Orientation of 1,3-Bisphosphoglycerate Analogs Bound to
Phosphoglycerate Kinase*
David L.
Jakeman
§¶,
Andrew J.
Ivory
,
G. Michael
Blackburn
, and
Michael P.
Williamson§**
From the
Department of Chemistry and
§ Department of Molecular Biology and Biotechnology, Krebs
Institute, University of Sheffield, Sheffield S10 2TN, United
Kingdom
Received for publication, November 19, 2002, and in revised form, December 23, 2002
 |
ABSTRACT |
We have previously reported dissociation
constants for a range of bisphosphonate analogs of
1,3-bisphospho-D-glyceric acid binding to yeast
phosphoglycerate kinase. Data for the unsymmetrical analogs were
difficult to interpret because it was not clear in which of the two
possible orientations these ligands bound. Here we report a novel NMR
method for quantifying orientation preference based on relaxation
effects induced by titration with CrADP, which is applied to these
ligands. It is shown that all ligands can bind in both orientations but
that the driving force for the orientational preference is to put the
,
-difluoromethanephosphonate group in the "basic patch"
(nontransferable phosphate) position. The relevance to the design of
phosphoglycerate kinase inhibitors is discussed.
 |
INTRODUCTION |
Phosphoglycerate kinase
(PGK)1 (EC 2.7.2.3) is a
glycolytic enzyme that catalyzes the following reaction, where MgADP
and MgATP represent the magnesium complexes of ADP and ATP,
respectively.
It is an attractive drug design target because trypanosomes rely
on glycolysis as their sole source of energy, and hence inhibition of
PGK could provide effective treatment for trypanosomal infections. The
enzymes from a wide variety of sources have been compared (1). They
show high sequence and structure similarity and consist of monomeric
proteins of 45 kDa with two distinct domains connected by a hinge (Fig.
1). The N-terminal domain (which also
contains the last 10 residues at the C terminus) has a "basic patch" containing a number of arginines and histidines that is the
binding site for the glycolytic substrates 1,3-BPG and 3-PGA. The
C-terminal domain is the binding site for ATP/ADP. The enzyme has a
number of crystal structures (2-13). In many the two domains are in an
open conformation with the substrates ~11 Å apart, which is much too
far for direct phosphotransfer. Crystal structures also exist in which
the enzyme forms a closed conformation with the two substrates close
enough for direct nucleophilic associative phosphotransfer (14). A
mechanism has been proposed based on these crystal structures of which
the key features are that Arg-38 binds to the transferred 1-phosphate
of 1,3-BPG and locates it close to the nucleophilic oxygen of ADP in
the closed form, whereas Lys-213 (Lys-197 in Thermotoga
maritima) (9) also binds to the phosphate, stabilizing the
negative charge that develops on it as it transfers, thereby catalyzing
the phosphoryl transfer. (Note: yeast PGK numbering has been used
throughout.) The hinge bending has been studied in detail. Another
subject of intense study is the kinetics of the reaction, which is
activated by anions (15). Although the issue has not been resolved, it
is likely that an anion binding site is formed when the two domains
close (16).

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Fig. 1.
A schematic picture of the binding sites on
PGK for 1,3-BPG and CrADP. No orientational or conformational
detail is implied. In the closed conformation, the two domains come
closer together such that the -phosphorus is close to the terminal
phosphate of ADP.
|
|
The crystal structures have provided considerable information on how
the substrates bind, both to the closed and to the open conformations.
1,3-BPG itself is very unstable, so no structures exist of bound
1,3-BPG. Crystals of 3-PGA show it bound with the 3-phosphate close to
Arg-168, Arg-121, and Arg-65 in the basic patch, with the
carboxylate close to Arg-38 and pointing toward the nucleotide.
Somewhat surprisingly, in crystals of Trypanosoma brucei PGK
grown in the presence of 3-PGA and MgADP from 2.5 M potassium phosphate, two of the four subunits in the asymmetric unit
have a phosphate anion bound in the basic patch but close to Arg-38,
i.e. close to the transferable phosphate site (11).
We have previously reported the synthesis and dissociation constants of
PGK ligands based on 1,3-BPG (17, 18), which inhibit PGK (19). The
phosphate linkages were replaced by non-scissile methanephosphonates,
giving ligands that bind as much as 50 times more tightly than 3-PGA.
