (32), 5-CHO-H4PteGlu and
5-CHO-H4PteGlu5 were activated by a
carbodiimide, and both were found to cross-link uniquely to
Lys3994 and to block further binding of folates. In the
second study (56), modification of the guanidinium groups of 2 Arg
residues in sheep liver SHMT, equivalent to Lys242 and
Arg412 in rcSHMT, blocked H4PteGlu binding.
These 3 residues are near the polyglutamate-binding site, but only
Lys3994 in the structure described here could possibly
interact with the polyglutamate tail of folate and only with
Glu5 of H4PteGlu5. Solution studies
of site mutants also did not implicate these residues in folate binding
(Table II). Cross-linking in solution of Lys3994 to the
activated polyglutamate chain most likely arises from collisions of the
entering, nonspecifically bound folate ligand with Lys3994,
whose main chain and side chain are flexible.
The site mutants that affect the affinity of
H4PteGlun are closely consistent with the crystal
structure. LysB1341 and LysB1342 could interact
with the
Isothermal titration calorimetry data show that the binding of
5-CHO-H4PteGlun is entropy-driven, most likely by displacement of ordered water from the pteroyl-binding site (32). In
the 2.1-Å structure of unliganded rcSHMT, there are ordered solvent
molecules in this site. The isothermal calorimetry titration experiments further suggest that the binding of the polyglutamate chain
occurs with a negative enthalpy and little change in entropy. This
suggests that the polyglutamate chain does not make specific interaction with groups on the enzyme, but binds electrostatically in
multiple conformations.
This inference is consistent with the crystal structure of
5-CHO-H4PteGlu3 complexed with rcSHMT, where
only a small number of the potential interactions between cationic
groups on the enzyme and anionic polyglutamyl carboxylates are actually
made. Other stabilizing electrostatic interactions could be made with
relatively minor changes in the orientation of lysine and arginine side
chains on the enzyme. These observations parallel those made on the
binding of tetraglutamylfolate analogs to thymidylate synthase and the conclusions drawn from those structures (16, 17). In those two
structures, the binding mode of the polyglutamate chain is very
similar, but few (if any) direct hydrogen bonds are made between the
tetraglutamyl chain and groups on the thymidylate synthase. The failure
to form specific interactions between the polyglutamyl carboxylates and
accessible groups on the enzymes in these two complexes is consistent
with the proposal that this binding is driven by electrostatic
attraction of the polyanion for a polycationic site that has been
identified at the active sites of both enzymes. In the
5-CHO-H4PteGlu3 complex with SHMT, we observed
multiple conformations, which were not observed in the thymidylate
synthase complex. The thymidylate synthase crystals were in a high
ionic strength solution, which may have left only the most stable of
multiple complex conformations in the crystal structure.
The occupancy by 5-CHO-H4PteGlu3 of subunits B
and D (but not A and C) is consistent with the calorimetric observation
that the stoichiometry of folate binding is 0.5/subunit. The binding of
5-CHO-H4PteGlu3 to subunits B and D disposes
the two polyglutamyl tails on opposite faces of the tetramer (Fig. 2),
thereby maximizing the distance between extended polyglutamyl chains.
This arrangement retains more of the cationic character of each of the
two tetramer surfaces centered at the pseudo-222 intersection than if
the ligands were bound pairwise to subunits A and D or B and C, where
the polyglutamate chains would lie on the same cationic face. It is noteworthy that the charge surface calculated with the
5-CHO-H4PteGlu3 ligand bound (data not shown)
is significantly altered, having diminished cationic character at some
distance from the ligand itself.
Comparison of rcSHMT with SHMT Proteins in Other
Studies--
The stoichiometry and subunit occupancy for folate
binding are different for each of the four SHMT structures that have
been determined with bound folate. In eSHMT, both monomers of the
homodimer contain bound 5-CHO-H4PteGlu, whereas in bsSHMT,
one monomer binding site of the homodimer is fully occupied by
5-CHO-H4PteGlu, but the other is only partially occupied.
