Identification and characterization of an allosteric inhibitory site on dihydropteroate synthase

Article abstract of DOI:10.1021/cb500038g

The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and quasi-harmonic analyses suggest that the effect of inhibitor binding is transmitted from the dimer interface to the active-site loops that are known to assume an obligatory ordered substructure during catalysis. Together with the kinetics results, these structural and dynamics data suggest an inhibitory mechanism in which binding at the dimer interface impacts loop movements that are required for product release. Our results potentially provide a novel target site for the development of new antibiotics.

Full text of DOI:10.1021/cb500038g

Articles
pubs.acs.org/acschemicalbiology
Open Access on 03/20/2015
Identification and Characterization of an Allosteric Inhibitory Site on
Dihydropteroate Synthase
†
†,#
†
†
‡
Dalia I. Hammoudeh, Mihir Date,
́
Mi-Kyung Yun, Weixing Zhang, Vincent A. Boyd,
†
‡
‡
,†
,†,§
Ariele Viacava Follis, Elizabeth Griffith, Richard E. Lee, Donald Bashford,* and Stephen W. White*
†
‡
Departments of Structural Biology and Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis,
Tennessee 38105, United States
§
Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee
8163, United States
3
*
S Supporting Information
ABSTRACT: The declining effectiveness of current anti-
biotics due to the emergence of resistant bacterial strains
dictates a pressing need for novel classes of antimicrobial
therapies, preferably against molecular sites other than those in
which resistance mutations have developed. Dihydropteroate
synthase (DHPS) catalyzes a crucial step in the bacterial
pathway of folic acid synthesis, a pathway that is absent in
higher vertebrates. As the target of the sulfonamide class of
drugs that were highly effective until resistance mutations
arose, DHPS is known to be a valuable bacterial Achilles heel
that is being further exploited for antibiotic development.
Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it
engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate
binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and quasi-harmonic
analyses suggest that the effect of inhibitor binding is transmitted from the dimer interface to the active-site loops that are known
to assume an obligatory ordered substructure during catalysis. Together with the kinetics results, these structural and dynamics
data suggest an inhibitory mechanism in which binding at the dimer interface impacts loop movements that are required for
product release. Our results potentially provide a novel target site for the development of new antibiotics.
he enzyme dihydropteroate synthase (DHPS) is encoded
by the folP gene and catalyzes the formation of 7,8-
catalysis via a complicated S 1 mechanism that involves the
transient folding of two flexible loops. The intermediate-state
N
7
T
dihydropteroate from p-aminobenzoic acid (pABA) and 6-
hydroxymethyl-7,8-dihydropterin-pyrophosphate (DHPP).
This reaction is a key step in the folate biosynthetic pathway
of bacteria and primitive eukaryotes, and the absence of the
folate pathway in higher eukaryotes makes it an attractive focus
structure that we characterized revealed that the pterin pocket
is designed to generate and then stabilize a cationic pterin
intermediate species and also revealed a transient pyrophos-
phate-binding pocket.
In light of these findings, we recently initiated a fragment-
based approach to identify new inhibitory scaffolds of DHPS.
Fragment based lead discovery (FBLD) is particularly useful for
identifying novel chemotypes for difficult targets such as DHPS
where traditional high-throughput methods have been challeng-
1
for antimicrobial drug discovery. DHPS is the target for the
highly effective sulfa drugs that have been used for over 70
years. However, the emergence of sulfa drug resistance has
2
3
,4
seriously compromised their utility and has spawned a5
number of efforts to develop new classes of DHPS inhibitors.
For a number of years, we have been pursuing novel
inhibitors of DHPS that target the conserved and structured
pterin-binding pocket with the goal of producing broad-
spectrum drugs that are less prone to generating resistance than
the flexible pABA binding site to which sulfonamide drugs bind.
Although we have identified a number of inhibitors, one
challenge has been that the pterin binding pocket is highly
selective for the pterin substrate and pterin analogues, and our
traditional screening and medicinal chemistry approaches have
8
,9
ing. In the current study, we screened a fragment library
against Bacillus anthracis DHPS (BaDHPS) with the goal of
identifying novel scaffolds that bind within the pterin pocket.
