'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site

Article abstract of DOI:10.1021/ja500616m

The binding of three closely related chelators: 5-hydroxy-2-methyl-4H- pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human ca

Full text of DOI:10.1021/ja500616m

Article
pubs.acs.org/JACS
‘
Unconventional’ Coordination Chemistry by Metal Chelating
Fragments in a Metalloprotein Active Site
†
†
‡
‡
ar A. F. de Oliveira,∥
́
David P. Martin, Patrick G. Blachly, Amy R. Marts, Tessa M. Woodruff, Ces
J. Andrew McCammon,† David L. Tierney,* and Seth M. Cohen*
,§,∥
,‡
,†
†
§
∥
Department of Chemistry and Biochemistry, Pharmacology, and Howard Hughes Medical Institute, University of California, San
Diego, La Jolla, California 92093, United States
‡
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States
S Supporting Information
*
ABSTRACT: The binding of three closely related chelators:
-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol,
5
ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol,
TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic
acid, TPMA) to the active site of human carbonic anhydrase
II (hCAII) has been investigated. Two of these ligands display
a monodentate mode of coordination to the active site Zn2+
ion in hCAII that is not recapitulated in model complexes of
the enzyme active site. This unprecedented binding mode in
the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the
steric restrictions of the active site force the ligands into a ‘flattened’ mode of coordination compared with inorganic model
complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of
the metal−ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be
significantly influenced by the protein active site. Diminishing the strength of the metal−ligand bond results in unconventional
modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and
understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.
10−16
INTRODUCTION
sites.
Although this approach can be effective, the
■
constrained interactions and nuances of a metalloprotein active
site cannot be readily recapitulated in such model scaffolds.
Several examples of metalloprotein active sites influencing
metal−ligand coordination have been reported. Specifically,
changes in coordination caused by interactions with the
surrounding active site have been observed with inhibitors of
carboxypeptidase A based on the N-hydroxyurea MBG as well
as inhibitors of thermolysin that utilize an α-mercaptoketone
Metalloproteins represent a significant fraction of the human
proteome, and many represent important therapeutic targets.
1
With respect to the latter, a large number of metalloprotein
inhibitors have been developed, with clinically approved
2
+
inhibitors available for the Zn -dependent histone deacetylases
HDACs), angiotension converting enzyme (ACE), and
(
2
,3
carbonic anhydrase (CA), among others. In the majority of
these examples, the small molecule inhibitors possess functional
groups that bind to the active site metal ion of the enzyme; a
relatively small selection of such groups, including carboxylates,
phosphates, and hydroxamic acids, are commonly employed as
the metal-binding groups (MBGs) of choice. Recently, a
number of efforts have focused on the development of
alternative MBGs to these commonly employed groups, and
indeed some newer metalloprotein inhibitors, such as
raltegravir and dolutegravir that target HIV integrase (HIV1
IN, Mg -dependent), employ more sophisticated heterocyclic
1
7,18
4
MBG.
In both cases, the changes in metal coordination are
the result of large aromatic groups on the inhibitor being
positioned to form significant interactions with hydrophobic
regions of the active site, and the coordination involved is
relatively weak.
5
6
Despite being a poor MBG for most metalloproteins, the
arylsulfonamide MBG (acetazolamide, Figure 1) dominates the
19
design of inhibitors of carbonic anhydrases (CAs). CA
2+
inhibitors are used in the treatment of glaucoma, epilepsy, and
3
7
−9
altitude sickness. The potency of sulfonamides for CAs is
MBGs.
These next-generation MBGs have the potential to
attributed largely to optimized protein−MBG interactions upon
binding the catalytic Zn2+ ion; the metal-bound amine and one
of the sulfonamide oxygen atoms both form strong hydrogen
bonds with nearby amino acid residues. In addition, the second
improve the potency, selectivity, and pharmacokinetics of
metalloprotein-targeted therapeutics.
As is the case with other forms of inhibitor and drug
development, the use of structure-aided design can be
invaluable to metalloprotein inhibitor design. In previous
efforts, inorganic model compounds have been utilized to
predict the binding of ligands to metalloprotein active
Received: January 23, 2014
Published: March 17, 2014
©
2014 American Chemical Society
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Article
concentration were plotted for three concentrations of inhibitor, and
the curves simultaneously fit for Ki using GraphPad Prism.
