Structural insights into a unique inhibitor binding pocket in kinesin spindle protein

Article abstract of DOI:10.1021/ja310377d

Human kinesin Eg5 is a target for drug development in cancer chemotherapy with compounds in phase II clinical trials. These agents bind to a well-characterized allosteric pocket involving the loop L5 region, a structural element in kinesin-5 family member

Full text of DOI:10.1021/ja310377d

Article
pubs.acs.org/JACS
Structural Insights into a Unique Inhibitor Binding Pocket in Kinesin
Spindle Protein
Venkatasubramanian Ulaganathan,§,†,‡ Sandeep K. Talapatra,§,‡ Oliver Rath,§ Andrew Pannifer,∥
David D. Hackney,*,⊥ and Frank Kozielski*,§
§The Molecular Motors Laboratory, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61
1BD, Scotland, U.K.
∥Drug Discovery Programme, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD,
Scotland, U.K.
⊥Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States
S
* Supporting Information
ABSTRACT: Human kinesin Eg5 is a target for drug
development in cancer chemotherapy with compounds in
phase II clinical trials. These agents bind to a well-
characterized allosteric pocket involving the loop L5 region,
a structural element in kinesin-5 family members thought to
provide inhibitor specificity. Using X-ray crystallography,
kinetic, and biophysical methods, we have identified and
characterized a distinct allosteric pocket in Eg5 able to bind
inhibitors with nanomolar Kd. This pocket is formed by key
structural elements thought to be pivotal for force generation
in kinesins and may represent a novel site for therapeutic
intervention in this increasingly well-validated drug target.
identification of a novel inhibitor-binding pocket presents the
opportunity to develop new series of inhibitors that could be
used either alone or in combination with existing Eg5
compounds. Using a benzimidazole series of Eg5 inhibitors
that was shown to bind to the enzyme in the presence of
ispinesib,9 we have used X-ray crystallography to characterize
an allosteric pocket formed by helices α4 and α6. Unexpectedly,
we found that these inhibitors also bind to the known ispinesib
pocket, and using Isothermal Titration Calorimetry (ITC),
Surface Plasmon Resonance (SPR), classical enzyme kinetics
and cell-based assays, we show that the observed inhibition by
the inhibitor is due to its binding at the novel site and that
binding at the ispinesib site is very weak.
1. INTRODUCTION
The kinesins are a superfamily of molecular motors that play a
key role in a diverse range of physiological functions by
transporting intracellular cargos along microtubule (MT)
filaments. Sometimes described as “nano-machines”, kinesins
convert the chemical energy of ATP into mechanical force,
driving their cargos in most cases toward the plus end of the
MT. Several kinesins play a critical role in the cell cycle. Eg5
(also known as KSP and a member of the kinesin-5 subfamily)
is required to form the mitotic spindle and inhibition of Eg5
results in distinctive monoastral spindles, cell cycle arrest and
cell death. Consequently, there is growing interest in the
kinesins as targets for cancer chemotherapy and there are now
potent inhibitors for two kinesin superfamily members, Eg5 and
CENP-E, in phase I and II clinical trials.1−4 The most advanced
drug target of the kinesin superfamily is Eg5 for which there are
several highly specific inhibitors in clinical development such as
ispinesib, SB-743921 and ARRY-520. Without exception they
all target an extensively studied allosteric site about 10 Å away
from the ATP binding pocket in a region formed by helix α2/
loop L5 and helix α3.5 These inhibitors show potent in vivo
activity but drug-resistant mutants have already been identified
in cell culture. The nucleotide-binding pocket of kinesins has
also been targeted for drug development through ATP-
competitive inhibitors,6,7 without binding to the ATP site,
and the existence of other putative sites on Eg5 for intervention
has been predicted by molecular modeling techniques.8 The
2. MATERIALS AND METHODS
Characterization and Purity of BI8. BI8 was synthesized in six
steps as previously described (Supporting Information section and
Scheme S1).10 The compound was judged to be 94.1% pure from
LC−MS. 1H NMR (400 MHz, MeOD) δ 8.12 ppm (s, 1H), 7.73 (d, J
= 8.4 Hz, 2H), 7.49 (m, 3H), 7.25−7.28(m 2H), 7.05 (t, J = 9.1 Hz,
1H), 6.91 (d, J = 8.3 Hz, 1H), 6.74 (d, J = 17.4 Hz,1H), 5.66 (s, 2H),
4.84(s, 1H), 3.83(s, 3H). 19F NMR (376 MHz, MeOD) Φ −61.55 (s),
−135.38 (s). 13C NMR (125 MHz, MeOD): δ 169.35, 152.98, 152.39,
143.21, 140.44, 136.32, 133.78, 133.15, 133.05, 132.08, 127.23, 126.82,
126.52, 125.69, 124.91, 122.75, 122.24, 117.18, 115.39, 113.59, 106.88
Received: October 20, 2012
Published: January 10, 2013
© 2013 American Chemical Society
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Figure 1. Biochemical characterization of the Eg5-BI8 complex. (a) Chemical structure and systematic name of the benzimidazole-based Eg5
inhibitor BI8. (b) Concentration−response plots for the inhibition of the basal (IC50 = 458.8 28.3 nM) and (c) MT-stimulated Eg5 ATPase
activities at 150 mM NaCl (IC50 = 590.2 26.0 nM; blue circles), 100 mM NaCl (1.3 0.1 μM; red), 50 mM (20.9 2.0 μM; olive) and without
addition of salt (partial inhibition; black). (d) Induction of characteristic monoastral spindles by BI8 in HCT116 cells. Immunostaining of α-tubulin
in green, phospho-Histon H3 in red and chromosomes in blue.
