From the
Biological NMR Centre, Department of
Biochemistry, University of Leicester, P.O. Box 138, University Road,
Leicester LE1 9HN, United Kingdom, the
Department of Mathematics and Physics, The Royal
Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871
Frederiksberg C, Denmark, and ¶The Oxford Centre
for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road,
Oxford OX1 3QY, United Kingdom
Received for publication, February 13, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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![]() |
INTRODUCTION |
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The structures of several MBLs have been solved by x-ray diffraction and
reveal two potential zinc ion binding sites at the active site
(616).
The zinc ligands are not fully conserved between the different subclasses of
MBL. In the subclass B1 enzymes, such as the B. cereus enzyme BcII,
which is the subject of the present work, the zinc in site 1 is coordinated by
the imidazole rings of three histidine residues (86, 88, and 149 in the B.
cereus enzyme or 116, 118, and 196 using the class B -lactamase
(BBL) numbering (17), given in
parentheses henceforth) and a solvent molecule. In site 2, the metal is
coordinated by a histidine (210, BBL263), an aspartic acid (90, BBL120), a
cysteine (168, BBL221), and one or two solvent molecules. The two metal ions
are relatively close to each other, but the apparent distance between them
ranges from 3.4 to 4.4 Å in different structures of the BcII and CcrA
(Bacteroides fragilis) enzymes
(811,
13,
15,
16). Several structures of the
CcrA enzyme show a bridging ligand
(9,
11,
13) between the two metals,
suggested to be an hydroxide ion; however, a bridging solvent molecule is not
universally present in structures of this enzyme
(7,
14). In a structure of BcII
containing two zinc ions determined at pH 7.5, a similar bridging solvent
molecule is seen (15), but in
structures of this enzyme at lower pH, this solvent molecule is much more
closely associated to the zinc in site 1 than to that in site 2
(8,
10). The second solvent
molecule at site 2 is carbonate
(8) or water
(7,
911,
13) but is missing in one
structure (15) (as well as in
structures with inhibitors bound)
(14,
16). The coordination of the
metal ions is thus quite variable, perhaps contributing to some of the
observed differences in substrate profiles and zinc affinities among MBLs
(1820).
The bridging hydroxide ion or water molecule has been proposed to be the
nucleophile responsible for
-lactam hydrolysis, but the precise role of
the two metals in catalysis remains unclear; mechanisms have been proposed in
which only site I plays a direct role in catalysis
(21) or in which the two zinc
ions are both involved as a binuclear center
(2,
12,
20). The BcII enzyme is active
with either one or two zinc ions bound, however, with different kinetic
characteristics (15,
22).2
Inhibitors of the class A serine -lactamases, such as clavulanic
acid, have been very widely used to protect penicillins from
-lactamase-mediated hydrolysis. However, since MBLs are resistant to all
clinically used serine
-lactamase inactivators, the search for a
clinically useful inhibitor of MBLs remains an important objective. We have
recently reported (23) that
thiomandelic acid (
-mercaptophenylacetic acid) is a broad spectrum and
reasonably potent inhibitor of MBLs. Structure-activity relationships show
that the thiol is essential for activity, and it was postulated that the
inhibitor thiol binds to the zinc ions. We now report direct studies of the
interaction of thiomandelate with the active site metals in
cadmium-substituted B. cereus MBL. In the majority of MBLs, zinc can
be exchanged with cadmium to yield catalytically active enzymes
(15), and in the case of CcrA
the structure of the cadmium-substituted enzyme has been shown to be
essentially identical to that of the zinc enzyme
(13). Isotopes of cadmium
provide very convenient probes for NMR and PAC spectroscopy, allowing direct
studies of the coordination and dynamics of the metal ion. We have reported
the use of a combination of NMR and PAC spectroscopy to study cadmium binding
to B. cereus MBL, revealing rapid intramolecular exchange of the
metal between the two sites in the monocadmium enzyme and negative
cooperativity in metal binding
(24). We have now used a
similar combination of 1H, 15N, and 113Cd NMR
and 111mCd PAC spectroscopy to study the interaction of the
R and S forms of thiomandelate with the metal ions in
cadmium-substituted B. cereus MBL.
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EXPERIMENTAL PROCEDURES |
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NMR SpectroscopyFor the NMR studies, the cadmium enzyme was
prepared at room temperature, by adding gradually a small volume (110
µl) of 0.1 M113CdCl2 (95.83% enriched) to
the -lactamase apoenzyme (0.81.8 mM) in 10
mM MES-Na, 100 mM NaCl, pH 6.4, 5%
2H2O to give [cadmium]/[enzyme] ratios of 1.02.0.