Representative examples of curve fitting are included in the Supporting
Information.
hCAII Crystallization. Crystals of hCAII were obtained by the
sitting-drop or hanging-drop vapor diffusion method. The protein
solution consisted of 20 mg/mL hCAII and 1 mM p-chloromercur-
ibenzoic acid in 50 mM Tris-SO , pH 8.0. The precipitant solution
4
contained 2.7−3.0 M (NH ) SO in 50 mM Tris-SO pH 8.15. Drops
4
2
4
4
consisted of 3 μL of protein solution plus 2.5−4.0 μL of precipitant
solution and were equilibrated at 18 °C against 750 μL of precipitant
solution. Crystals roughly 0.3 × 0.3 × 0.3 mm in size appeared after 2
days to 3 weeks. Once formed, crystals were transferred to 15 μL of
soak solutions containing inhibitor (at saturation, ∼1 mM), 1.5 M
sodium citrate, 50 mM HEPES pH 8.15, 5% glycerol, and 2−5%
DMSO. Crystals were taken directly from the soak solutions for data
collection. Due to potential interference from the high concentration
of DMSO necessary for ligand solubility, cocrystallization of the
ligands with hCAII was not attempted.
Crystal Structure Determination. X-ray diffraction studies on
hCAII crystals were carried out at 100 K with a Bruker D8 Smart 6000
CCD detector and utilizing Cu Kα radiation (λ = 1.5478 Å) from a
Bruker-Nonius FR-591 rotating anode generator. The data were
integrated and scaled using the Bruker APEX software suite. All
Figure 1. Structures of ATM, TM, TPMA, and several previously
reported hCAII inhibitors: acetazolamide, based on the arylsulfona-
mide MBG, 1,2-HOPTO, 2-mercaptophenol (2-MP) and its
methylated analog thioguaiacol (TG).
sulfonamide oxygen and the aromatic ring are positioned to
occupy the substrate binding pocket of the enzyme active site.
In order to elucidate the binding of MBGs to metalloproteins
as compared to model compounds, this report describes the
binding of a series of closely related heterocyclic chelators to
the active site of human carbonic anhydrase II (hCAII) and an
inorganic model complex. Because it has many characteristics
that make it a suitable model system including its rigid structure
and ease of crystallization, hCAII has been widely used to
crystals belonged to the monoclinic space group P2 . The data were
1
phased by molecular replacement using a previously reported hCAII
2
4
structure (PDB 3KS3) with water molecules removed. Models were
2
5
built by alternating refinement using REFMAC5 and manual
2
6
2
0,21
visualization and model building in Coot. Ligand topologies were
examine protein−ligand interactions.
In the present study,
27
generated using the PRODRG server. The structures have been
deposited in the Protein Data Bank (PDB IDs 4MLX and 4MLT for
ATM and TM, respectively). Mercury salts are commonly used in
hCAII crystallization to increase crystal quality and size. Both
the MBGs of interest are O,S-donor ligands based on a
hydroxythiopyrone scaffold. Specifically, the three ligands, 5-
hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-
hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-
hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA),
which differ only by the presence and/or position of a single
methyl group, are examined (Figure 1). In model complexes
based on a tris(pyrazolyl)borate (Tp) platform, these chelators
structures contain p-mercuribenzoic acid bound to Cys206. Complete
Ph,Me
data collection details and refinement statistics for the Tp
Zn-
(ATM) and hCAII crystal structures can be found in the Supporting
Information.
X-ray Absorption Spectroscopy. Samples for XAS (∼2 mM in
protein) were prepared from lyophilized CA (Sigma Aldrich),
dissolved in 50 mM phosphate buffer (pH 7.5) that was dialyzed
overnight against the same buffer to remove salts and adventitious
metals. MBG complexes were prepared by addition of a buffered
solution of the MBG to the hCAII solution to a final concentration of
15
bind in an identical, bidentate manner. In contrast, in the
active site of hCAII these chelators display a variety of
2
+
coordination modes with the active site Zn ion, including an
unprecedented monodentate mode of binding by thiomaltol.