55.29, 47.08. HR-MS (m/z): [M]+ calculated for C23H17N3F4O3,
460.1279; found, 460.1272. Analysis (% calculated, % found for
C23H17N3F4O3): C (60.13, 58.42), H (3.73, 3.74), N (9.15, 8.27).
Protein Crystallization. A total of 50 μL of Eg5 (10 mg/mL) was
mixed carefully with 2 μL of BI8 (50 mM) and the mixture was
incubated at 4 °C for 2 h. The mixture was then centrifuged at 13 000g
for 5 min to remove any undissolved inhibitor. Diffraction quality
single crystals of Eg5-BI8 complex were obtained by sitting drop, vapor
diffusion using 1 μL of protein complex and 1 μL of the reservoir
solution (0.1 M 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid
(HEPES) buffer at pH 7.5, 20% (w/v) polyethylene glycol (PEG)
3350 and 0.1 M MgCl2) in VDX plates (Hampton Research) at 19 °C.
The crystal was cryoprotected using 0.12 M HEPES buffer at pH 7.5,
22% (w/v) PEG 3350, 0.12 M MgCl2 and 15% (v/v) PEG-400 and
flash frozen in liquid nitrogen.
Data Collection and Refinement. Data were collected at
Diamond Light Source at beamline I02 and processed using XDS.11
The integrated data were scaled using SCALA. The Eg5-BI8 complex
belonged to space group C2 with one molecule in the asymmetric unit.
The structure was solved by molecular replacement (Molrep12) using a
single chain of Eg5 in complex with STLC (PDB ID 2WOG). A single
subunit was positioned and refined using REFMAC513 and PHENIX
refine.14 The Rfree was calculated by using 5% of data. The σA weighted
electron density map and difference map were inspected and the
model was improved using COOT.15 The ligand coordinates and cif
dictionaries were prepared using the PRODRG server.16 Data
collection and refinement statistics are given in Table S1. The
structure is very well refined with an Rfree of 23.3% with 97.6% of all
residues in the preferred region of the Ramachandran plot, 1.7% in the
allowed region and 0.7% outliers. The final model comprises residues
18−363, one molecule of Mg2+ADP, one HEPES moiety and two BI8
molecules. Residues 1−17 preceding the motor domain, 174−181
(loop L7) and 272−286 (loop L11) are missing due to high flexibility
in these regions. Mg2+ADP is bound in the nucleotide-binding region
with the Mg ion being octahedrally coordinated. Figures were
prepared using Pymol.17 Coordinates and structure factors of the
Eg5-BI8 complex have been deposited with the PDB under 3ZCW.
Surface Plasmon Resonance Competition Assays. Experi-
ments were performed at 25 °C using a Biacore T100 (Biacore) in
running buffer (50 mM TRIS, pH 7.0, 200 mM NaCl, 1 mM MgCl2,
0.5 mM EDTA and 0.05% surfactant P20) supplemented with 1%
DMSO before each run with a flow rate of 30 μL/min. Experiments
were performed at least in duplicate. Double His-tagged Eg5 (200 nM)
was immobilized on the surface of the NTA sensor chip charged with
Ni2+ to approximately 2000 RU. The sensor surface was regenerated
between each experiment with a 60 and 120 s injection of 1 M
Imidazole and 10 mM HEPES, pH 8.3, 150 mM NaCl, 350 mM
EDTA and 0.005% P20, respectively. For analyte preparation, the
compounds were prepared in DMSO as a 50 mM stock solution.