Samples of the inhibitor complexes were prepared by the addition of microliter
volumes of stock inhibitor solution (50 mM in 0.5 M MES
buffer, 100 mM NaCl, pH 6.4) to the enzyme solution. The final
sample volume was 450 µl, and the temperature during acquisition was 298 K.
The same enzyme preparation and similar sample preparation procedures were
used in PAC spectroscopy (see below).
All NMR experiments were performed on Bruker Avance DRX/DMX instruments. 1H spectra were obtained at 600 MHz by using a waterflip-back pulse combined with Watergate (26, 27). Backbone NH resonances were observed by 1H,15N heteronuclear single quantum coherence (HSQC) with States-TPPI and Watergate. Observation of imidazole 15N(C)H resonances was by 1H,15N HMQC (heteronuclear multiple quantum coherence) with a refocusing delay of 16.7 ms (1/2JN(C)H), as described previously (25). The same pulse sequence was used for 1H,113Cd HMQC with 10, 16.7, or 33.3 ms (1/2JCd-H) as refocusing delay. Selective decoupling experiments were performed using G3 Gaussian cascade shaped pulses within an MLEV sequence (28). All 113Cd experiments were performed at 133 MHz. The external reference was Cd(ClO4)2. For the direct detection of 113Cd, 100,000 free induction decays were recorded, acquiring 4096 complex points in 51 ms, with a 30° observe pulse (4 µs), a spectral width of 80,000 Hz (600 ppm), and a relaxation delay of 0.5 s. A 200-Hz exponential multiplication was used prior to Fourier transformation, followed by base-line correction.
PAC SpectroscopyThe necessary amounts of the nuclear probe
111mCd (49-min half-life) were produced by a 24-MeV
particle bombardment of 98.9% enriched metallic 108Pd (Oak Ridge
National Laboratory). The radiochemical separation of the target and the
purification of 111mCd have been described previously
(29). The resulting
carrier-free solution (50 µl) was split in two with volume ratios of 1:4,
allowing two consecutive PAC counting sessions with equal statistics to be
performed from one 111mCd production batch. The tracer solution for
each experiment was subsequently adjusted to the desired cadmium concentration
by the addition of small volumes of CdCl2 solution in
metal-depleted MES buffer. The added amounts of cadmium (0.25190 nmol)
were large compared with both the 111mCd content and other
contaminating metals in the solution, even at the lowest concentrations used
([Cd]/[enzyme] = 0.01). Control experiments indicated that any zinc
contamination was <0.005 molar equivalent. The apoenzyme was added, giving
a final enzyme concentration of 0.5 mM in the PAC samples. The pH
was adjusted to pH 7.0 (room temperature) by the addition of small amounts of
15 M HCl or NaOH. The final sample conditions were 50
mM HEPES, 130 mM NaCl, with volumes ranging from 50 to
200 µl. After a 10-min waiting period at room temperature, to ensure
equilibrium for metal binding to the enzyme, pure crystalline sucrose (ACS
reagent from Sigma) was added to give a 55% (w/w) solution in order to slow
the rotational diffusion of the enzyme. The PAC sample preparation (tracer and
metal addition, incubation, pH adjustment, and sucrose addition) was done
immediately before each measurement, allowing a direct transfer of the sample
to the temperature-controlled environment of the PAC sample holder. Unless
otherwise noted, PAC experiments were performed at 1 °C.
The differential time PAC spectrometer
(30) uses six 2''
BaF2 scintillation detectors located in a face-centered cube around
the sample to absorb the 151 + 245-keV -cascade of the
111mCd decay. The FWHM time resolution of the instrument is 1.0 ns,
the single detector count rate is
60 kcps, and the total data acquisition
lasts 100200 min, giving 50100 million recorded
coincidences.
PAC Data AnalysisThe perturbation function,
G2(t), of the angular correlation pattern is
derived from the measurements as described previously
(2932).
This function depends on the local electric quadrupole interaction at the site
of the cadmium nucleus and contains the structural information about the
co-ordination geometry that can be deduced from a PAC experiment. In the case
of identical, static, and randomly oriented molecules, the perturbation
function denoted G2(t) can be expressed as
follows,
![]() | (Eq. 1) |
In the liquid state, the NQI in itself is time-dependent because of the
Brownian motion of the protein, giving an exponential damping of the observed
correlation pattern with a time constant equal to the rotational diffusion
time R. Additional time-dependent variation in the local NQIs
can be induced by, for example, an exchange process between two coordination
geometries. A distribution of coordination geometries is allowed for by
introducing a Gaussian frequency spread
=
0/
0. The perturbation function
A2G2(t), where
A2 is the amplitude, was analyzed by a
conventional nonlinear least-squares fitting routine, giving the frequency
0, the asymmetry
, the relative Gaussian frequency
distribution
, and the rotational diffusion time
R for
each of the observed NQIs.