The results not only show the utility but also the limitations of
bioinorganic modeling while highlighting the subtle influence of
active site structure on metal−ligand bonding. Such subtle
effects on coordination chemistry are not readily predicted by
current paradigms in bioinorganic chemistry and, consequently,
are not implemented in standard drug design efforts directed at
metalloproteins. Taken together, the findings presented here
demonstrate that metal coordination by an exogenous ligand in
a metalloprotein active site is strongly influenced by the protein
active site. Existing drug design protocols for metalloproteins
will need to be adapted to account for these perturbations.
6
mM (3-fold molar excess). All samples contained 20% (v/v) glycerol
as a glassing agent and were loaded in Lucite cuvettes with 6 μm
polypropylene windows before being frozen rapidly in liquid nitrogen.
X-ray absorption spectra were measured at the National Synchrotron
Light Source (NSLS), beamline X3B, with a Si (111) double-crystal
monochromator; harmonic rejection was accomplished using a Ni
focusing mirror. Fluorescence excitation spectra for all samples were
measured with a 31-element solid-state Ge detector array. Samples
were held at ∼15 K in a Displex cryostat. Detailed data collection and
reduction procedures can be found in the Supporting Information.
Density Functional Computations. All geometry optimizations
28
were performed using the Gaussian 09 suite of programs, utilizing
Becke’s three-parameter hybrid method with the Lee, Yang, and Parr
2
9−32
EXPERIMENTAL SECTION
correlation functional (B3LYP)
and CPCM solvation (ε = 10).
with the 6-311+G(d,p) basis set
The B3LYP functional has
■
3
3−35
Synthesis. MBGs and TpPh,MeZn(ATM) (TpPh,Me = hydrotris(5,3-
previously been used to successfully recapitulate geometric parameters
methylphenylpyrazolyl)borate) were synthesized using modified
of model active sites for Zn2 metalloproteins as well as free energies
+
36
1
5,22
reported procedures.
Information.
Details can be found in the Supporting
37
of water-chloride exchange in zinc chloride complexes. Additional
details and explanations can be found in the Supporting Information.
23
hCAII Activity Assay. As previously reported, hCAII was
expressed and purified, and a detailed procedure can be found in
the Supporting Information. Assays were performed in 50 mM HEPES
pH 8.0 containing Na SO to an ionic strength of 100 mM. Enzyme
Linear transit calculations were performed with the phenyl groups of
Ph,Me
Tp
Zn omitted; this modified system is referred to as TpZn.
2
4
(
100 nM final concentration) was incubated with varying concen-
RESULTS
■
trations of inhibitor for 10 min at room temperature before the
addition of substrate (p-nitrophenyl acetate, final concentration
between 50 μM and 10 mM). The reaction was monitored by the
increase in absorbance at 405 nm. Initial reaction rates vs substrate
hCAII Inhibition. Previously reported data from screening a
library of MBGs against hCAII revealed TM and TPMA as
moderately potent fragments. Determination of K values for
3
i
5
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ATM, TM, and TPMA reveals inhibition constants consistent
with these early reports (Table 1). The effect of the inhibitors
In the active site of hCAII, ATM adopts the expected
bidentate coordination mode to the Zn2+ ion of hCAII,
resulting in trigonal bipyramidal geometry around the metal
(Figure 3). The S−Zn (2.57 Å) and O−Zn (2.28 Å) bonds are
Table 1. K Values (mM) of Compounds Against hCAII
i
compound
Ki
compound
Ki
ATM
THM
TPMA
0.65 ± 0.06
1.4 ± 0.2
1.1 ± 0.2
ATM-OMe
THM-OMe
6.9 ± 1.0
>50
on the K curve of hCAII-catalyzed hydrolysis of p-nitrophenyl
m
acetate is consistent with competitive inhibition (Figure S1).
While TM and TPMA have similar K values (1.4 ± 0.2 and 1.1
i
±
0.2 mM, respectively), ATM is roughly 2-fold more potent
0.65 ± 0.06 mM). When the hydroxyl group of ATM is
(
methylated (ATM-OMe, Figure 1), the molecule drops ∼10-
fold (K = 6.9 ± 1.0 mM) in potency. In contrast, methylation
i
of TM (i.e., TM-OMe) results in a complete loss of inhibitory
activity against hCAII (K ≫ 50 mM).
i
Binding Mode Analysis. In order to explain the variation
in inhibitory activity for this series of molecules, the X-ray
crystal structures of ATM and TM bound to TpPh,MeZn model
compounds and the hCAII active site were examined. The
crystal structure of TM bound to this model complex has been
Figure 3. Crystal structure of ATM bound in the active site of hCAII.