Concentration series for each inhibitor were prepared ensuring a
consistent final DMSO concentration of 1%. To correct for the minor
DMSO volume effect, a DMSO calibration series was prepared using
0.5% and 1.8% of DMSO in running buffer. A set of 8 aliquots of 700
μL each was prepared using the two DMSO concentrations for the
DMSO standard curve. Protein immobilization on the chip was carried
out at a flow rate of 5 μL/min, whereas for the experiments, a flow rate
of 30 μL/min was used. The steady state analyses of molecular
interactions were calculated after subtraction of backgrounds (inhibitor
binding to a control flow cell) using the BIAcore T100 Evaluation
software and Kaleidagraph 4.0. The binding constant KD was
determined according to the respective binding model. To investigate
two-site binding and to analyze the affinity for both sites, BI8 was
measured in a range from 0 to 528 μM. BI8 was first injected to
determine the binding affinity of the two sites followed by a single
ispinesib injection at 10 μM. As hypothesized, ispinesib competitively
removed BI8 from the allosteric pocket saturating the site at a
concentration of 10 μM. BI8 was then injected at the same range to
determine the KD for the second novel site. Similarly, as a negative
control, monastrol was used at a concentration series ranging from 0
to 40 μM. Two independent runs of 200 nM Eg5 immobilized at 2000
RU over the same NTA sensor surface charged with Ni2+ for 12 h had
a loss of only ∼100−150 RU providing evidence for the stability of
binding of double histidine tagged Eg5 (Eg51−368-12His) over time on
the sensor surface. The sensorgram was allowed to proceed for a
longer period after each injection to ensure that the injection had
completely finished.
Biophysical Experiments. All fluorescence experiments were
performed in A25 buffer with 25 mM KCl and 125 mM NaCl.
Fluorescence spectra and titrations were performed at room
temperature with a Tecan Saphire2 fluorimeter with band widths of
10 and 5 nm for excitation and emission. Transient experiments were
performed with an Applied Photophysics SX20 stopped flow
fluorimeter at 25 °C using excitation at 280 nm and a 305 nm long
pass filter for intrinsic fluorescence of tyrosine and tryptophan.
3. RESULTS
A series of benzimidazole inhibitors of Eg5 were disclosed
which were able to bind Eg5 in the presence of ispinesib,9
suggesting the presence of a second binding site in the enzyme.
We synthesized a set of benzimidazole analogues and
determined their IC50 values for the inhibition of the basal
and MT-stimulated ATPase activity. One particular compound,
named BI8 (BenzImidazole number 8) (Figure 1a; Supple-
mentary Methods and Scheme 1), demonstrated nanomolar
potency inhibiting the basal ATPase activity (Figure 1b) and
reasonable physicochemical properties. Interestingly, the IC50
for the inhibition of the MT-stimulated ATPase activity varied
depending on the ionic strength (Figure 1c), a behavior
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Figure 2. Characterization of the α4/α6 inhibitor-binding pocket in Eg5. (a) Back view of Eg5 showing the allosteric binding pocket occupied by BI8
(colored in green), formed by helix α4 of the switch II cluster and helix α6 (both colored in red), preceding the neck-linker. (b) Electron density
omit map (colored in blue) contoured at 3 σ for BI8 bound in this pocket. (c) Conformational changes induced upon binding of BI8 in the helix α4/
α6 pocket (purple), compared to native Eg5 (light blue). (d) Magnification of the inhibitor-binding pocket. BI8 is shown in green and residues
forming the inhibitor-binding pocket are colored in purple. Water molecules are shown as red spheres and hydrogen-bond interactions are shown as
dotted lines.