However, a significant subset of the binding molecules was
found to bind elsewhere on the protein, and we identified this
novel ligand binding site at the dimer interface using X-ray
crystallography. A derivative of one of the interfacial binding
molecules displayed significant inhibitory activity, and kinetic
Received: January 17, 2014
Accepted: March 20, 2014
Published: March 20, 2014
6
tended to yield pterin-like molecules with limited solubility. In
addition, we recently demonstrated that DHPS performs
©
2014 American Chemical Society
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studies, NMR analyses, and molecular dynamics (MD)
calculations suggest that ‘breathing motions’ within the
DHPS dimer and communication between the dimer interface
and the active-site loops create this allosteric inhibition.
three DHPS enzymes at 250 μM. To verify that this was not
simply due to a lack of binding, we used surface plasmon
resonance (SPR) and WaterLOGSY titration to measure the Kd
value of the pure compound. Both methods verified that 1
binds with very similar K values: 187 ± 2 μM using SPR and
d
RESULTS AND DISCUSSION
1
68 ± 35 μM using WaterLOGSY (Figure 2A−C). In contrast,
■
Initial Identification of an Allosteric Inhibitor. Using
the Maybridge Ro3 library of 1,100 compounds, we performed
a fragment screen of BaDHPS using the WaterLOGSY (water-
when 11 was synthesized in-house, it showed 100% inhibition
of all three enzymes at 250 μM, and the IC50 values for
BaDHPS, YpDHPS, and SaDHPS were measured as 50, 31, and
10
ligand observed via gradient spectroscopy) NMR protocol.
A
17 μM, respectively (Figure 3). Measuring the K value of 11
d
total of 74 fragments were revealed as potential binders. Using a
previously characterized high affinity pterin pocket-binding
inhibitor (2-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-
using SPR proved to be technically challenging due to solubility
issues, but it was possible to measure a value of 130 ± 1 μM
using isothermal titration calorimetry (ITC) (Figure 2D). This
11
tetrahydropyrimido[4,5-c]pyridazin-3-yl)propanoic acid), as
K value was measured in the absence of substrates, but ITC
d
well as the substrate pABA, we then used competitive-
experiments in the presence of substrates revealed that 11
enables pABA to bind DHPS with a 2-fold increase in binding
affinity (Figure 2E and F).
To further understand the way in which 11 exerts its
inhibitory effect, two Michaelis−Menten kinetic experiments
were conducted on all three enzymes in which the rates of
DHPS catalysis were measured at increasing concentrations of
12
WaterLOGSY (c-WaterLOGSY) to determine which of
these binders access active site pockets. c-WaterLOGSY
revealed that 10 fragments clearly bind to regions of DHPS
other than the active site (compounds 1−10, Figure 1). The
11. In the first experiment, the concentration of DHPP was
varied and the pABA concentration was held constant, and this
was reversed in the second experiment. The results were
difficult to interpret for the first experiment, and this was not
surprising because we have demonstrated that the DHPS
catalytic mechanism involves an ordered S 1 process in which
N
DHPP is the compulsory lead substrate without which the
7
pABA-binding pocket fails to form. However, the second
experiment revealed that both the K and V values decrease
m
max
with increasing concentrations of 11 (Table 1 and Supple-
mentary Figure S1). In the presence of 11 and with DHPP
present in the pterin-binding pocket, pABA binds DHPS with
increased affinity (K decreases with increasing concentrations
m
of 11). The actual K and V values vary somewhat between
m
max
the three orthologs, but the trend is clear and consistent. One
interpretation of this result, consistent with the enzyme
mechanism, is that the binding of 11 to an allosteric site of
DHPS promotes the ordering of the two active site loops. This
would enhance the binding of pABA to the active site, thereby
Figure 1. 2D structures of the fragment hits that were identified to
bind at the dimer interface of DHPS.
decreasing K , but would also slow the release of product,
m
thereby decreasing Vmax
.
fragments that do appear to access the active site are currently
Crystallographic Analyses. To determine whether
compounds 1−11 engage the same site on DHPS as suggested
by their similar structures, selected compounds were soaked
into BaDHPS crystals and the complex structures were
determined. Previous studies have shown that BaDHPS crystals
being studied and will be reported elsewhere. Compounds 1−
1
0 all share the common structural feature of a trifluoro group
attached to a phenyl group, which suggests that they access the
same site on DHPS.