The |Fobs| electron density map (gray) is shown contoured at 1.5σ for
protein residues, while the omit |Fobs − Fcalc| map (red) is shown
contoured at 3.0σ for ATM. A schematic representation of the
interactions between ATM and hCAII can be found in Figure S12.
1
5,16
previously reported,
and ATM shows a very similar binding
mode to the Zn2 ion. The ligands bind in a bidentate fashion,
+
2+
resulting in trigonal bipyramidal geometry around the Zn ion
Ph,Me
(Figure S2). The S−Zn and O−Zn distances of Tp
Zn-
(ATM) (2.35 and 2.09 Å, respectively) are similar to those in
the complex with TM (2.34 and 2.06 Å). In both cases, the
oxygen donor atom occupies an axial coordination site, while
the sulfur atom is an equatorial donor. The two ligands
coordinate the Zn2+ with nearly ideal “head-on” binding; the
plane formed by the ligand atoms is essentially normal to the
plane formed by the three pyrazole nitrogen donors. This angle
both ∼0.2 Å longer than those in TpPh,MeZn(ATM). As
predicted by the model complex, the oxygen donor occupies an
axial coordination site, while the sulfur is in an equatorial
position. The hydroxyl group of the ligand, in addition to
binding the Zn2+ ion, is in close proximity to the hydroxyl
groups of Thr199 (O−O distance of 3.51 Å) and Thr200 (O−
O distance of 3.71 Å), and the methyl group is positioned to
interact with the side chains of Val121 and Phe131 of the
hydrophobic region of the active site. Due to the steric
restrictions of the hCAII active site, the ligand cannot bind in
the ideal head-on fashion (ϕ = 180°); it is forced to tilt more
than 20° relative to that of the model complex (Figure 2, ϕ =
(ϕ, Figure 2) will be quantified by the torsion angle between
1
43°).
The crystal structure of TM bound in the active site of hCAII
reveals an unconventional coordination mode: the ligand acts
as a monodentate donor through the sulfur atom with a bond
length of 2.4 Å, resulting in a distorted tetrahedral geometry
around the Zn2+ ion. The ligand electron density is best fit as a
combination of two binding modes, both with 50% occupancy,
in which the coordinated sulfur atoms overlay (Figure 4). In
one orientation, the exocyclic hydroxyl and methyl groups are
oriented toward the hydrophobic wall of the active site formed
by Val143, Leu141, Val121, and Phe131. In the second
conformation, the ligand is flipped so that the hydroxyl group is
directed toward hydrophilic residues of the active site, allowing
for a hydrogen bond with the side chain of Thr200 (O−O
distance of 2.85 Å). In this conformation the ring of TM is
positioned ∼1.1 Å closer to the to the side chains of Val121 and
Val143, allowing for enhanced hydrophobic contacts with these
groups. The average B factor of TM is significantly greater than
that of ATM (40.5 vs 21.5), consistent with its lower affinity
and disordered binding. Efforts to soak TPMA into hCAII
crystals repeatedly resulted in poorly defined electron density
for the MBG that could not be suitably modeled.
2
+
Figure 2. For ATM, TM, and TPMA, ϕ is defined as the Zn −S−O-
C dihedral angle (A, shown labeled on ATM with red arrows). This
parameter defines the angle between the planes formed by the Zn−S−
O and S−O−C atoms, representing the tilt of the MBG. MBGs
Ph,Me
assume different binding modes in Tp
Zn (B, ϕ
) and hCAII
Model
(
C, ϕProtein) complexes. |ϕModel| ranges from 166 to 174°, while |ϕProtein
|
ranges from 90 to 143° depending on the MBG.
the Zn2+ ion, the sulfur donor, the oxygen donor, and the
endocyclic carbon bound to the oxygen; these angles show
Ph,Me
absolute values of 166° and 174° for the Tp
complexes of
ATM and TM, respectively. Given the similarity of the TM and
ATM complexes, it is expected that the TPMA structure would
show an identical coordination geometry.