It was observed that replacement of methanephosphonate by
,
-difluoromethanephosphonate increased the affinity, and it
was suggested that this was because of the lower pKa induced by the fluorines (18, 20). However, replacement of both methanephosphonates by difluoromethanephosphonates led
to no further increase in affinity, suggesting that only one end of the
molecule required the lower pKa and consequent greater charge density at neutral pH. Affinities could not be interpreted further because it was not possible to tell in which orientation the unsymmetrical ligands were binding. We have therefore developed a method to distinguish the orientation of the ligands and
report its application with an analysis of the implications for design
of PGK ligands.
 |
EXPERIMENTAL PROCEDURES |
Yeast PGK was obtained from Roche Molecular Biochemicals as an
ammonium sulfate suspension, resuspended in 0.1 M Tris, 0.1 mM EDTA, 6 mM 2-mercaptoethanol, 10 µM phenylmethylsulfonyl fluoride, and 5% ammonium
sulfate, then concentrated into 40 mM KCl, 10 mM triethanolamine, pH 7.1 ± 0.05, in
D2O. The protein concentration was typically 0.3 mM. PGK ligands were synthesized as described previously
(18). CrADP was prepared as described (21).
One-dimensional 31P NMR spectra were acquired at 300 K on a
Bruker AMX-500 spectrometer at 203.462 MHz using 60,000 scans over a
spectral width of 8196 Hz and 2048 complex data points. A pulse angle
of 60 ° (6.5 µs) was used with a relaxation delay of 0.5 s
between pulses. Simultaneous decoupling of 1H and
19F was achieved during signal acquisition using a
directional coupler. The probe was tuned to 19F, and
optimal decoupling without disturbance of the signal was achieved using
1H decoupling (26 db attenuation of normal pulse power or
~330 Hz decoupling field at the sample) applied directly through the directional coupler and 19F decoupling (10 db attenuation
or ~500-Hz field) into the side of the directional coupler. Both
channels used Waltz-16 composite pulse decoupling. It proved
particularly important to decouple the 31P signals
efficiently from both 1H and 19F. In spectra
where 19F was not decoupled, addition of CrADP gave rise to
differential broadening of the 19F-coupled triplet with the
outer lines broadening much more rapidly than the central line (22).
This effect is caused by different relaxation rates of different
elements of the scalar-coupled density matrix (23), possibly
exacerbated by cross-correlation effects (24). Although these effects
are of interest from a theoretical perspective, they make analysis of
the relaxation data much more complicated. Indeed, simple analysis of
the width of the central line gave results completely different from
those obtained using complete decoupling. The free induction decays
were zero filled to 4096 complex points, and then sensitivity enhanced
by mild exponential multiplication of 2-3 Hz. A control spectrum was
recorded with 1 molar eq of 1,3-BPG analog to PGK and no CrADP.
Subsequent spectra were recorded after the addition of 12 mM CrADP. Three or four points were obtained for each
analog with up to 1.5 eq of CrADP. The spectra were analyzed using
Felix (Accelrys Inc., San Diego, CA) to measure the line width of the
31P signals by fitting to Lorentzian lines. The fitting was
typically truncated at 10% of peak height. Errors were estimated by
repeated fitting using different processing, base-line correction, and starting values for the fit. Simulations showed that the mild linebroadening applied had no significant effect on the line width ratios.
 |
THEORY |
The magnetic moment of an unpaired electron is
103
times greater than that of a nucleus. Fluctuation of local magnetic
fields leads to relaxation, and the larger magnetic fields resulting from the presence of a paramagnetic species give rise to particularly efficient nuclear spin relaxation. The strong local magnetic field produced by an unpaired electron can be coupled to nuclei by
dipole-dipole interactions. The dipolar effect of the unpaired electron
on surrounding nuclei is inversely proportional to the sixth power of
the distance between electron and nucleus, implying that the distance
from a nucleus to an unpaired electron can be measured by its effect on
nuclear transverse relaxation times (T2), which
are proportional to line widths. In the experiments described here we
measure the effect of Cr3+ on the relaxation of the two
31P nuclei in the ligand, here denoted
and
, and use
the relative broadening to measure the distance to each nucleus and
hence the orientation of the ligand.