Activity and Kinetic Assays. Using previously described
are amenable to the soaking protocol for crystallographic
7
,11
6,7,13
inhibition assays,
these 10 compounds were tested for
analysis,
and we were able to obtain high quality crystal
inhibitory activity against BaDHPS and the DHPS enzymes
from Yersinia pestis (YpDHPS) and Staphylococcus aureus
structures of BaDHPS in complex with 4, 5, 6, and 11 (Figure
4). Data collection and refinement statistics are shown in
(
SaDHPS). Compound 1 was found to effectively inhibit all
three DHPS enzymes at 250 μM (BaDHPS, 86%; YpDHPS,
1%; SaDHPS, 46%), whereas binders 2−10 displayed only
Supplementary Tables S1 and S2. In the BaDHPS P6 22 crystal
2
lattice, there are two monomers in the asymmetric unit that
create two dimers around 2-fold symmetry axes, and clear
electron density was visible for all four compounds at these
dimer interfaces. The compound density sits exactly on the
crystallographic 2-fold symmetry axis and formally corresponds
to a pair of 2-fold related molecules, each with half occupancy.
Electron density on crystallographic symmetry axes can be
misleading, but we are confident that this density corresponds
to the bound molecule for three reasons. First, the shapes of the
densities closely match those of the bound compounds with the
common trifluorophenyl group of each compound buried at the
6
modest inhibitory activities. Compound 1 in the Maybridge
fragment library was reported to be 4-(trifluoromethyl)
benzylamine, but when it was chemically and structurally
characterized, the sample was found to contain a mixture of 1
and the ‘dimeric’ Schiff base N-(4-(trifluoromethyl)-
benzylidene)-1-(4-(trifluoromethyl)benzylamine (compound
1
1, Figure 1), with the latter in excess. Consequently,
compound 1 was repurchased (Sigma Aldrich) and verified as
7% pure, and this sample showed no inhibition of any of the
9
1
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Figure 2. Binding affinities of compounds 1 and 11 to BaDHPS. (A) SPR sensorgram and (B) binding isotherm for the BaDHPS:1 complex. The
fragment was injected as a 3-fold dilution series starting at 200 μM, and the data were fit to a 1:1 interaction model. (C) WaterLOGSY titration of
compound 1: solid triangle, compound 1; solid diamonds, compound 1 in the presence of BaDHPS; solid circle, difference. (D, E, F) ITC binding
experiments of (D) compound 11 titrated into a solution of BaDHPS, (E) pABA titrated into BaDHPS in the presence of PtPP, and (F) pABA
titrated into BaDHPS in the presence of PtPP and compound 11.
Table 1. Kinetic Analyses of DHPS in the Presence of
a
Compound 11
concn of compd 11 (μM)
0
50
Vmaxobs
100
Vmaxobs Kmobs
Vmaxobs
Kmobs
Kmobs
(
nM/min) (μM) (nM/min) (μM) (nM/min) (μM)
BaDHPS
YpDHPS
SaDHPS
125.8
177.3
583.6
1.2
2.1
7.7
106.7
131.3
211.2
1.0
1.8
2.7
70.81
40.95
31.19
0.74
1.2
0.38
a
In these experiments, the concentration of the pterin substrate
DHPP) was held constant (20 μM), and the concentration of pABA
(
was increased from 0 to 10 μM.
Figure 3. IC50 determination of compound 11 versus BaDHPS (blue),
YpDHPS (red), and SaDHPS (green). The enzyme activities were
measured in the presence of 0−125 μM compound 11.
and Supplementary Figure S2). Second, identical crystals
soaked in small molecules that bind within the pterin pocket
have previously never displayed this electron density. A specific
example of a published crystal structure was carefully re-
evaluated to confirm this (Supplementary Figure S2). Finally,
6
center of the interface in the same location making hydro-
phobic contacts with Leu235/235′, Met264/264′, and the
methylene groups of Glu236/236′ and Glu260/260′ (Figure 4
the electron density is present on both of the 2-fold axes in the
1
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to optimally interact with and stabilize loop7. This, in turn,
would compromise the binding of the distal half of 11 to loop7
and explain its weak electron density. To explore this model
further, we used NMR and computational approaches.