In all cases where more than a single NQI is observed, the perturbation
function is assumed to be the sum of the individual perturbation functions
weighted by a population factor
(30). However, the fitting
procedure will not always succeed in giving all five parameters for each of
several NQIs in a given spectrum. Additional constraints are added to assist
the fitting in these cases. In the case of experiments with
S-thiomandelate, it was possible (judged by acceptable values for the
reduced 2 function) to fit the spectra obtained at different
[cadmium]/ [enzyme] ratios simultaneously. This global fit gave four distinct
NQIs, each having individual
0 and
values, but with
identical values of
R and
. Each
S-thiomandelate measurement is assumed to contain these four NQIs
with different amplitudes (possibly zero). The spectrum obtained at
[cadmium]/[enzyme] = 1.9 must necessarily arise primarily from the binuclear
enzyme, since no free cadmium is observed, with both sites almost equally
populated, and it was thus fitted under the constraint of equal amplitude, and
the amplitudes of the mononuclear NQIs were fixed to zero. The goodness of the
fit was judged by the overall
2 value.
The fitting procedure was more complicated in the case of the spectra
obtained in the presence of R-thiomandelate, where distinct dynamical
features in the 1 °C spectrum prohibit an overall fit of all
stoichiometries. The spectra of R-thiomandelate-inhibited samples at
20 °C and +30 °C were fitted assuming four NQIs, two of which
are found and fitted at 30 °C and three at 20 °C. The
temperature difference introduces differences in R, and this
parameter was not constrained in the fitting process. We assumed the same
value of
as found in the S-thiomandelate experiments in these
fits.
Molecular ModelingThe starting point for the modeling was the crystal structure of the B. cereus enzyme containing two zinc atoms (8) (Protein Data Bank accession number 1bvt [PDB] ) with hydrogens added using the AMBER package (33) and the bicarbonate ion and water molecules coordinating the zinc ions removed; the charges were determined for all atoms using the program Divcon with a PM3 Hamiltonian coupled with a dielectric continuum model for the solvent effect (34, 35). The ligands R- and S-thiomandelate were built using AMBER, and the geometry optimization and charges determination were conducted using Divcon. Docking was carried with AutoDock (36), allowing flexibility about rotatable bonds of the inhibitor using the AutoTors utility but keeping the protein rigid. A large population size of 200 was used to ensure that conformational space was exhaustively searched; the maximum number of energy evaluations was set to 0.5 million (in tests, using 1 or 3 million did not improve the results), the number of trials was 200, the number of generations was 0.04 million, and all other parameters were set according to the defaults in the AutoDock manual (36). In additional test calculations, thiomandelate was docked into structures where Lys171 is in an extended conformation (see "Discussion"); the inhibitors were removed from the published structures, and the protein and ligand were parameterized as described by Hanessian et al. (37).
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RESULTS |
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Effects of Thiomandelate Binding on the 113Cd Spectrum The addition of one molar equivalent of either R-or S-thiomandelate to the dicadmium enzyme leads to a substantial downfield shift of both of the 113Cd resonances (Fig. 2). The addition of 0.5 molar equivalents of thiomandelate leads to a spectrum containing both resonances corresponding to the free enzyme and those corresponding to the complex, with approximately equal intensities, indicating that thiomandelate binding is in slow exchange on the 113Cd NMR time scale. The further addition of the inhibitor from 1 to 2 molar equivalents does not change the NMR spectra. The direction and magnitude of the inhibitor-induced change in chemical shift is consistent with that expected for the coordination of a sulfur atom to each of the cadmium ions and hence with the idea that the inhibitors bind directly to both metal sites.
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Direct evidence for this and the assignment of the two 113Cd
resonances in the inhibitor complexes can be obtained from
1H,113Cd HMQC spectra, shown for the
R-thiomandelate complex in Fig.
3. Both the 113Cd and the imidazole 1H
resonances of the inhibitor complexes are sharper than those of the free
enzyme, and cross-peaks are observed from the 343 ppm 113Cd
resonance for three imidazoles, His88, His86, and
His149 (BBL118, -116, and -196). (The latter two histidines have
not been individually assigned in the cadmium-substituted enzyme but can be
tentatively assigned, as shown in Table
I, by comparison with the assigned resonances in the zinc enzyme
(23).) The 343 ppm
113Cd resonance can thus be unambiguously assigned to the cadmium
in site 1. There is also a strong cross-peak to a signal at 5.36 ppm, which
can be assigned to the CH (benzylic proton) of the bound
R-thiomandelate (chemical shift 4.72 ppm for the free inhibitor in
C2HCl2). This inhibitor resonance also gives a strong
cross-peak to the low field 113Cd resonance
(Fig. 3), which in turn shows
cross-peaks to the imidazole protons of His210 (BBL263) and the
-proton(s) of Cys168 (BBL221) and can thus be assigned to the
metal in site 2. The observation that, in a complex containing 1 molar
equivalent of inhibitor, the R-thiomandelate resonance at 5.36 ppm
gives a cross-peak in the 1H,113Cd HMQC spectrum to both
113Cd resonances is clear evidence that R-thiomandelate
coordinates through its sulfur atom to both cadmium ions.