5
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EXAFS data (Figure 5B) reveals that all three MBGs cause a
similar shift in the third oscillation of the EXAFS (k ∼7 − 9
−1
Å ). Comparison of the theoretical EXAFS for Zn−N and
Zn−S interactions shows this is where the two patterns are
most likely to show visible divergence (Figure S7), suggesting
that all three MBG complexes include a Zn−S scattering
interaction. Each MBG causes a similar shift in the shape of the
Zn X-ray absorption near edge structure (XANES, Figure S8),
also consistent with S-coordination.
The curve fits are consistent with the qualitative assessment
given above. The ATM complex with hCAII appears 5-
coordinate, with the sulfur of the MBG directly coordinated
(
fits that excluded the Zn−S bond gave fit residuals that were 3-
fold larger, Figure S4 and Table S4). The Zn−N/O distance is
also slightly longer than for the resting enzyme and the other
two MBG complexes, suggesting higher coordination in the
ATM complex, and it is this XANES spectrum that shows that
largest energy shift. In contrast, fits to the TM complex data
suggest that the total coordination number remains at four with
the MBG coordinated through only the sulfur atom (fits that
excluded the Zn−S bond gave fit residuals that were 2- to 3-fold
larger, Figure S5 and Table S5). This is consistent with the
change in the outer shells, where more linear MBG
coordination could potentially amplify multiple-scattering
interactions, although a deeper analysis is outside the scope
of the present study. TM also produces a change in shape in the
XANES but a smaller energy shift than ATM. The TPMA
complex is strikingly similar to the resting enzyme with only the
shift in principal frequency noted above being readily apparent.
Fits to these data also suggest retention of a total coordination
of four in the TPMA complex (as observed for TM), with the
MBG S-bound (fits that excluded the Zn−S bond gave fit
residuals that were approximately 2-fold larger, Figure S6 and
Table S6). This is supported by the XANES, which clearly
show a change in shape on addition of TPMA.
Figure 4. Crystal structure of TM bound in the active site of hCAII.
The ligand has two conformations, shown in green and cyan. Omit
maps (|Fobs|, gray; |Fobs − Fcalc|, red) are shown contoured at 1.5σ. A
diagram of the interactions between THM and hCAII can be found in
Figure S12.
X-ray Absorption Spectroscopy. X-ray absorption spec-
troscopy (XAS) data suggest the binding modes observed in
the crystal structures are representative of those present in
frozen solution. The data for hCAII with and without the
MBGs (ATM, TM, and TPMA) are shown in Figure 5.
Computational Analysis. To assess one possible cause for
the switch to monodentate coordination of TM and TPMA in
the active site of hCAII, linear transit computations employing
density functional theory (DFT) were conducted along ϕ from
1
80° to 90° for TM complexed to the simplified TpZn scaffold.
Near ϕ = 180°, the plane formed by TM is perpendicular to the
plane defined by the three Zn -coordinating pyrazole nitro-
gens. Similar to the Tp
2+
Ph,Me
Zn(TM) crystal structure, this
computed structure adopts a trigonal bipyramidal geometry
with the hydroxyl group as an axial donor and the sulfur donor
coordinating equatorially. The calculated O−Zn and S−Zn
3
Figure 5. Fourier transforms (A) of the k -weighted EXAFS (B) of
hCAII with ATM, TM, and TPMA. In each case, the data for the
resting enzyme are shown as a thin line overlay.
distances (2.08 and 2.45 Å, respectively) are similar to those
Ph,Me
observed in the crystal structure of Tp
S9).