NMR Analyses. To study the effects of 11 on loop
conformation and dynamics, we investigated whether BaDHPS
would be suitable for NMR analyses. We first performed a 2D
1
1
15
H− N HSQC-TROSY NMR experiment with perdeuterated
5
N-labeled BaDHPS, and approximately 40% of the residues
were observable in the spectrum (Figure 5A). We therefore
2
13 15
prepared H, C, N-labeled BaDHPS and performed 3D
HNCA and HN(CO)CA experiments with the goal of
assigning these observable resonances. Not counting 16
assigned peaks within the N-terminal His tag, 72 resonances
1
15
in the H− N HSQC-TROSY spectrum of the protein were
assigned (Supplementary Figure S3 and Supplementary Table
S3). These include the residues within loops1, 2, 4, 5, and 6, the
N-terminal two-thirds of helix α1, strand β2, the C-terminal half
of helix α8, and the C-terminus. These assignments encompass
the active site and dimer interface and showed that NMR
would be an ideal tool to monitor loop conformational changes
and to investigate whether the active site and the interface are
indeed dynamically linked.
Figure 4. Crystal structures of BaDHPS in complex with fragment
molecules bound at the dimer interface. (A) Compound 4. (B)
Compound 5. (C) Compound 6. (D) Compound 11; the distal half of
the compound is shown in transparency to reflect that its position is
derived from weak electron density and docking studies (see text). The
two DHPS monomers are shown in gray and wheat, and key residues
that interact with the fragments are shown as sticks. The F − F
We first confirmed that the active site loops become more
ordered in the presence of substrate as previously observed by
7
crystallography. To prevent substrate turnover in these
experiments, we used the inactive DHPP analogue 6-
hydroxymethyl-pterine-pyrophosphate (PtPP) that we and
o
c
13,15
simulated annealing omit electron density maps are contoured at 2σ.
others have shown to bind within the DHPS pterin pocket.
Weak peak perturbations were observed by the addition of 0.4
mM PtPP, but more profound perturbations were observed
when 0.4 mM pABA was subsequently added (Figures 5B). In
general, the spectrum of the BaDHPS/PtPP/pABA ternary
complex is more dispersed, and many more resonances appear
unit cell that create the two independent DHPS dimers, and
their shapes are very similar.
Although the electron density for 11 clearly shows that the
proximal half of the molecule binds at the dimer interface, the
very weak electron density for the distal half precluded a
reliable interpretation in terms of a molecular model. Liquid
chromatography−mass spectrometric analysis showed that 11
is stable in conditions that recapitulate the crystallization
conditions (pH and time), which excludes the possibility that
the weak electron density for the distal half reflects compound
cleavage. To generate a potential docked conformation of the
entire molecule, we used the docking program ‘Glide’ from the
1
15
compared to the H− N HSQC-TROSY of the free enzyme.
This is consistent with the formation of a folded loop1−loop2
substructure within the ternary complex within which pABA
binds, as observed in the intermediate-state crystal structure of
7
DHPS. Importantly, the addition of PtPP and pABA also
elicits major perturbations in signals of residues at the dimer
interface including the C-terminus (Figure 5C). This result
supports our proposal that the dimer interface and the loop1/
loop2 pABA binding site are dynamically linked.
Upon addition of 11, further perturbations of resonances
from loop1, loop2, the dimer interface, and the C-terminus are
observed (Figure 5D). Specifically, more well-dispersed
resonances appear stemming from ordered regions of the
protein, and many of the existing resonances increase in
intensity. These changes strongly suggest that the addition of
14
̈
Schrodinger software suite. In all 10 top-scoring poses, the
proximal half of 11 is buried within the dimer interface as
observed in the electron density, while the distal half is bent
toward loop7 on one of the two monomers in a manner
consistent with the weak electron density. It is noteworthy that
in the MD simulations described below, the distal part is mobile
as suggested by the weak electron density and moves between
bent conformations that engage one or the other monomer.
The bent conformation is very attractive because loop7
11 further stabilizes the substrate-bound substructure formed
by loop1 and loop2. These NMR analyses have demonstrated a
communication between the dimer interface and the active site
such that molecules bound at one site perturb the other. The
observation that many resonances in the free BaDHPS were
unobservable suggested that BaDHPS undergoes motions on
the intermediate chemical shift time scale (i.e., μs−ms).
Motions on this time scale have been shown to be important
7
interacts with loop1 in the intermediate-state structure, and
this suggests that 11 functions as an inhibitor by physically
linking the interface with the active site loops. This model is
consistent with three observations. First, the kinetic data
described above indicate that 11 stabilizes the pABA-binding
pocket that is created by the ordering of loop1 and loop2.