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This argument is further strengthened by detailed analysis of the resonance at 5.36 ppm in the 113Cd-edited 1H spectrum of the R-thiomandelate complex. This resonance is shown in Fig. 4, where it can be seen to consist of four lines. These could in principle arise from two doublets,5 reflecting two separate thiomandelate-cadmium interactions, or a doublet of doublets, reflecting the simultaneous interaction of one thiomandelate with both cadmium ions; these two possibilities can be distinguished by 113Cd decoupling experiments. Broadband 113Cd decoupling gives rise to a single 1H resonance, not the two separate resonances that would be predicted for two separate thiomandelate-cadmium interactions. Second, selective 113Cd decoupling at 343 ppm leads to collapse of both the two outer pairs of lines, revealing a doublet of 3JH-Cd = 31 Hz, whereas selective decoupling at 372 ppm leads to collapse of the four-line pattern into a doublet of 3JH-Cd = 15 Hz. This behavior is exactly what would be predicted for a doublet of doublets arising from the interaction of one thiomandelate with both cadmium ions and provides unambiguous evidence that R-thiomandelate binds simultaneously to both cadmium ions in the enzyme. The 31-Hz 3JH-Cd scalar coupling can be assigned to the cadmium in site 2, and the 15-Hz scalar coupling can be assigned to the cadmium in site 1.
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In the 1H,113Cd HMQC spectra of the
S-thiomandelate complex (not shown), only a single correlation is
observed for the 1H resonance at 5.2 ppm assigned to the
thiomandelate CH, to the 113Cd resonance at 375 ppm assigned
to the cadmium in site 2, and the 3JH-Cd scalar
couplings are clearly much smaller and are not resolved in spectra such as
those in Fig. 4. In this case,
there is a degree of ambiguity in the 1H,113Cd HMQC
spectrum, since the imidazole resonance that appears at 5.2 ppm, upfield of
the thiomandelate C
H signal, in the R-thiomandelate complex
partly overlaps the latter signal in the S-thiomandelate complex.
However, the similar downfield shifts of both the 113Cd resonances
on the binding of both isomers of the inhibitor, in particular the large
downfield shift of the resonance of the cadmium in site 1, make it highly
probable that the sulfur of S-thiomandelate, like that of
R-thiomandelate, coordinates to both metals. The chemical shifts and
scalar couplings to the metal ligands in the free enzyme and the two inhibitor
complexes are summarized in Table
I.
Thiomandelate Binding to the Monocadmium EnzymeWe have
earlier reported that the 113Cd spectrum of the enzyme having 1
cadmium equivalent bound contains a single resonance. Comparison with PAC
spectra under identical conditions showed that this reflects a rapid
intramolecular exchange of the single metal between the two sites
(24). The addition of 1 molar
equivalent of either R- or S-thiomandelate to the
monocadmium enzyme led to a 113Cd spectrum having two resonances at
the same chemical shifts as those observed for the complexes of the dicadmium
enzyme (Fig. 5). Furthermore,
the 113Cd-edited 1H spectra of the
R-thiomandelate complex showed that the thiomandelate CH
resonance at 5.35 ppm had the same 1H,113Cd scalar
coupling as observed for the dicadmium enzyme (Fig. S1, Supplementary
Material), demonstrating that the thiomandelate-enzyme complex contains two
cadmium ions.
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With this being the case, given that the overall [cadmium]/[enzyme] ratio
is 1, the sample must also contain apoenzyme. This was confirmed by using the
1H,15N HSQC spectrum of the 15N-labeled
enzyme as a "fingerprint" of the different states of the enzyme
(Fig. 6). It is clear that the
spectrum of the sample containing enzyme, cadmium, and
R-thiomandelate in a 1:1:1 ratio contains more amide cross-peaks in
the HSQC spectrum than can be accounted for by the number of residues in the
protein, indicating that this sample must consist of a mixture of species.