previously observed for monastrol and which was explained by
altering the strength of the electrostatic interactions between
the Eg5 motor and MTs.18 In HCT116 cells, BI8 shows a GI50
the trifluoromethylbenzyl group of the inhibitor. Analysis of the
structure using the Glide module from the Schrodinger
modeling software suite identified these aromatic residues as
having particularly favorable interactions with the ligand. In
addition, further hydrophobic interactions are formed. The 3-
fluoro-4-methoxyphenyl group is within van der Waals distance
of the side chain of Leu292 (α4) and Leu295 is buried upon
binding of BI8. BI8 also forms a network of polar interactions
to the protein and to solvent molecules. The carboxylate group
makes a direct hydrogen bond interaction with Thr300 and also
to a water molecule, mediating an interaction with the side
chains of Arg297 (both helix α4). The solvent exposure of the
carboxylate may attenuate the binding energy associated with
these polar interactions but the interatomic distances are in the
range expected for a strong hydrogen bond. The solvent-
exposed nitrogen atom of the imidazole ring makes another
water-mediated hydrogen bond interaction to the phenolic
moiety of Tyr352. The amino group of the methoxyphenyl
moiety forms a hydrogen bond interaction with the hydroxyl
group of Tyr104 (β3). The 3-fluoro substituent of the
methoxyphenyl group shows weak van der Waals interactions
with the side chain of Ile288 and the main chain oxygen of
Asn289 (both α4). Finally, the secondary amino group of BI8
forms a strong hydrogen bond interaction with the hydroxyl
group of Tyr104 (β3) and a weak hydrogen bond is formed
between the methoxy group of BI8 and Asn289.
value of 25
3 μM, indicating weak cell permeability. BI8
induces monoastral spindles (Figure 1d), the typical phenotype
observed for Eg5 inhibitors. To understand the novel binding
characteristics of BI8 identified in kinetic experiments, we
determined the structures of the Eg5-BI8 complex to 1.7 Å.
Data collection and refinement statistics are summarized in
Table S1. Clear well-defined electron density was observed for
BI8 in a novel site but unexpectedly BI8 was also observed to
bind in a pocket partially overlapping with the known ispinesib
site.19
An Allosteric Inhibitor-Binding Site Formed by
Helices α4 and α6. The BI8 inhibitor-binding pocket is
formed by helix α4 of the switch II cluster, helix α6 preceding
the neck-linker region and strand β3 (Figure 2a). The electron
density map for the inhibitor is very well-defined with the entire
molecule apparent (Figure 2b). Both helices α4 and α6 move
outward by about 2 Å to accommodate the inhibitor (Figure
2c). The neck linker region is undocked but structured
representing the intermediate inhibitor-bound state. The most
striking feature of the complex between the inhibitor and
residues of this binding pocket is the extensive network of
aromatic interactions (Figure 2d): The side chain of Tyr352
forms a face-to-face stacking interaction with the benzimidazole
ring while Tyr104 forms another face-to-face interaction with
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Figure 3. Characterization of the second allosteric binding pocket. (a) Eg5 in complex with Mg2+ADP (pink) and BI8 (green) bound in the allosteric
inhibitor-binding pocket formed by helices α2 and α3 and loops L7 as well as L9. BI8 bound in the novel site is colored in gray. (b) Overlay of Eg5-
BI8 and Eg5-ispinesib complexes in the inhibitor-binding region. There is a significant overlap of BI8 (green color) with ispinesib (shaded in yellow).
(c) Fo − Fc omit map of BI8 bound in the inhibitor-binding pocket, contoured at 3σ (colored in blue). The methoxy group has no density in the
omit map indicating that its position is flexible. (d) Binding of BI8 to the allosteric pocket leads to significant proximal structural changes in loop L5
and helix α3. (e) Stereoplot of the magnification of the inhibitor-binding pocket showing residues involved in BI8 binding.
The Second Inhibitor-Binding Site Overlaps with the
Established Allosteric Ispinesib Pocket. The second
pocket is formed by helices α2 and α3 and loops L7 and L9
(Figure 3a) and partially overlaps with the binding site of well-
characterized Eg5 inhibitors such as ispinesib (Figure 3b) and
monastrol. Electron density for this site appeared only toward
the end of the refinement and parts of the molecule, such as the
methoxy group, are disordered (Figure 3c) suggesting either
greater flexibility or lower occupancy for BI8. Although loop L5
does not seem to be directly involved in inhibitor binding, it
displays a new conformation that differs from the ones
described for the native Eg5 structure or the conformation
described for other Eg5-inhibitor bound complexes (Figure
3d). Trp127, which is solvent exposed in the native Eg5
structure, moves by 7.2 Å (Cα−Cα) and now interacts with
Asp130 (loop L5) and Glu215 (α3). The upper part of helix α3
is rotated by approximately 14° around an axis close to the apex
of the helix to accommodate the inhibitor leading to a shift of
about 7 Å at the top of helix α3 (Figure 3d). These radical
changes on BI8 binding have not been observed previously with
any other Eg5 structure and are most likely due to BI8 binding.