Second, compounds 1−10 would be expected to be poor
inhibitors of DHPS as observed because they lack the distal half
and merely occupy the dimer interface without engaging loop7.
Third, loop1 and loop2 are trapped in nonfunctional
conformations in the BaDHPS crystal lattice and are unable
16−20
in catalysis and allostery.
The increased intensity of some
resonances and the appearance of new resonances upon
addition of 11 may indicate that loop and interface motions
are slowed from the invisible intermediate time scale to the
slow-exchange time scale, freezing out catalysis-supporting
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1
15
1
15
Figure 5. NMR analyses of BaDHPS and its complexes. (A) H− N−TROSY-HSQC spectrum of 300 μM apo BaDHPS. (B) H− N−TROSY-
HSQC spectrum of 300 μM BaDHPS in the presence of PtPP and pABA. (C) Residues that shift in the presence of substrates PtPP and pABA
7
mapped onto the intermediate-state crystal structure of the closely similar YpDHPS. The residues are shown as spheres and color-coded according
to position on the structure; loop1, orange; loop2, cyan; dimer interface, red; other locations, green. The active site is shown only for one monomer
for clarity and is indicated by pABA and DHPP represented by sticks (yellow carbon atoms) at the N-terminus of helix α-loop7 (yellow). Note the
1
15
close association between loop1 and the tip of loop7 (yellow) in this structure. (D) H− N−TROSY-HSQC spectrum of 300 μM BaDHPS in the
presence of PtPP, pABA, and compound 11. Compared to panel B, resonances in panel D are generally more intense, and three of the most
prominent additional resonances are circled.
motions and inhibiting the enzyme. To gain more insight into
the dynamics of the system, we performed MD simulations.
Computational Analyses. MD simulations were per-
formed on four systems: the BaDHPS enzyme alone (E), the
enzyme with 11 bound (IE), the enzyme with the substrates
bound (ES), and the enzyme with both inhibitor and substrates
bound (IES). The basic starting structure for the enzyme and
for the inhibitor were from the X-ray structure of the
compound 11 complex reported here. The active site loop1
and loop2, the substrates, and the catalytically essential Mg2+
ion are not present in this structure, and these were modeled
In the E simulation, the largest fluctuations occur in loop1
and loop2 at the active site, and this is largely captured in the
highest-amplitude quasi-harmonic mode (Figure 6), although it
also persists in the next several modes (Supplementary Figure
S4A). An animation of this mode reveals that these loop
motions are coupled to an overall breathing motion in which
the monomers rock toward and away from each other, pivoting
about the symmetric dimer interface (Supplementary Movie
S1). In the presence of the inhibitor (IE simulation), this
intermonomer motion is strongly damped, and the mode
changes to one in which active-site movements are correlated
to those in the inhibitor binding site (Supplementary Movie
S2), particularly the loop7 residues 231−235 (Figure 6B).
These residues are indeed adjacent to the distal half of 11 in
our interpretation of the corresponding crystal structure
(Figure 4D). Inspection of the IES model shows that contacts
between these loop7 residues, including the highly conserved
Arg234, and residues Phe33 and Ser34 of loop1 may participate
in transmitting these motions (Figure 7).
from the structure of YpDHPS in complex with the S 1
N
+
7
intermedates, DHP , pABA, and PPi. This is consistent with
the scenario in which inhibition occurs at a later step of
catalysis, as suggested by the kinetic and NMR data. The
resulting trajectories were analyzed by the quasi-harmonic
2
1,22
approach
that identifies sets of correlated motions grouped
together as “quasi-harmonic modes” analogous to harmonic
vibrational modes.
1
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Figure 6. Molecular dynamics simulations of the BaDHPS dimer and its complexes. Shown are (A) the root-mean-square fluctuations (RMSF) of
the α-carbons at each residue position during the simulations and (B) the contribution of the highest-amplitude quasi-harmonic mode to those
fluctuations. The analysis was performed for all the α-carbons of the homodimer, but only one monomer is shown here. Blue line, enzyme alone
simulation (E); red line, enzyme plus 11 (IE); green line, enzyme plus substrates (ES); magenta line, enzyme plus substrates plus 11 (IES). Loop1 is
highlighted in orange, loop2 in cyan, and loop7 (which includes the small helix α-Loop7) in yellow.