Overlays with the spectrum of the apoenzyme
(Fig. 6A) and with the
spectrum of the R-thiomandelate complex of the dicadmium enzyme
(Fig. 6B) show
unequivocally that the sample contains a mixture of roughly equal amounts of
these two species. All the resonances in the spectrum of the sample containing
enzyme, cadmium, and R-thiomandelate in a 1:1:1 ratio can be
accounted for by the sum of the spectra of apoenzyme and of the
R-thiomandelate complex of the dicadmium
enzyme.6 No resonances
were observed that could correspond to a thiomandelate complex of the
monocadmium enzyme; we estimate that this complex would have been detected if
it had been present at levels corresponding to 5% of the total enzyme.
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These observations clearly demonstrate that the addition of thiomandelate to a sample of 1.8 mM enzyme containing cadmium at a [cadmium]/[enzyme] ratio of 1 leads to positive cooperativity in metal binding such that only the R-thiomandelate complex of the dicadmium enzyme (with a corresponding amount of apoenzyme) is observed. This is in marked contrast to the negative cooperativity in cadmium binding observed in the absence of inhibitor (24).
PAC Spectroscopy
The Enzyme-S-Thiomandelate
ComplexFig. 7 shows
the Fourier transforms of PAC spectra recorded at different [Cd(II)]/[E]
ratios in the presence of 1 molar equivalent of S-thiomandelate. The
results of fitting the three spectra for this complex simultaneously, as
described under "Experimental Procedures," are presented in Tables
II and
III. For the sample having
[Cd(II)]/[E] = 1.9 and 1 molar equivalent of S-thiomandelate, both
metal sites will be almost equally occupied, giving two NQIs of equal
amplitude (NQI-1 and NQI-2 in Table
II). Calculations using the angular overlap model (see
"Discussion") suggest that NQI-1 corresponds to cadmium in site 2
and that NQI-2 corresponds to cadmium in site 1 and are consistent with the
presence of a sulfur atom from the ligand in the coordination sphere of both
metals. The presence of the peak at 0.26 gigarads/s at all stoichiometries
shows that the binuclear cadmium enzyme must in fact be present in all cases.
The substantial changes as a function of [cadmium]/[enzyme] at 0.2
gigarads/s must reflect the presence of additional species, most likely
monocadmium enzyme(s), at lower stoichiometries. This is reflected in the
fitted parameters (Tables II
and III), where a substantial
proportion of NQI-4 is present at the lower stoichiometries (together with a
small amount of NQI-3 at the lowest stoichiometry). However, from
Table III it is clear that the
binuclear species (NQI-1 and NQI-2) still predominates even at [Cd(II)]/[E] =
0.01, indicating cooperativity in metal ion binding.
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The Enzyme-R-Thiomandelate ComplexThe Fourier transforms of
PAC spectra recorded at different [Cd(II)]/[enzyme] ratios in the presence of
1 molar equivalent of R-thiomandelate at 1 °C are shown in
Fig. 8. In this case, the
spectrum of the complex of the R-isomer at [Cd(II)]/[enzyme] = 1.9,
which should be a simple spectrum with two NQIs at equal abundance, could not
be satisfactorily fitted by the procedure used to analyze the corresponding
spectrum of the complex of S-thiomandelate, since a reduced
2 sum of 1.9 was obtained. The most likely explanation of this
failure to fit the spectrum with a simple model is the presence of dynamic
features (in addition to rotational diffusion). PAC spectra of a sample
containing enzyme, cadmium, and R-thiomandelate at a ratio of 1:1.9:1
were therefore obtained at temperatures of 20 and 30 °C
(Fig. 9). These two spectra
could be analyzed satisfactorily with three or two NQIs, respectively (Tables
IV and
V). In the spectrum obtained at
20 °C, two of the three NQIs (NQI-2 and NQI-3 in Tables
IV and
V) together account for about
50% of the spectral intensity. This suggests that in the complex of
R-thiomandelate with the binuclear cadmium enzyme, one of the two
metal sites exists in an equilibrium between two coordination geometries that
interconvert with a rate sufficiently slow at 20 °C to produce two
static NQIs. In agreement with this hypothesis, the spectrum at 30 °C can
be explained by the sum of only two NQIs, each accounting for
50% of the
intensity. NQI-1 at 30 °C is the same as NQI-1 at 20 °C,
whereas NQI-4 at 30 °C is intermediate between NQI-2 and NQI-3 at
20 °C. This suggests that at 30 °C, the rate of interconversion
between the two coordination geometries has increased to a value such that
only one NQI is observed as the average of the two NQIs detected at 20
°C, making an interesting PAC analogue of the effects of chemical exchange
in NMR. Note that the fitted value of
0 for NQI-4, from the
30 °C spectrum, is identical to the numerical average of the fitted values
for NQI-2 and NQI-3 from the 20 °C spectrum. At 1 °C, the rate
appears to be in an "intermediate exchange" range, and the
spectrum could be fitted as a broadening of the average NQI fitted at 30
°C. In addition to these dynamic complexities, the spectra in
Fig. 8 demonstrate that, for
the complexes of R-thiomandelate as for those of
S-thiomandelate discussed above, weak new features appear at low
metal ion stoichiometry, which we attribute to one or more species of the
mononuclear complex.