Tetrameric rcSHMT has 2 molecules of bound
5-CHO-H4PteGlu/tetramer, one in each tight dimer, whereas
mocSHMT has 5-CHO-H4PteGlu bound to both subunits of one
tight dimer and only one subunit of the other tight dimer. No obvious
reason for these differences is evident from the structures.
SHMT belongs to the fold type I family of PLP enzymes, which have a
core amino acid sequence of ~45 kDa that is conserved in the >50
SHMT sequences available (57, 58). The amino acid- and PLP-binding
sites are highly conserved in all SHMTs, and the canonical SHMT
structure supports the inference that the folate interaction site
evolved from a fold type I PLP precursor enzyme through sequence
insertions and not by domain swapping from other folate-requiring
enzymes (27). This may be the reason why anti-folate compounds
developed as chemotherapeutic agents are ineffective as inhibitors of
SHMT and suggests that an effective anti-folate inhibitor of SHMT might
not inhibit other folate enzymes.
Previous studies have shown that increasing the chain length of
H4PteGlun from 1 to 5 lowers the
Kd by ~2 orders of magnitude for mammalian
rcSHMTs. In E. coli, only the first 2 glutamate residues are
linked through the -glutamyl tail of at least 3 glutamyl residues that
is required for physiological activity. This study combines solution
binding and mutagenesis studies with crystallographic structure
determination to identify the extended binding site for
tetrahydropteroylpolyglutamate on rabbit cytosolic SHMT. Equilibrium
binding and kinetic measurements of H4PteGlu3
and H4PteGlu5 with wild-type and Lys
Gln or
Glu site mutant homotetrameric rabbit cytosolic SHMTs identified lysine residues that contribute to the binding of the polyglutamate tail. The
crystal structure of the enzyme in complex with
5-formyl-H4PteGlu3 confirms the solution data
and indicates that the conformation of the pteridine ring and its
interactions with the enzyme differ slightly from those observed in
complexes of the monoglutamate cofactor. The polyglutamate chain, which
does not contribute to catalysis, exists in multiple conformations in
each of the two occupied binding sites and appears to be bound by the
electrostatic field created by the cationic residues, with only limited
interactions with specific individual residues.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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3.5 M) of the crystallization medium (2.5 M
ammonium sulfate), which limited occupancy of the binding site. This
inference is supported by the second thymidylate synthase-folate analog crystal structure (folate analog LY231514-Glu4), in which
Glu1-Glu3 are defined, but the electron
density for Glu4 is poorer. Crystals of this complex were
obtained at lower ionic strength (I
2.0 M).
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strain of E. coli and purified as described for wild-type rcSHMT (35).
Km and kcat values with
L-serine as substrate were determined at pH 7.3 and
30 °C by coupling to the NADP-dependent oxidation of the
product 5,10-methylene-H4PteGlu (36). Kd values for (6S)-H4PteGlu1-5 were
determined by a spectrophotometric method and analyzed by the method of
Scatchard as described (32). Tm values were determined in a
Microcal differential scanning calorimeter.
dFc map that was visualized using the program O
(41). In this initial map, broken density corresponding to
5-CHO-H4PteGlu was observed in monomer B of the dimer in
the asymmetric unit, but density was not observed for either of the two
remaining glutamyl groups. The model was iteratively refined via high
temperature torsion angle dynamics and a maximum likelihood target
function (42) using CNS Version 1.0, with each refinement cycle
followed by manual rebuilding in program O using SIGMAA-weighted 2mFo
dFc cross-validated maps
or composite omit maps (43). Non-crystallographic symmetry was enforced
via positional NCS restraints between monomers comprising the
dimer. During iterative rebuilding, residue geometries and interactions were monitored with the programs OOPS (44), WHATCHECK (45), and PROBE
(46). We noted that for the 2.1-Å unliganded rcSHMT structure, for
which diffraction data had been acquired under similar conditions, the
value of Rwork plateaued near 24% in
P41212, even though visual inspection of both
SIGMAA-weighted and composite omit maps did not indicate significant
errors in the model. By re-merging the integrated intensity data in the
lower P41 symmetry space group and expanding the asymmetric
unit to a pair of dimers with 222 symmetry, both
Rwork and Rfree dropped
significantly, which led us to conclude that the correct space group is
P41. In the lower symmetry space group, additional NCS
restraints were added between monomers in the two tight dimers
comprising the asymmetric unit. After several rounds of rebuilding and
refinement, during which the weight of the NCS restraints was reduced
from 300 to 37.5 kcal/mol and 390 water molecules and 3 glycerol
molecules were added to the model,
Rfree and
Rwork improved to 29.4 and 22.0%, respectively.