In contrast to the novel site, BI8 binding at this site does not
demonstrate the network of aromatic interactions and this
would be expected to reduce the binding energy by
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Figure 4. ITC analyses of ligand binding to Eg5. Raw isothermal titration calorimetry data (upper panels) and normalized ITC data for titrations
plotted versus the molar ratio of titrant/protein (lower panels) demonstrating saturable exothermic and endothermic evolution of heat upon
sequential additions of (a) monastrol (500 μM) and (b) ispinesib (500 μM) to Eg5. The reverse experiment was performed by sequential additions
of (c) ispinesib (500 μM) and (d) monastrol (500 μM) to Eg5. Additions of (e) BI8 (500 μM) followed by (f) ispinesib (500 μM) to Eg5. The
reverse experiment with sequential additions of (g) ispinesib (500 μM) and (h) BI8 (500 μM) to Eg5. (i) Thermodynamic cycle for ligand
(ispinesib/monastrol) binding to Eg5, showing the relationship between the enthalpic changes of binding on monastrol, ispinesib and ispinesib
competitively replacing monastrol from the allosteric binding site. (j) Summary of ITC data for BI8 in combination with two other Eg5-specific
inhibitors, ispinesib and monastrol. Data analysis indicates that the binding data fit well to a two binding site model for BI8 and a single binding-site
model for ispinesib.
comparison. There are some hydrophobic interactions, notably
with the side chains of Leu160 and Leu172 and also a stacking
interaction between Arg221 and the methoxybenzene group of
BI8. Hydrogen bonding interactions are also present with the
anilinic nitrogen forming a water-mediated hydrogen bond to
Ser237. The 5-carboxylic acid group of the benzimidazole
moiety is solvent exposed and displays several hydrogen bonds
with nearby water molecules (Figure 3e) but these water
molecules do not appear to mediate interaction with the
protein.
The observation that BI8 binds at the known ispinesib site
raises the possibility that the potent inhibition measured in
biochemical assays could be due to binding at this site and the
binding at the novel site could be weak. To distinguish which
site was generating the potent inhibition, we used two label-free
biophysical approaches and fluorescence techniques to
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Figure 5. SPR competition studies of BI8, ispinesib and monastrol to double hexahistidine tagged Eg5 immobilized on NTA surface. (a) SPR
response curves for increasing BI8 concentrations (0−528 μM) monitored on a surface with 2000 RU of double histidine tagged Eg5. (b) SPR
response curves for various concentrations of BI8 from 0 to 528 μM monitored on a surface with 2000 RU of double histidine tagged Eg5 after
injection of a saturating concentration of ispinesib indicating BI8 binding to a novel second site. (c) Ispinesib injection at 10 μM before (black) and
after (red) second BI8 injection. The first ispinesib injection competitively removes BI8 from the mutual site which allows BI8 to bind only to the
second novel site, whereas the second ispinesib injection with low or no response confirmed that ispinesib remains bound to Eg5 through second
BI8 injection. d) SPR response curves for various concentrations of monastrol injection from 0 to 40 μM monitored on a surface with 2000 RU of
double histidine tagged Eg5. (e) SPR response curves for various concentrations of second BI8 injection from 0 to 40 μM monitored on a surface
with 2000 RU of double histidine tagged Eg5 after injection of a saturating concentration of ispinesib indicating that monastrol cannot bind due to its
lower affinity compared to ispinesib to the same binding site. (f) Ispinesib injection at 10 μM before (black) and after (red) second monastrol
injection. The first ispinesib injection competitively removes monastrol from the allosteric inhibitor site. Therefore, monastrol shows no response
upon second injection. Second ispinesib injection with low or no response confirmed that ispinesib remains bound to Eg5 throughout second
monastrol injection. (g) The table represents Kd for BI8 and monastrol binding, using the steady state affinity model, experimental Rmax for each
measurement and χ2 distributions around 10 suggesting a good fit of the model to the experimental data. All measurements for SPR binding were
reference corrected.
demonstrate that there is a single high affinity site and binding
at this novel site accounted for the measured inhibition.
Isothermal Titration Calorimetry Competition Assays.