The MD simulations support the notion that natural long-
range coupled motions within the dimer mediate communica-
tion between the dimer interface and the monomer active sites.
Specifically, the simulations have shown that an intermonomer
rocking motion is reduced upon inhibitor binding and also that
correlations are increased between the flexible loops in the
active site and the region that spans the N-terminal part of helix
α7 and the preceding loop7 residues in the inhibitor-binding
site. These analyses suggest a mechanism of allosteric inhibition
in which inhibitor binding both rigidifies the protein and alters
the pattern of dynamic correlations between dimer-interface
and active-site movement. This appears to inhibit a step
subsequent to the binding of the second substrate, and the
kinetic data suggest that this step is product release.
The apparent connection between DHPS catalysis and the
dimer interface prompted us to look more closely at our
Figure 7. A model showing how the distal half of compound 11
previous YpDHPS crystal structures of the dimer in the apo
(
magenta carbons, stick and surface representations) is proposed to
7
conformation, the product-analogue-bound conformation, and
exert a long-range inhibitory effect on the active site of BaDHPS via its
interaction with the ordered loop1−loop7 substructure (orange-
the intermediate-state conformation in which DHPP and pABA
had bound, PPi had been cleaved but not released, and the
pterin-pABA bond had not been formed. This comparison
revealed that the monomers rotate around the axis of helix α7
in transitioning between the apo and intermediate structures,
with the product-bound structure midway between these
extremes (Supplementary Figure S5). Examination of loop1
and the loop7 region immediately preceding helix α7 in these
structures also shows that the latter moves closer to loop1 on
going from the apo state to the intermediate state and moves
back in the product-bound state (Supplementary Figure S6).
Finally, in the product analogue-bound structure, loop1 rotates
out of the active site to form an “open” conformation that
allows the product to be released. Together with the MD and
NMR results, these observations suggest that when 11 interacts
with loop7, it leads to stronger loop1−loop7 interactions that
restrict the movement of loop1 out of the active site, thereby
inhibiting product release. A similar phenomenon has been
7
yellow) that is known to be required for catalysis. In this model, the
structures of loop1 and loop2 (cyan) at the active site and the bound
+
substrates DHP and pABA (shown as sticks) are based on the known
7
structure of the YpDHPS intermediate-state catalytic complex.
Analysis of the ES simulations shows that substrate binding
significantly decreases the motion of loop1 but leaves loop2
mobile, while the additional binding of inhibitor (IES)
significantly reduces the loop2 motion (Figure 6A). Animations
of the largest quasi-harmonic modes from the ES and IES
simulations (Supplementary Movies S3 and S4) show that
overall rocking or twisting motions, already reduced by
substrate binding, are further reduced by inhibitor binding.
These largest modes capture most of the overall RMS
fluctuation (Figure 6A vs B). A more detailed examination of
these modes shows that a motion of residues 256−258 in the
ES simulation is strongly damped upon inhibitor binding
23
(
Figure 6B, green vs magenta). This region includes His256
observed in ribonuclease A.
that coordinates the leaving PPi and is important for the SN1
catalytic mechanism. These results suggest that inhibition
occurs at a step after both substrates have bound, such as a
chemical step or subsequent product release, consistent with
the kinetic and NMR data.
Concluding Remarks. Allosteric sites on enzymes provide
novel opportunities for drug discovery, and a major advantage
of exploiting these sites is the ability to avoid mechanistic
aspects of the active site that hamper the discovery and
24
development of small molecule inhibitors. Allosteric sites on
1
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1
DHPS potentially offer a new approach to therapeutically
inhibit this valuable antimicrobial target that has a particularly
complex and mobile active site. Our studies have revealed that
the DHPS dimer interface offers an excellent opportunity for
drug discovery. Although DHPS has never been shown to be an
allosteric enzyme or to have allosteric effectors, it appears that
the natural motions of the dimer are linked to the active site
and can be manipulated with small molecules to affect catalytic
activity. This is actually supported by and explains mutations
that are found in several sulfonamide resistant strains of S.
aureus in which lysine and glutamic acid residues are inserted
spectra were recorded for each sample, a 1D H reference, a 1D
WaterLOGSY in the absence of protein, and a 1D Water-
LOGSY in the presence of 10 μM BaDHPS. Samples for c-
WaterLOGSY contained 20 μM concentration of the high
affinity pterin-binding inhibitor reported previously (2-(7-
amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]-
11
pyridazin-3-yl)propanoic acid) or 20 μM pABA in the
presence of 100 μM pyrophosphate, 10 μM DHPS, and 300
μM concentration of the fragment to be screened. Water-
LOGSY titration experiments were performed in the presence
of 10 μM DHPS and decreasing concentrations of compound 1
15
into the distal end of helix α8 at the dimer interface.