Zn(TM) (Figure
Detailed fitting results for each data set are given in Figures
S3−S6 and Tables S3−S6. Comparison of the Fourier
transforms (FTs) in Figure 5A shows that each MBG (bold
lines) leads to only minor overall perturbations relative to the
resting enzyme (thin lines). ATM provides the most striking
changes in the first shell, with both a shift to higher R and
narrowing of the main peak (chiefly Zn−N/O scattering),
together with increased amplitude at R + α ∼2.1 Å (chiefly
Zn−S). The TM complex shows similar, although more subtle,
changes in the first shell scattering and substantial changes in
the outer shell scattering pattern. The TPMA complex is most
similar to the resting enzyme, with only subtle changes in the
first shell apparent in the extended X-ray absorption fine
structure (EXAFS) FTs. However, examination of the k-space
As TM is tilted along ϕ, the Zn donor atom distances
increase gradually until ϕ reaches 120°, which is the last point
along the linear transit where the ligand coordination is
bidentate (Figure 6). For values of ϕ < 120°, no stationary
2
+
states corresponding to bidentate coordination of the Zn ion
are obtained; monodentate coordination by the thione becomes
the favored binding mode. At these points, the O−Zn distance
is >3.4 Å, while the S−Zn bond length, having a value of 2.30 −
2+
38
2.32 Å, more closely resembles a thiolate−Zn bond. Similar
trends in coordination mode along the ϕ reaction coordinate
are also present for TM and TPMA (Figure 6).
For all three MBGs, the lowest energies are achieved at
values of ϕ near 180°, where the ligand assumes its ideal “head-
on” binding mode as observed in the model complexes.
5
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perturb MBG coordination. The inhibitory activity of ATM-
OMe suggests that the interaction between the hydroxyl group
and the Zn2+ ion is not essential for activity, and the
monodentate binding of TM further supports this. While the
methyl group of ATM is ideally positioned for hydrophobic
interactions when the ligand adopts bidentate coordination, this
is not the case for TM. It appears that in order to maximize
other protein−ligand interactions, the O−Zn binding inter-
action is sacrificed. The conformation in which the hydroxyl
and methyl groups are oriented toward the hydrophobic wall of
the active site is very similar to a previously reported structure
40
of 2-mercaptophenol (2-MP) bound to hCAII (Figure 8).
Figure 6. Calculated O−Zn distances (top) and relative binding
energies (bottom) as a function of ϕ from linear transit computations.
Distorting the ligand coordination from ϕ = 180° leads to an
increase in energy relative to the head-on binding geometry,
and the energy of the complex appears to follow a parabolic
path up to the point where the coordination mode shifts from
bidentate to monodentate (Figure 6). Shifting to monodentate
2+
coordination of Zn through the sulfur atom causes the relative
energy of the complex to level out at ∼12−15 kcal/mol above
the energy of the “head-on” binding mode.
Figure 8. Overlay of the crystal structure of TM and inhibitors that
show similar binding modes. Left: The conformation with the hydroxyl
group of TM facing the hydrophobic pocket occupies a space similar
to that of 2-mercaptophenol (PDB 2OSM, shown in green). Right:
When the hydroxyl group of TM is oriented toward the hydrophilic
side of the active site, the ring nearly overlays with that of 2-
mercaptophenol.
DISCUSSION
■
Aside from being tilted away from ideal “head-on” binding, the
coordination of ATM to the active site Zn ion of hCAII is
similar to that predicted by the Tp
S2). This bidentate coordination mode has been previously
reported for 2-mercaptopyridine-N-oxide (1,2-HOPTO), a
similar MBG (Figure 7). The previously reported K of 1,2-
2
+
Ph,Me
model complex (Figure
39
i
Although both TM and 2-MP are monodentate ligands in
Ph,Me
hCAII, only the Tp
model complex of 2-MP recapitulates
41
Ph,Me
this monodentate binding mode. In contrast, the Tp
Zn-
(
TM) complex shows strong bidentate coordination from both
the sulfur and oxygen donor atoms. This suggests that while the
monodentate binding of 2-MP to hCAII is driven largely by the
properties of the ligand itself, the monodentate binding mode
of TM is a direct result of interactions with the hCAII active
site environment.
In contrast to TM, which loses all inhibitory activity when
methylated (TM-OMe), when the hydroxyl group of 2-MP is
methylated (TG, Figure 1), the activity against hCAII is
unaffected (K =3.2 ± 0.3 μM for TG vs 3.0 ± 0.7 μM for 2-
i
3
MP). This suggests that the binding mode of TM that is
relevant to inhibition is the conformation in which the hydroxyl
and methyl groups are oriented toward the hydrophilic residues
of the active site. The loss in potency for TM-OMe is
consistent with this binding mode, as the interaction between
TM and Thr200 would be diminished and the methoxy group
would likely have a steric clash with either neighboring protein
residues or well-ordered active site water molecules. With the
hydroxyl group oriented toward the hydrophilic side of the
pocket, a hypothetical bidentate coordination mode would
position the methyl group of TM very close to the hydrophilic
side of the active site, where well-ordered water molecules
interact with protein residues. Consequently, TM rotates
Figure 7. Overlay of the crystal structures of ATM and 1,2-HOPTO
(
PDB ID 3M1K, shown in yellow) bound to hCAII. Although the
donor atoms are positioned similarly (left), a view along the plane of
the ligands (right) reveals that ATM is ∼10° closer to ideal “head-on”
binding.