To dissect the contribution made to the potent biochemical
inhibition by each of the inhibitor-binding pockets, we
investigated the interaction between Eg5 and BI8 using ITC
competition assays. By blockading the ispinesib site using
saturating concentrations of ispinesib and then titrating in BI8,
the binding of BI8 to the novel pocket can be isolated and
characterized thermodynamically. We first carried out a proof
of principle experiment of this “ispinesib blockade” approach by
testing whether ispinesib could block the binding of monastrol,
a moderately potent inhibitor known to bind at the ispinesib
site (Figure 4a−d). Monastrol was shown to bind the Eg5-ADP
complex with a Kd of 1.4 μM, in agreement with recently
published data. Monastrol binding exhibited a strong enthalpy
contribution to the Gibbs Free Energy of binding. The Kd of
ispinesib cannot be accurately estimated due to the step-like
curve but is approximately 10 nM and is entropically
dominated. In both cases, a 1:1, ligand/protein stoichiometry
was observed, in agreement with existing crystal structures. We
then saturated the Eg5-ADP complex with ispinesib and titrated
monastrol into the saturated Eg5-ADP-ispinesib complex and
observed no monastrol binding, consistent with the site being
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Figure 6. Intrinsic fluorescence titration of BI8 binding. (a) Emission spectra of Eg5 with BI8 and ispinesib. Spectra of 1.7 μM Eg5 with 0.2 mM
ADP alone (black), 4 μM ispinesib (orange), 4 μM BI8 (green) or 4 μM ispinesib and 4 μM BI8 (blue). Excitation was at 280 nm. (b) Fluorescence
titration of Eg5 with BI8 and ispinesib. Eg5 (6.4 μM) with 0.2 mM ADP was titrated with BI8 or ispinesib. Excitation was at 280 nm. Emission was at
310 nm for titration with BI8 alone (triangles) and at 380 nm for sequential addition of ispinesib (squares) followed by BI8 (circles). The theoretical
curves were fit using the full quadratic expression for mutual depletion. (c) Kinetics of BI8 binding to Eg5. Eg5 with 0.2 mM ADP was mixed with
BI8 for final concentrations of 0.5 μM Eg5 and 5 or 10 μM BI8. Excitation was at 280 nm with a 305 long pass emission filter. The first-order rate
constant for the decrease in fluorescence on binding of BI8 is plotted versus the average concentration of free BI8 during the transient. The intercept
on the Y-axis (solid triangle) is fixed at 0.14 s−1 for the dissociation rate constant. (d) Influence of BI8 on binding kinetics of ispinesib. Eg5 (1 μM)
with 0.2 mM ADP and either 0 or 20 μM BI8 was mixed with an equal volume of 8 μM ispinesib. Excitation was at 280 nm with a 305 long pass
emission filter.
inaccessible due to blockade by the more potent compound.
We then performed the reverse experiment by saturating the
Eg5-ADP complex with monastrol and titrating in ispinesib. As
expected, ispinesib binding was observed, consistent with
ispinesib displacing the weaker-binding monastrol. The
endothermic binding observed in this second competition
assay is also consistent with displacement of monastrol (Figure
4i). Having demonstrated the validity of the ispinesib blockade,
we then investigated the binding of BI8 to the Eg5-ADP
complex in the presence of ispinesib (Figure 4e−h).
to the preformed Eg5-BI8 complex showed that ispinesib could
bind to Eg5 with a ratio of 1:1, consistent with it replacing BI8
from one binding site only, probably the overlapping site (helix
α2/loop L5/helix α3) (Figure 4f).
The reverse experiment showed that after injection of
ispinesib (Figure 4g), BI8 could still bind to the preformed
Eg5-ispinesib complex with a binding stoichiometry of 1:1
(Figure 4h). The data indicate that because the first allosteric
binding pocket (helix α2/loop L5/helix α3) is occupied by
ispinesib, BI8 with its significantly higher Kd value can only
bind to the second, still unoccupied inhibitor-binding pocket
(helices α4 and α6). The Kd for this binding event is 3.3 μM,
and although not in exact agreement with either of the values
obtained when titrating BI8 alone, we suggest that this
corresponds to the site with the lower dissociation constant
of 0.65 μM since affinity at the weaker site is not possible even
to estimate accurately with ITC. Therefore, the physiologically
relevant site is the novel allosteric pocket. The stoichiometry of
binding (n), Kd values, entropic and enthalpic values are
First, we titrated in BI8, and obtained data that was best
modeled using a sequential model with two sites in agreement
with our crystal structure (Figure 4e). The data indicate the
existence of one strong (Kd = 0.65 0.01 μM) and one weaker
(Kd = 27.5
3.9 μM) Eg5 inhibitor-binding pocket. This
second value is an estimate as the concentrations of BI8 and
protein are too low to accurately estimate the true value and it
could therefore be significantly higher and the limiting
solubility of BI8 prevents a suitable experimental setup to
obtain a more accurate value. Subsequent injection of ispinesib
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represented in Figure 4i. To add more evidence to this
conclusion, two further orthogonal techniques were used.