(
2, 1, 0.5, 0.25, 0.125, 0.0625, 0 mM).
Compound 11 inhibits all three of the DHPS enzymes that we
have studied, and this suggests that future antimicrobials that
target the interface are likely to be broad-spectrum. Compound
NMR Spectroscopy: 2D and 3D Experiments. See
Supporting Methods.
X-ray Crystallography. BaDHPS crystals were grown as
described previously. The four complex cocrystal structures
11 currently lacks antimicrobial activity and has limited aqueous
13
solubility, and the metabolic stability of the scaffold is a
potential concern. However, using the structural and molecular
dynamics information presented here, structure-guided syn-
thetic efforts are currently underway to increase the affinity,
physicochemical properties and potency of derivative small
molecules. The bipartite nature of the binding site, with a
proximal deep aryl pocket at the dimer interface adjacent to
multiple potential H-bond donors/acceptors and a distal site
comprising exposed residues within loop7, implies that each
can be independently optimized. An important initial step in
this process is ‘fragment hopping’ to identify new and
were obtained by soaking the compounds into BaDHPS
crystals overnight at ∼10 mM in mother liquor. Soaked crystals
were briefly immersed in cryoprotectant (a mixture of 50%
mineral oil and 50% Paratone-N) before flash freezing in liquid
nitrogen. Diffraction data were collected at the SER-CAT
beamlines 22-ID and 22-BM at the Advanced Photon Source
26
and processed using HKL2000. Structures were refined using
27
28
29
REFMAC and PHENIX and optimized with COOT.
Enzyme Inhibition Assays and Enzyme Kinetics. The
enzyme activities of BaDHPS, YpDHPS, and SaDHPS were all
determined by a linked assay in which the released
pyrophosphate was converted to inorganic phosphate by
pyrophosphatase. The inorganic phosphate was detected
using the PiColorlock Gold Kit (Innova Biosciences) with a
NanoDrop 2000C spectrophotometer (Thermo Scientific).
The 100-μL reaction mixtures contained 5 μM pABA, 5 μM
8
,9
potentially tighter binding scaffolds for each of these sites.
This is currently underway for the proximal pocket where
crystallography can readily be used to monitor progress. For the
distal site where structural analysis has proven to be more
challenging, we are using the scaffold present in compound 11
as a proximal ‘anchor’ and synthesizing a library of derivative
compounds to screen for more potent distal scaffolds that can
be subsequently characterized by crystallography. The potential
of allosteric inhibitors as antimicrobial agents is well validated
clinically; for example, anti-HIV drugs Evavirenz, Nevirapine,
and Delaviridine all target an allosteric site within the viral
DHPP, 10 mM MgCl , 5% DMSO, 50 mM HEPES pH 7.6,
2
0
.01 U yeast inorganic pyrophosphatase (Fermentas Life
Sciences), and 5 nM DHPS. Inhibitor compounds were
dissolved in DMSO, and inhibition was tested at 250 μM.
Inorganic phosphate was measured after 20 min of incubation
at 37 °C. To determine the half maximal inhibitory
24
reverse transcriptase. This bodes well for the development of
allosteric modulators of DHPS, particularly those that can be
combined with other antifolates to minimize the risk of
resistance development.
concentration (IC ) values, DHPS activities were measured
50
in the presence of various concentrations of the compounds.
The mechanism of inhibition by compound 11 was studied
with all three DHPS enzymes. At three different concentrations
of the inhibitor (0, 50, 100 μM), the initial catalytic rates were
determined at 0, 0.5, 1, 2, 4, 6, 8, 10 μM pABA with a fixed
concentration of 20 μM DHPP. Data were analyzed using
GraphPad Prism software.
METHODS
■
Protein Production. BaDHPS, YpDHPS, and SaDHPS
7
,13
were overexpressed and purified as previously described.