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![]() |
DISCUSSION |
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The NMR data reported in the present paper provide unequivocal evidence
that R-thiomandelate indeed binds through its thiolate sulfur to both
the metal ions in the cadmium-substituted enzyme. The observation in the
1H,113Cd HMQC spectrum of cross-peaks from both cadmium
signals to the resonance of the -proton of R-thiomandelate,
together with the quartet structure of this proton resonance, demonstrates
that each cadmium has a molecule of R-thiomandelate bound to it. The
results of the selective cadmium decoupling experiment demonstrate clearly
that a single R-thiomandelate
-proton is scalar coupled to
both cadmiums and hence that a single R-thiomandelate molecule binds
to the enzyme, binding to both metals simultaneously. The inhibitor clearly
binds to the metals through its sulfur, as demonstrated by the very large (up
to 200 ppm) downfield shift of the 113Cd resonances on inhibitor
binding, characteristic of sulfur coordination, and by the observation of
JH-Cd scalar couplings of 1530 Hz, typical of
three-bond couplings through the sulfur rather than the four-bond couplings
that would be involved if the binding was through the inhibitor's carboxylate
group.
With the information that the sulfur of R-thiomandelate
coordinates to both cadmium ions, we attempted, as described under
"Experimental Procedures," to produce a model for the complex by
computationally docking the inhibitor into the crystal structure of the B.
cereus enzyme with two zinc ions bound
(8), assuming that, as for the
B. fragilis enzyme
(13), the structures of the
cadmium and zinc enzymes are closely similar. Initial attempts failed to
identify a low energy structure in which the inhibitor sulfur was bound to
both metals. Examination of the structures obtained indicated that the side
chain of Lys171 (BBL 224), which interacts with the carboxylate
group of the inhibitor, plays a crucial
role.7 In the recent
crystal structure of a mercaptocarboxylate inhibitor bound to the IMP-1 MBL
(16), in which the sulfur of
the inhibitor binds to both metals, the side chain of the corresponding
Lys161 is essentially fully extended. We therefore repeated the
docking calculations after changing the side chain of Lys171 to an
extended conformation and obtained a low energy structure in which the sulfur
was bound to both metals; this is shown in
Fig. 10. The sulfur atom of
the inhibitor is positioned essentially equidistant from the two metals,
whereas one oxygen of the carboxylate group of the inhibitor forms a good
hydrogen bond with the of
Lys171 and the other interacts with the zinc in site 2. Docking
thiomandelate into other structures where Lys171 is in an extended
conformation (14,
16) supports this binding
mode. Thus, it appears that the inhibitor displaces both water molecules bound
to the metals in the free dizinc enzyme; its sulfur atom displaces the
"bridging" water, whereas the carboxylate displaces the additional
water (or carbonate ion in one structure)
(8) bound to the metal in site
2. Whereas this is only a model (it does not, for example, reflect the changes
in the
3-
4 loop on inhibitor binding)
(23), it is likely that it
contains some of the essential features of R-thiomandelate binding.
Thus, in the model, H
of the bound R-thiomandelate is 1.95
Å from H
of histidine 149, and a strong nuclear Overhauser effect
between these two protons is indeed
observed,8 suggesting
that the position of R-thiomandelate relative to the metal ions shown
in Fig. 10 is basically
correct.
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The S-isomer of the inhibitor also appears to bind to both metals
simultaneously, although in this case the evidence, while strong, is not
entirely unequivocal. Only one of the two cadmium resonances, assigned to the
metal in site 2, shows a cross-peak to the thiomandelate -proton
resonance in the 1H-113Cd HMQC spectrum. However, the
resonance from the cadmium in site 1 also shows a very large downfield shift
(140 ppm) on S-thiomandelate binding, with the direction and
magnitude expected for coordination of a sulfur atom and almost as large as
that seen on the binding of R-thiomandelate. Further support for the
idea that the sulfur of S-thiomandelate binds to both cadmium ions comes from
calculations of the NQIs using the angular overlap model (AOM)
(38), the results of which are
shown in Fig. 11. Site 1 was
modeled with tetrahedral geometry with three histidine ligands and a
mercaptosulfur ligand; from Fig.