Surface Plasmon Resonance Analysis of BI8 Binding
to Eg5. A similar ispinesib blockade approach was used to
distinguish binding affinities for BI8 at the two sites using SPR
(Figure 5a−f). First, monastrol/ispinesib control experiments
were carried out to demonstrate the validity of this approach.
Then, two-site binding to Eg5 by BI8 was demonstrated by a
series of BI8 injections with a maximum concentration of 0.528
mM. Fitting the data with a steady state affinity model
generated Kd values of 470 nM and ∼34 μM, respectively
(Figure 5g) comparable to the affinities estimated from ITC
measurements (Figure 4i). The ispinesib site was then blocked
by injection with 10 μM ispinesib and subsequent extended
periods of injection with ispinesib-free buffer demonstrated a
very slow dissociation from Eg5, resulting in this site being
blocked for the duration of subsequent experiments. BI8
injection following ispinesib binding showed a maximal
response approximately half that seen in the initial experiment
without ispinesib, indicating a 1:1 stoichiometry. Fitting the
data with a one site steady-state affinity model resulted in a Kd
of 640 nM, similar to the more potent of the two values
measured in the absence of ispinesib and to the more potent
value observed in ITC.
Eg5 Has One Tight Binding Site for BI8 That Can Be
Detected by Quenching of Intrinsic Fluorescence.
Ispinesib quenches the intrinsic fluorescence of Eg520 as
illustrated by the spectra in Figure 6a. Titration of the
fluorescence of Eg5 with ispinesib-serves as a control for tight
binding with a stoichiometry of 1:1 (Figure 6b, squares) using
excitation at 280 nm and emission at 380 nm. At an Eg5
concentration of 6.4 μM, ispinesib titrates with an equivalence
point at 8.4 μM that is in reasonable agreement with the
concentration of 6.4 μM determined by the Bradford assay.
Figure 6a also indicates that BI8 significantly quenches the
fluorescence of Eg5 at emission wavelengths <370 nm, but has
little effect at longer wavelengths. The decrease in fluorescence
emission at 310 nm can be used to titrate BI8 binding to Eg5
(Figure 6b, diamonds). An unrestrained fit to a full mutual
depletion model with one site yielded an Eg5 concentration, Kd
and maximum decrease of 7.5 μM, 0.71 μM and 60%,
respectively. This establishes a 1:1 stoichiometry for tight
binding of BI8, but cannot provide a fully accurate Kd because
of the high degree of mutual depletion and the uncertain partial
contribution of binding of BI8 to the weaker site. The weaker
site for BI8 that is observed by ITC apparently does not titrate
in this range, although the continued slow decrease in
fluorescence at higher BI8 concentration may represent
increasing partial occupancy of the weak site. At a lower Eg5
concentration of 1.07 μM Eg5, the influence of mutual
depletion is decreased and the fit yields an Eg5 concentration
and Kd of 0.96 and 0.34 μM, respectively (Figure 6b insert).
The decrease in fluorescence on BI8 binding can also be used
to determine the rate constant, kon, for BI8 binding by stopped
flow. Mixing Eg5 with an excess of BI8 produces a rapid
decrease in intrinsic fluorescence that obeys first-order kinetics.