1
5
2
13 15
2
N, H-Labeled BaDHPS and C, N, H-labeled BaDHPS
25
Computational Methods. Starting structures for the
molecular dynamics simulations were based on the crystallo-
graphic structure of the complex with compound 11 reported
here, with additional modeling of loops and substrates based on
were overexpressed according to established procedures and
were purified using the same procedures as unlabeled BaDHPS.
NMR Spectroscopy: WaterLOGSY Screen. The May-
bridge Ro3 fragment library (1100 compounds at the time of
7
our previously reported structures, as described in Supple-
purchase) was dissolved in DMSO-d into 50 mM stock
6
mental Methods. Simulations were done in explicit solvent with
solutions. The fragments were screened in pools of 5 at a
concentration of 150 μM each, and the 216 samples were
prepared in 20 mM phosphate buffered saline (PBS), pH 7.0,
periodic boundary conditions and the Amber ff99SB force
30
field. For each of the four complexes, E, IE, ES and IES, 16 ns
production simulations were carried out. For E and IE, longer
simulations were also run (150 and 40 ns, respectively), but
these showed patterns of fluctuation and correlation very
similar to those reported for the 16 ns runs. Simulation details,
as well as the methods of quasi-harmonic mode analysis, are
provided in Supporting Methods.
1
0% D O, 0.01% Triton-X 100. All spectra were recorded with
2
a Varian Inova 600 MHz NMR spectrometer equipped with a
triple resonance inverse probe at 298 K. The pulse sequence
used was the homonuclear one-dimensional WaterLOGSY with
10
a water flip-back pulse. Data collection parameters for the
WaterLOGSY experiments were 200 scans, mixing time = 1.25
s, relaxation time = 2.6 s, and spectral windows of 13 ppm. All
data were processed using TopSpin (Bruker Biospin). Three
Surface Plasmon Resonance (SPR) Experiments. See
Supporting Methods.
1
300
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ACS Chemical Biology
Articles
Isothermal Titration Calorimetry (ITC). BaDHPS was
dialyzed against 50 mM HEPES, pH 7.6, 5 mM MgCl2.
Experiments were performed using an ITC200 (Microcal)
calorimeter at 298 K. All titrations were measured in 40 mM
and Z. Li, J. Bollinger, B. Waddell. and the St. Jude Molecular
Interactions Core for technical assistance. X-ray diffraction data
were collected at the Southeast Regional Collaborative Access
Team (SER-CAT) 22-ID and 22-BM beamlines at the
Advanced Photon Source, Argonne National Laboratory, and
we thank SER-CAT staff for their assistance. Supporting SER-
CAT institutions may be found at www.ser-cat.org/members.
html. Use of the Advanced Photon Source was supported by
the U.S. Department of Energy, Office of Science, Office of
Basic Energy Sciences, under Contract No. W-31-109-Eng-38.
HEPES, 4 mM MgCl , 5% DMSO (10% in the titration of 11)
2
at 298 K. Titration of 2.7 μL of 1 mM compound 11 into a
solution of 10 μM BaDHPS was performed over 14 injections.
pABA titrations in the presence or absence of 11 were
performed over 19 injections of 2 μL each. Cell and syringe
concentrations were 20 μL BaDHPS, 0.1 mM PtPP (with and
without 0.5 mM 11) and 0.25 mM pABA, 0.1 mM PtPP (with
and without 0.5 mM 11, respectively).
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Chemical Synthesisof Compound 11, (E)-N-(4-
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ASSOCIATED CONTENT
Supporting Information
■
*
S
Methods for 2D and 3D NMR Spectroscopy experiments,
computational simulations and quasi-harmonic mode analysis,
and Surface Plasmon Resonance (SPR) experiments. X-ray
crystallography data collection and refinement statistics,
BaDHPS NMR assignments, Michaelis−Menten kinetics,
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pubs.acs.org.
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Accession Codes
Coordinates for compound 4 bound BaDHPS: 4NHV;
coordinates for compound 5 bound BaDHPS: 4NIL;
coordinates for compound 6 bound BaDHPS: 4NIR;
coordinates for compound 11 bound BaDHPS: 4NL1.
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AUTHOR INFORMATION
Corresponding Authors
E-mail: Don.Bashford@stjude.org.
E-mail: Stephen.White@stjude.org.
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Notes
#
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
Grant AI070721 (S.W.W. and R.E.L.), Cancer Center (CORE)
Support Grant CA21765, and the American Lebanese Syrian
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