11A one can observe that AOM calculations for this site
fit reasonably well with the NQIs with values for
0 of about
200 megarads/s, although
is somewhat high. Site 2 was modeled either
with tetrahedral geometry with a histidine, an aspartic acid, a cysteine and a
mercaptosulfur ligand (Fig.
11B) or with a five-coordinate geometry including a water
ligand positioned opposite the histidine ligand
(Fig. 11C). For site
2, the calculations for the five-coordinated geometry fit the data well. Thus
AOM calculations support the idea that the mercaptosulfur of
S-thiomandelate binds both cadmium ions.
|
We thus conclude that both isomers of this simple inhibitor bind through
their sulfur atom to both cadmium atoms simultaneously. There are differences
between the two isomers in their precise mode of binding, manifest in the
different parameters for the cadmium NQIs derived from the PAC spectra of the
two inhibitor complexes of the dicadmium enzyme, in the different
113Cd chemical shifts, and in the 1H and 15N
chemical shifts of the metal ligands. These differences most probably reflect
differences in the cadmium-sulfur bond lengths and angles in the two
complexes, imposed by the necessarily different interactions of the two
isomers with nearby protein residues, but the chemical shifts are not
sufficiently well understood to allow us to draw clear cut structural
conclusions. The docking calculations for S-thiomandelate (not shown)
suggest that for this isomer, unlike the R-isomer shown in
Fig. 10, the carboxylate group
binds to Lys171 (BBL 224) but not to the metal in site 2, so that
in this complex the fifth ligand to the cadmium in this site may be a water
molecule. This difference would certainly be expected to lead to differences
in cadmium and imidazole chemical shifts. The
3JH-Cd scalar coupling constants to the
inhibitor C proton are notably different between the complexes with
R-thiomandelate (15 and 31 Hz) and S-thiomandelate (<15
Hz). The magnitudes will depend on the orientation of the inhibitor in the
complex, through the dihedral angle about the thiomandelate C
S
bond, but also on the electron distribution on the metals and their ligands,
and clear-cut relationships to conformation can rarely be discerned
(25,
39). We earlier noted that the
changes in backbone amide 1H and 15N chemical shifts on
inhibitor binding to the zinc enzyme are generally very similar for the two
isomers (23), suggesting that
the structural differences between the two complexes are local ones.
A striking difference between the two inhibitor complexes formed with
R- and S-thiomandelate is the observed influence of a dynamic process
at one of the two metal sites on the PAC spectra of the
R-thiomandelate complex. This was unambiguously established by the
temperature dependence of the spectra but was not observed for the
S-thiomandelate complex. This may reflect a local conformational
difference between the R- and S-thiomandelate complexes such
that a dynamic process occurs uniquely in the former. On the other hand, it
may also be that the same process occurs in both complexes but much more
rapidly (with a time constant less than 1 ns corresponding to rates of
>109 s1) in the
S-thiomandelate complex so that only an average is observed. The
nature of this dynamic process cannot be identified with certainty, but some
possibilities can be ruled out. First, the fact that the
1JH-Cd scalar couplings are observed for all of
the histidine metal ligands (and cross-peaks are observed for these and for
Cys168 in the 1H,113Cd HMQC spectrum) in the
R-thiomandelate complex rules out the possibility that any of these
are dissociating from and reassociating to the metals at rates >90
s1, much too slow to produce dynamic effects in
the PAC spectra. Furthermore, the observation of two
3JH-Cd scalar coupling constants of 15 and 31
Hz between the metals and the thiomandelate -proton, together with the
analysis of the PAC spectra demonstrating that only one of the two metals is
affected by this dynamic process, rules out the possibility that the thiol
group of the inhibitor is "jumping" between the two metals at
>10 s1. One possibility is that it arises from
the dissociation and reassociation of the carboxylate group of the inhibitor,
which our modeling suggests binds to the cadmium in site 2 in the
R-thiomandelate complex but not in that of the S-isomer.
Comparison of the results of the AOM calculations for site 2 in
Fig. 11, B and
C, illustrates the change in the frequency parameter
0 that would arise from such an exchange of an oxygen
ligand. However, this simple model does not account quantitatively for the
measured NQIs of the R-thiomandelate-enzyme complex, indicating that
other changes in the coordination sphere must occur in this dynamic
process.
One of the most striking observations in the present work is the clear
demonstration of positive cooperativity in metal binding in the presence of
either isomer of the inhibitor. We have recently demonstrated that in the
absence of the inhibitor there is negative cooperativity in cadmium binding to
BcII from strain 569/H/9 (24).