In the latter stages of the refinement, positional restraints were
removed for regions of the structure that differed significantly
between NCS-related monomers, and model adjustments were made in
SIGMAA-weighted 3mFo
2dFc
maps. In the final cycles of simulated annealing via high temperature torsion angle dynamics, the positions of the
5-CHO-H4PteGlu3 residues and several amino
acids in partially disordered loops that interact with
5-CHO-H4PteGlu3 were restrained via harmonic
restraints. Coordinate errors in the refined structure were estimated
using the formalism of Luzatti (47) as implemented in CNS.
Refinement statistics are summarized in Table
I. Of the 470 residues in the rcSHMT
sequence, there is no interpretable electron density for the
amino-terminal 13 residues. Also, as noted below, there is variable
missing density in the different subunits for residues in the insert
just after residue 244 and broken or localized missing density around
residues 134, 355, and 399.
Data collection and refinement statistics for the
rcSHMT·5-CHO-H4PteGlu3 complex
-carboxyl groups of the glutamate residues and to the side chain carboxyl group for the terminal glutamate residue. In the rcSHMT structure, residues for which
density was not observed, as well as side chains of residues modeled as
alanine due to missing side chain density, were added to the structure
with the aid of a side chain rotamer data base (49) in O Version 8.0. The calculation was carried out on a cubic grid with 165 points along
each axis, corresponding to a grid spacing of 3 Å. To investigate the
effect of binding of the triglutamate chain on the surface charge
distribution in rcSHMT, the bound folate residues were excluded from
one set of calculations.
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Gln or Glu and Arg
Gln or
Glu point mutants in and near this channel were made, and the mutant
proteins were purified and assayed with serine and H4PteGlu
to determine specific activity, stoichiometry of H4PteGlu
binding, and affinity with serine for both H4PteGlu and
H4PteGlu5. All mutants exhibited Km values for serine that were the same as those of
the wild-type enzyme and a stoichiometry for
H4PteGlu5 of 0.5/subunit. Many of the mutant
enzymes also showed no significant changes in either
kcat or Kd for
H4PteGlu and H4PteGlu5 (Table II). However, some mutations did affect
the affinity for H4PteGlun, with the relative
affinity being dependent on the number of glutamate residues in the
folate (Table II). The catalytic activity for these mutants with serine
and H4PteGlu as substrates was also essentially unchanged
(<2-fold) when extrapolated to infinite H4PteGlu
concentration.
Affinity of H4PteGlun for rcSHMT site mutants as a
function of glutamate chain length
2dFc
electron density map of the soaked
rcSHMT·5-CHO-H4PteGlu3 complex showed density
with several breaks in this putative H4PteGlu3
site at the 0.95
level in two (subunits B and D) of the four
crystallographically independent monomers of the rcSHMT tetramer that
constitute the tetrameric asymmetric unit. This density in one subunit
of each tight dimer coincided with the site at which
5-CHO-H4PteGlu binds in the
eSHMT·5-CHO-H4PteGlu·glycine and
mocSHMT·5-CHO-H4PteGlu·glycine ternary complex
structures and extends into the cationic channel hypothesized to be the
binding site of the polyglutamate tail (Fig.
1). The electron density in the putative
polyglutamate-binding site could be fit with a triglutamyl chain bound
in two distinct conformations in each of subunits B and D (see below).