HOPTO against hCAII (0.85 mM) is close to that of ATM and
3
coincides with the similar binding mode. Although the
2
+
interaction between ATM and the Zn ion appears to be
bidentate, the residual inhibitory activity of ATM-OMe
suggests that the electrostatic interaction between the oxygen
donor and the Zn2 ion is not essential for binding.
+
The coordination mode of TM to the active site Zn2+ ion of
hCAII demonstrates that a protein environment can strongly
toward a monodentate coordination of Zn to preserve the
preexisting interactions in the pocket. The microscopic pKa
2+
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values of TM in the active site of hCAII are computed to be 4.1
and 7.2 when the hydroxyl group is oriented toward the
hydrophilic and hydrophobic pockets, respectively (Figure
scohen@ucsd.edu
Notes
4
2
The authors declare no competing financial interest.
S11). Coupling these data with the observation of the two
conformations in a ∼1:1 ratio in the crystal structure
determined at pH 8 suggest that at low pH, the predominant
species of TM is protonated and oriented toward the
hydrophobic pocket (attempts to verify this crystallographically
were unsuccessful); at high pH (pH > 7.2), the deprotonated
form of TM is dominant with the ligand hydroxyl group
oriented in the hydrophilic pocket, which is likely the
conformation responsible for the observed inhibitory activity.
From the structural data acquired for TM, particularly when
compared to the analogous O,S-donor ATM, it is evident that
ACKNOWLEDGMENTS
■
J.A.M. acknowledges support from the National Institute of
Health (NIH GM31749), National Science Foundation (MCB-
020765), Howard Hughes Medical Institute, National
Biomedical Computation Resource and NSF supercomputer
centers. P.G.B. acknowledges support from the National
Institute of Health Molecular Biophysics Training Grant
1
(
2T32GM008326-21). D.L.T. acknowledges support from the
National Institutes of Health (P30-EB-009998 to the Center for
Synchrotron Biosciences from the NIBIB, which supports
beamline X3B at the NSLS) and the National Science
Foundation (CHE-1152755). S.M.C. acknowledges support
from the National Institutes of Health (R01 GM098435).
D.P.M. is supported by a SMART scholarship from the Office
of the Secretary of Defense - Test and Evaluation (N00244-09-
2+
the Zn −MBG interaction is not the sole dictator of ligand
binding. Ligand acidity is likely not a major driving force in the
change in coordination, as ATM and TM have relatively close
43
acidities (pK = 7.64 and 8.06, respectively). The DFT-
a
derived geometric and energetic analyses of TpZn(MBG)
complexes show that over the ligand orientations available to
MBGs in hCAII (|ϕ| = 90−143°), there can be a very small
energy difference between monodentate and bidentate
1
-0081).
2
+
coordination of Zn . For example, between ϕ = 125−115°,
bidentate and monodentate coordination modes for the ligands
considered in this study differ in energy by <5 kcal/mol. From
this observation, it is reasonable that the orientation of TM can,
in some circumstances, be altered by interactions with the
active site of hCAII. This finding implies that de novo or
fragment-based approaches to inhibitor development must take
care to elucidate circumstances where protein effects alter the
coordination mode of an MBG.
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CONCLUSION
■
The rational design of metalloprotein inhibitors requires
knowledge as to how those inhibitors coordinate the active
site metal ion. While small molecule model complexes have
been used as proxies for coordination in enzyme active sites, the
results presented here demonstrate that the active site
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ASSOCIATED CONTENT
Supporting Information
■
*
S
(
Full experimental details for ligand synthesis, protein expression
and purification, crystal structure collection details and
refinement statistics, and complete computational procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(
́
T.; Roques, B.
́
1
(
2
AUTHOR INFORMATION
Corresponding Authors
dtierney@miamioh.edu
■
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