Chromium(III) is a high spin (3d3 ion) dipolar
relaxation probe. The contribution to the nuclear transverse relaxation
rate (1/T2,p) from the paramagnetic probe is
given by equations derived by Solomon and Bloembergen and presented by
Mildvan and Gupta (Ref. 25 and references therein) as Equation 1.
|
(Eq. 1)
|
fq is the mole fraction of nuclei bound, C
is a constant dependent on nuclear and electronic magnetic moments,
r is the distance from Cr3+ to the relaxing
nucleus, and F(
c) is a function of
c (correlation time for dipolar relaxation)
and
(Larmor frequency of phosphorus). The line width at
half-height (
1/2,obs) in the presence of
the paramagnetic ion is inversely proportional to the observed
T2 (T2,obs, Equation 2).
|
(Eq. 2)
|
Under conditions of fast exchange of the Cr3+ on and
off the protein, the observed relaxation rate can be considered to
arise from two contributions (Equation 3), namely, the relaxation rate in the absence of the paramagnetic ion 1/T2,abs,
and the relaxation rate arising from dipolar interaction with the bound
paramagnetic ion, 1/T2,p.
|
(Eq. 3)
|
Combining Equations 1, 2, and 3 for a 31P nucleus
gives Equation 4 (
c is the assumed constant).
|
(Eq. 4)
|
k is a constant assuming all
1/T2,abs values are the same for all observed cases.
A plot of peak width at half-height versus [CrADP] has a
gradient of
C'
r
.
Thus, a ratio of gradients allows us to estimate the relative distances of the 31P nuclei
and
(r
/r
) in the basic
patch from the paramagnetic probe CrADP in the nucleotide binding site.
The ratio of gradients is calculated according to Equation 5,
|
(Eq. 5)
|
because C'
= C'
. Thus, the 31P signal that
broadens most on the addition of CrADP is the one closest to the
Cr3+.
If the enzyme remains in its open conformation when the ligands bind,
then the ratio r
/r
is fixed and can be estimated from crystal structures (3) as 1.8. Any
ratio of gradient
/gradient
less extreme
than
1/(r
/r
)6 = 1:34 can therefore be interpreted as caused by the ligand binding in
both possible orientations. If the ligand binds in conformation I with
probability p and in conformation II with probability 1
p (Fig. 2), then the
results are determined by Equations 6 and 7.
|
(Eq. 6)
|
|
(Eq. 7)
|
Substituting Equations 6 and 7 into Equations 2 and 3, we obtain
Equation 8.
|
(Eq. 8)
|
After rearrangement and using Equation 5, the apparent distance
ratio is given by Equation 9, thereby allowing us to estimate conformational preference.
|
(Eq. 9)
|
This function is shown in Fig. 3,
which shows that relatively small proportions of binding in the
alternative conformation drastically reduce the apparent distance
ratio.
If the enzyme conformation is altered in the direction of the closed
form when the ligands bind, then both r
and
r
are reduced and the ratio
r
/r
increases. The
conformational preference derived above (Equation 9) is therefore a
maximum limit, and any closed form population brings the conformational
preference closer to 50%.
 |
RESULTS |
31P NMR spectra were acquired for a range of PGK
ligands in 1:1 complexes with PGK, and line widths were measured as a
function of added CrADP. The addition of CrADP caused a monotonic
increase in line width as expected. From the 1H spectra
acquired on titration of the ligands with PGK, it is clear that no
large conformational changes occur in PGK, and thus the enzyme is
expected to remain in the "open" conformation seen in the majority
of crystal structures. Typical spectra
are shown in Figs. 4 and 5 for a ligand
containing only methanephosphonates and a ligand containing both a
methanephosphonate and an
,
-difluoromethanephosphonate, respectively. Differential broadening exists of the two 31P
signals, which indicates a conformational preference of the ligand.
Line widths were measured by line fitting, and the gradients of line
width versus CrADP were compared to derive an effective distance ratio of the two phosphorus atoms from the chromium, as in
Equation 5. A typical result is shown in Fig.
6 for compound 6 (data from
Fig. 5). The results are summarized in Table II, which also presents
the affinities measured previously. None of the distance ratios
measured approached the theoretical limit of 1.8 as presented above
(assuming that the open enzyme conformation is the same as that in the
crystal). The largest ratio found was 1.49. We therefore used the
distance ratios to calculate the approximate conformational preference
of the ligands, which are listed in Table II. Conformational
preferences range between 29 and 94%, with some analogs showing almost
no orientational preference. We note that nonspecific binding of the
ligand to PGK would reduce the ratio toward a 50% orientational
preference; therefore, the values given in Table II represent minimum
values for the conformational preference.