In subunit B, there are alternate conformations for Glu3 of
5-CHO-H4PteGlu3, and in subunit D, there is
bifurcation from Glu1 to give two alternate conformations
of Glu2-Glu3 (Fig.
2).
View larger version (117K):
[in a new window]
Fig. 1.
Surface charge distribution of the tetrameric
rcSHMT complex showing 5-CHO-H4PteGlu3
(green space filling) extending into the cationic
region centered on intersecting 2-fold rotation axes at the center of
the rcSHMT homotetramer. Blue is + and red
is charge field.
View larger version (67K):
[in a new window]
Fig. 2.
Orthogonal views of the rcSHMT tetramer
complexed with 5-CHO-H4PteGlu3. Subunits A + B and C + D (green and gold, respectively, in
each pair) make up each tight dimer. Arrows and plus
signs indicate pseudosymmetry dyad axes. The glycine-linked PLP
groups in subunits A and C are rendered as red sticks, and
the 5-CHO-H4PteGlu3 in subunits B and D as
space-filling models. The two different conformations of the
polyglutamate chain are indicated by magenta and
cyan space-filling models.
2dFc)
contoured at the 0.95
level for the triglutamyl groups in the
subunit B- and D-binding sites (Fig. 3)
was modeled in two conformations in each site. The two conformations in
each site differ from those in the other site and in their interactions
with the enzyme (Fig. 4). The
5-CHO-H4PteGlu group is oriented almost identically in subunits B and D, with the
-carboxylate of the first glutamyl group
oriented toward the HO-
of Tyr64 (2.64 and 2.80 Å). The
interactions of the Glu1
-carboxylate with the HO-
of
Tyr64 are slightly shorter than those observed in the
E. coli and mocSHMT complexes with
5-CHO-H4PteGlu (26, 27).
View larger version (41K):
[in a new window]
Fig. 3.
3Fo 2Fc omit map electron
density contoured at 0.95
for the
5-CHO-H4PteGlu3 ligand in subunit B with
several proximal binding site residues.
View larger version (35K):
[in a new window]
Fig. 4.
Schematic Ligplot (50) figure of the two
5-CHO-H4PteGlu3 conformers in subunits B
(A and B) and D (C
and D).
5-CHO-H4PteGlu3 is shown by green
bonds. Not all possible interactions are shown because some were not
observed in the structure and require side chain reorientation to be
formed.
-carboxylate of Glu2 is oriented
toward the side chains of LysB1341 and
LysB1342, the interaction with LysB1341 being
the tighter one. Neither carboxylate of Glu3 in both
conformers in subunit B is close enough to any groups in the enzyme to
make strong interactions. In subunit D, both the
- and
-carboxylates of Glu3 of one conformer can interact with
LysD1341 and LysD1342. In the other conformer,
either the
- or
-carboxylate can interact with
LysD346.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-carboxylate of Glu1 in subunit B and with the
Glu3 carboxylates of one conformer in subunit D. These lysines could also hydrogen-bond to the carbonyl oxygen of the Glu1-Glu2 peptide bond in polyglutamate chain
lengths greater than 1. H4PteGlu3 and
H4PteGlu5 showed higher Kd
values when either of these residues was mutated. LysD346
makes a close approach to both carboxylates of Glu3 in
subunit D and could approach Glu1 in subunit D if
LysD346 adopted a different side chain conformation.
Mutation of LysD346 similarly increased the
Kd values for H4PteGlu3 and H4PteGlu5. LysB1341 and
LysB1342 approach Glu2 in subunit B, but
mutation of any of the lysines only weakly affected the
Kd for the binding of
5-CHO-H4PteGlu2 and
H4PteGlu2. This insensitivity to the loss of
the positive charges at Lys1341, Lys1342, or
Lys346 for the diglutamate form of the folate may arise
from alternative interaction(s) between the
-carboxylate group of
Glu2 and these residues that are absent in the mono- and
triglutamate forms of 5-CHO-H4PteGlu. Extrapolation of the
5-CHO-H4PteGlu3 structure to Glu4
and Glu5 would lead in both sites to interactions with Lys3991 and Lys3994, mutation of which
increased the Kd for
H4PteGlu3 and H4PteGlu5. A longer polyglutamate chain might
also interact with the region in which Lys335,
Lys346, Lys354, and Lys358 and also
the more distal Arg3963 lie.