The two macroscopic dissociation constants for cadmium binding differ by a
factor of 1000 (26), but
at [cadmium]/[enzyme] < 1, the PAC spectra show that the metal occupies two
sites with roughly equal probability. Thus, the two macroscopic constants do
not reflect metal binding to two distinct sites with different intrinsic
affinities but rather reflect negative cooperativity in metal binding in the
absence of inhibitor. Studies of the wild-type and mutant enzyme by optical
spectroscopy and extended x-ray absorption fine structure experiments
indicated that there is a similar negative cooperativity in the binding of
zinc and cobalt to the enzyme
(40). However, the present
experiments demonstrate that in the presence of the inhibitor thiomandelate
there is positive cooperativity in cadmium binding. NMR experiments carried
out at an enzyme/cadmium/inhibitor ratio of 1:1:1 demonstrated that the sample
contained an approximately equimolar mixture of the inhibitor complex of the
binuclear cadmium enzyme and apoenzyme. These were clearly identified from
their characteristic 1H,15N HSQC spectra, which also
showed that any other species, such as enzyme with only one cadmium bound,
represented
5% of the enzyme. These observations cannot be explained by
equal macroscopic dissociation constants for binding the two cadmium ions in
the presence of the inhibitor. PAC spectra were obtained over a wide range of
[cadmium]/[enzyme] ratios in the presence of S-thiomandelate, and
analysis of these spectra showed that even at a [cadmium]/[enzyme] ratio as
low as 0.01, the binuclear cadmium enzyme represented 64% of the total cadmium
present, again inconsistent with equal Kd values
for the two metals and indicating a marked cooperativity in metal binding. The
observed relative amounts of mono- and binuclear cadmium species as a function
of [cadmium]/[enzyme] ratio (from 0.01 to 2.0) can only be satisfactorily
simulated by a macroscopic dissociation constant for the binding of one
cadmium ion about 100-fold greater than that for the binding of the second
cadmium ion. The observed positive cooperativity clearly results from the fact
that the inhibitor coordinates simultaneously to both metals, and one would
thus expect a similar cooperativity with the physiological metal zinc,
although direct measurements of this do not exist.
Recent measurements suggest that at physiological metal ion concentrations,
most metallo--lactamases may exist as the apoenzyme and that the
presence of substrate leads to enhanced zinc binding in the mononuclear form
of the enzyme (41). The
present experiments suggest a parallel in the sense that the addition of the
inhibitor markedly changes the relative populations of the mono- and binuclear
enzyme forms. Depending on the metal concentration in the milieu in which the
enzyme exists (cf. Ref.
41), the requirement for two
metal ions for optimal inhibitor binding might be considered a disadvantage of
this class of inhibitors for clinical applications. However, the existence of
marked inhibitor-induced cooperativity in metal ion binding that we have
demonstrated here means that, even at low metal concentrations, the binuclear
enzyme will be a substantial proportion of the total, and the catalytic
activity will be effectively inhibited by converting the mononuclear enzyme
into a mixture of inhibited binuclear enzyme and inactive apoenzyme.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains an additional figure.
|| To whom correspondence should be addressed. Tel.: 44-116-252-2978; Fax: 44-116-223-1503; E-mail gcr{at}le.ac.uk.
1 The abbreviations used are: MBL, metallo--lactamase; BBL, class B
-lactamase; MES, 4-morpholinoethanesulfonic acid; NQI, nuclear
quadrupole interaction; AOM, angular overlap model.
2 C. Damblon, V. Nandwani, and G. C. K. Roberts, unpublished data.
3 C. Damblon and G. C. K. Roberts, manuscript in preparation.
4 This resonance is assigned to the cysteine rather than to the aspartate in
site 2, since the -protons of the cysteine are separated from the
113Cd by three bonds rather than the four in the case of the
aspartate.
5 Two thiomandelate CH resonances with different chemical shifts, each
split into a doublet by coupling to one 113Cd; either two doublets
of 31 Hz separated from one another by 0.113 ppm (15 Hz) or two doublets of 15
Hz separated from one another by 0.233 ppm (31 Hz).
6 Three or four cross-peaks of the sample containing enzyme, cadmium, and
R-thiomandelate in a 1:1:1 ratio show slight differences in chemical
shift from the obviously corresponding cross-peaks in the spectrum of the
apoenzyme. This can most probably be ascribed to slight differences in the
conditions used for recording the two spectra.
7 These calculations indicate that our earlier suggestion
(25) that the carboxylate of
thiomandelate might interact with Arg91 is incorrect; it was not
possible to find a low energy structure in which this interaction was formed,
and the large change in amide NH chemical shift observed for Arg91
(25) must be an indirect
effect, perhaps via the metal ligand Asp90.
8 C. F. Damblon, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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