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Fig. 4.
CrADP titration of 1. The left-hand peak
is the peak labeled in Table I. Protein and 1 concentration is 300 µM. The spectra represent no CrADP
(a), 28 µM CrADP (b); 56 µM CrADP (c).
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Fig. 5.
CrADP titration of 6. The
left-hand peak is the peak labeled in Table I. Protein
and (6) concentration is 300 µM. The spectra
represent no CrADP (a), 28 µM CrADP
(b); 56 µM CrADP (c).
|
|

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Fig. 6.
Line widths measured for the CrADP titration
of 6. Data are from Fig. 5. Triangles,
P ; squares, P . The lines are
best fit lines to the data, with gradients of 1.2 and 0.58, respectively. The ratio
r /r is then
obtained as (1.2/0.58) 1/6 = 0.89.
|
|
The ligands were designed to have a similar electronic distribution to
1,3-BPG and 3-PGA in having either a simple ketone or an amide group in
analogous positions to the substrates (Table I). Therefore it was expected that the
non-symmetrical ligands would bind such that the carbonyl oxygen would
occupy a location similar to that of the carboxylate of 3-PGA. The
results presented in Table II for the
nonfluorinated analogs 1, 4, and 5 show that they do indeed bind preferentially in the expected
orientation but that the conformational preference is not strong.
We have previously shown that substitution of the methanephosphonate by
the
,
-difluoromethanephosphonate group almost always gave a large
increase in affinity for PGK (18). (One of the few exceptions is
7, which is mainly in the diol form and therefore has steric
problems in binding.) This was true whether the fluorines were placed
at what was expected to be the 1-phosphate end (2) or the
3-phosphate end (3), yet substitution at both ends gave
binding that was no stronger and indeed often weaker. The results
(Table II) indicate that in all cases the difluoromethanephosphonate
group is positioned at the 3-phosphate end. This finding makes sense of
the affinities as discussed below.
 |
DISCUSSION |
A quantitative use of paramagnetic relaxation as done here
is only applicable if certain conditions are met. Many of the
NMR-specific requirements have been addressed by Gupta and co-workers
(25, 26), who showed in a study of pyruvate kinase that CrADP can be
used as a substitution-inert paramagnetic analog of MgADP and that the relaxation effects are dominated by the paramagnetism of the
chromium in a simple r
6
relationship. CrATP has been shown to be a suitable analog of MgATP
when binding PGK, and its effects on longitudinal relaxation rates were
used to probe substrate binding (27). It is also of course necessary
that CrADP binds at a single site on PGK. In this case chromium is in a
very similar position to magnesium (28, 29). Considerable debate has
occurred as to whether 1,3-BPG and its analogs bind at more than one
site on PGK (16), and it is now reasonably clear that at least in the
open form it only has a single major binding site (13). Finally, by
using a low concentration of both enzyme and CrADP along with tight
binding PGK ligands we can ensure that essentially all of the
paramagnetic relaxation of the ligand occurs while bound to the enzyme,
and no significant relaxation occurs in a CrADP·ligand binary
complex. This fact has been confirmed by measurements in the absence of enzyme (data not shown).
Apparent 31P-chromium distance ratios were obtained from
measuring the change in line width of the 31P signal with
the addition of CrADP. In all cases the ratios were substantially
smaller than the theoretical limit of 1.8. This result could in
principle be explained if the geometry of the bound 1,3-BPG analogs
relative to CrADP is very different from that of 1,3-BPG so that the
31P-chromium distance ratios become more similar. This
circumstance is unlikely. In PGK crystal structures (2-13) and in a
modeling study (30) the only significant change is a complete or
partial closure of the hinge between the two domains that brings the
transferable 1-phosphate closer to the bound nucleotide while
maintaining the same orientation and therefore makes the ratio
r
/r
greater not
smaller. The two most likely explanations for the reduced distance
ratios are either that CrADP or the analogs bind weakly in different
binding sites or that the 1,3-BPG analogs can bind in the correct
binding site but with both possible orientations. The first option can
be ruled out as discussed above. Thus, binding in two possible
orientations is the most likely explanation for the low
r
/r
ratio.