-carboxylate group, additional glutamates being
linked through the
-carboxylate (59). The affinities of cognate
monoglutamylfolates versus pentaglutamylfolates (with the
-linkage for the last 2 glutamate residues) for eSHMT differ by only
a factor of 2, indicating that eSHMT does
not have a well defined
polyglutamate-binding
site.5,6
There are several insertions of amino acids in the mammalian enzymes
compared with eSHMT, three of which are located in the polyglutamate-binding site. The insertion at position 134 introduces a
KKK sequence that makes interactions with Glu2 and
Glu3 in subunits B and D, respectively, and the insertion
at Lys399 is in position to interact with Glu5
of an extended polyglutamate. Site mutants of Lys3991
confirm this prediction in exhibiting a significantly lower affinity for 5-CHO-H4PteGlu5, and the nearby
Lys3994 is the residue found to cross-link with activated
folates (Table II). The 9-residue insertion at Gly244,
which contains both an Arg and a Lys residue, has incomplete density in
the crystal structure. Although removal of this loop lowered the
affinity of 5-CHO-H4PteGlu5 by a factor of only
2, suggesting that it does not play a critical role in binding the polyglutamate chain, it may play a role in either channeling of the
reduced folate from other folate-binding enzymes or guiding the folate
substrate into the active site, without directly affecting the binding.
In contrast to PLP-, amino acid-, and pteridine-binding sites of SHMTs,
which are highly conserved, the residues identified in this work that
interact with the polyglutamate tail of
H4PteGlu3 are less conserved in many SHMTs.
This suggests that other groups may interact with the polyglutamate
chain or have different binding loci in the enzymes from different organisms.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK55648 (to V. S.).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 1LS3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Present address: Dept. of Medical Technology, Yuanpei Inst. of
Science and Technology, Hsin-Chu City, Taiwan.
§ To whom correspondence should be addressed: Inst. for Structural Biology and Drug Discovery, Virginia Commonwealth University, 800 East Leigh St., Suite 212, Richmond, VA 23219-1540. Tel.: 804-828-6139; Fax: 804-827-3664; E-mail: xrdproc@hsc.vcu.edu.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M210649200
2 J. N. Scarsdale, G. Kazanina, V. Schirch, and H. T. Wright, unpublished data.
3 The numbering system is based on the amino acid sequence of eSHMT (25), with suffix extensions denoting insertions in rcSHMT relative to eSHMT (e.g. 3991 is the first inserted residue after residue 399) and uppercase letter prefixes (A-D) identifying the subunit (e.g. B3991).
5 S. Angelaccio and R. Contestabile, personal communication.
6 V. Schirch, unpublished data.
4 S. Angelaccio and R. Contestabile, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: H4PteGlu, tetrahydropteroylglutamate; H4PteGlun, tetrahydropteroylglutamate with n glutamyl residues; SHMT, serine hydroxymethyltransferase; bsSHMT, B. stearothermophilus serine hydroxymethyltransferase; rcSHMT, rabbit cytosolic serine hydroxymethyltransferase; eSHMT, E. coli serine hydroxymethyltransferase; mocSHMT, mouse cytosolic serine hydroxymethyltransferase; 5-CHO-H4PteGlu, 5-formyltetrahydropteroylglutamate; 5-CHO-H4PteGlun, 5-formyltetrahydropteroylglutamate with n glutamyl residues; MES, 2-(N-morpholino)ethanesulfonic acid; PABA, p-aminobenzoic acid; PLP, pyridoxal phosphate; NCS, non-crystallographic symmetry.
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