Equation 9 provides a crude but useful characterization of the
approximate conformational preference of the 1,3-BPG analogs tested.
The most striking result is that all of the
,
-difluoromethanephosphonate analogs bind with the more highly
charged difluoromethanephosphonate at the 3-phosphate end. This finding
is perhaps surprising in view of the location of the phosphate anion at
the 1-phosphate end in the T. brucei crystal
structure, but it allows us to interpret the ligand affinities in a
straightforward manner. We assume that the greater charge density
induced by the difluoro substitution is the main driving force for the
conformational preference seen. It may also be the case, however, that
the fluorines can act as mimics of the lone pairs of the
phosphate-bridging oxygen of the substrate and participate in hydrogen
bonds to the enzyme, as seen in a crystal structure of glycerol
phosphate bound to AMP-PCF2P (31). However, the result does
agree with the long standing findings that the 3-phosphate groups
should be doubly ionized to bind effectively (32) and that the binding
of the carboxylate of 3-PGA is less important (33). We note that the
strongest conformational preference was for 3, which has
the amide functionality in a "substrate-like" location.
The results indicate that the location of the
difluoromethanephosphonate dominates the orientation of binding and
that any functionalization of the carbon chain between the phosphorus
atoms has only a secondary effect. Thus, a
difluoromethanephosphonate group at the 3-phosphate end is always
favorable and will make the ligand bind in this orientation even if
this means disrupting other substrate-like interactions elsewhere. The
effect can be seen from a comparison of 2 and 3,
both of which bind with the fluorines at the 3-phosphate end, despite
putting the carbonyl group of 2 in the "incorrect"
location. However, such "incorrectness" only reduces the affinity
from 2 µM (3) to 6 µM
(2). This conclusion makes sense of our previous study (18),
which showed that substitution by a single difluoromethanephosphonate always improved affinity but that substitution of
difluoromethanephosphonates at both ends of the ligands conferred no
additional affinity and often reduced the affinity. We infer that the
presence of a difluoromethanephosphonate group at the transferred
1-phosphate position must be unfavorable either for steric reasons or
because the greater charge density on the difluoromethanephosphonate
cannot easily be stabilized in the open form of the enzyme. The enzyme
functions by stabilizing the developing negative charge on the
transferring phosphate in the closed form (11), and therefore we
suggest that inhibitors should only have a charge of
1 at the
1-phosphate end but a charge of
2 at the 3-phosphate end.
Our results suggest that the basic patch is important mainly as a
region of high positive charge to locate the 3-phosphate of the
substrate (34). This finding may explain why mutations in the basic
patch have relatively little effect on the kinetics of the reaction (1,
35).
In summary, we have shown that the results obtained on the orientation
of PGK ligands allow us to interpret the affinities measured
previously. We suggest that an effective PGK inhibitor should have a
high charge density at the non-transferred 3-phosphate end but a lower
charge density at the transferred 1-phosphate end. Steric restrictions
at the 3-phosphate end are not strong. We have not identified strong
requirements for functionality within the ligand, although
substrate-like functionality provides weak additional affinity.
 |
FOOTNOTES |
*
This work was supported by a studentship from the
Biotechnology and Biological Sciences Research Council (BBSRC)
(to D. L. J.) and BBSRC Grant GR/H/32780. The Krebs
Institute is a BBSRC Biomolecular Sciences Centre.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.
¶
Current address: College of Pharmacy, 5968 College St.,
Dalhousie University, Halifax, Nova Scotia B3H 3J5, Canada.
Current address: Environment Australia, GPO Box 787, Canberra
ACT 2601 Australia.
**
To whom correspondence should be addressed: Dept. of Molecular
Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Tel.: 44-114-222-4224; Fax: -44-114-272-8697; E-mail: m.williamson@sheffield.ac.uk.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211769200
 |
ABBREVIATIONS |
The abbreviations used are:
PGK, yeast
phosphoglycerate kinase;
1, 3-BPG, 1,3-bisphosphoglycerate;
3-PGA, 3-phosphoglyceric acid;
AMP-PCF2P,
,
-difluoromethyleneadenosine 5'-triphosphate;
mgATP, magnesium
complex of ATP.
 |
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.