The observed rate increases linearly with BI8 concentration
(Figure 6c) with a slope of 0.24 M−1 s−1 that defines the
bimolecular association rate constant for binding of BI8 to the
tight site. The rate of BI8 dissociation can be calculated as
∼0.14 s−1 using Kd = koff/kon and values of 0.34 μM and 0.24
M−1 s−1 for Kd and kon, respectively. The intercept in Figure 6c
is plotted at this value of 0.14 s−1, but the intercept is not
significantly different from zero by extrapolation. Additionally,
preloading of BI8 at the tight site has little effect on the rate of
ispinesib binding (Figure 6d)
4. DISCUSSION
The crystal structure of the Eg5-ADP-BI8 complex reveals a
second druggable inhibitor binding pocket ∼19 Å from the
extensively studied allosteric ispinesib site. The existence of this
site, located between helices α4 and α6 of the Eg5 motor
domain, has previously been proposed for biaryl Eg5 inhibitors
using biochemical and modeling techniques.6,7 These biaryl
inhibitors have the unusual property of being both allosteric
and competitive with ATP. This is in marked contrast to the
BI8 class of benzimidazole inhibitors that have been shown to
be uncompetitive with ATP. It would be of considerable
interest to obtain detailed structural information on the binding
of biaryl inhibitors to determine how these two classes of
inhibitors can bind to the same region on Eg5, yet alter the
kinetics in such different ways, however biaryl compounds do
not seem to bind to Eg5 in the absence of MTs. The existence
of a similar site (and other putative novel binding pockets) has
also been suggested using molecular modeling simulations.8 We
now provide the structural basis for this pocket and a detailed
understanding of the Eg5-inhibitor interactions. Unexpectedly,
inhibitor binding was also observed in the ispinesib site, but
using three orthogonal biophysical and biochemical techniques,
we have shown that there is a single high affinity site, that high-
affinity binding is the result of binding to the α4/α6 site, and
that this site is responsible for the observed inhibition. High
affinity binding at this site appears to be driven by both a
network of aromatic interactions and hydrogen bonding with
the carboxylate group of BI8. These interactions are much less
prominent in the second pocket, perhaps explaining the weaker
electron density and lower affinity of the inhibitor at this site.
The binding mode of ispinesib and other allosteric inhibitors
to the pocket formed by helix α2/loop L5/helix α3 and the
sequence of structural changes leading to the final inhibitor
bound state have been revealed in considerable detail using
steady state and presteady state kinetics as well as biophysical
and structural methods.18−23 The location of the site between
helices α4 and α6 immediately suggests a mechanism for the
inhibitory effect of the compound. Small changes in the
nucleotide-binding pocket induced by ATP hydrolysis are
thought to lead to amplified structural changes in the switch II
cluster and neck-linker region which generate the molecular
motion responsible for shifting the two antiparallel MTs apart
to form the bipolar spindle. By binding between helix α4 of the
switch II cluster and helix α6 preceding the neck-linker, BI8
may lock Eg5 in a conformation that is no longer able to
undergo the nucleotide-dependent conformational changes
required for the completion of the catalytic cycle. The
mechanism by which this inhibitor can exert its physiological
effect on ATPase activity is emphasized by molecular dynamics
simulations, which demonstrated that residues in this pocket
directly interact with loop L13. This loop is highly correlated
with the Switch-I and Switch-II regions whose movement is
critical allowing movement of the neck linker region.
Furthermore, the neck linker region in the Eg5-BI8 complex
is locked in the intermediate state that was also observed for
inhibitors binding in the ispinesib allosteric binding pocket.22 In
the Eg5-STLC complex, the transition from the native to the
inhibitor bound conformation goes through a sequence of
structural changes from the native state without inhibitor to the
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intermediate state in which helix α4 is in the obstructive
position and as a consequence the neck-linker is undocked.
Finally, in the final inhibitor-bound state, the switch II cluster is
in the permissive position allowing the neck linker to dock to
the motor domain from the initial perpendicular orientation
that involves the swinging of the neck-linker of about 32 Å.21
Although allosteric inhibitors such as ispinesib, monastrol, and
STLC as opposed to BI8 target two distinct sites on Eg5, these
complexes reveal the same intermediate state; in both
complexes, ADP-release is inhibited and the monoastral spindle
phenotype is observed in cells. Therefore, we hypothesize that
inhibitors like BI8, which target a different site, act through a
similar mechanism. Further investigations will be necessary to
provide insights into the sequence of conformational changes
occurring in the catalytic domain in support to our hypothesis.
during SPR experiments. This project was supported by CR-
UK.
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5. CONCLUSION
This structural characterization of the α4/α6 inhibitor-binding
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benzimidazole series of inhibitors, creates the opportunity for
both structure-based and ligand-based approaches to develop
novel inhibitor series for Eg5. The significant conformational
changes on ligand binding generate a well-defined “druggable”
pocket and the crystal structure now enables the application of
structure-based design and screening approaches. Importantly,
the development of inhibitors at this site has the potential for
delaying the onset of resistance to drugs targeting the ispinesib
site. This could be particularly relevant since mutants highly
resistant to ispinesib treatment have been observed in cell
culture and combination therapy is a well-established approach
to slow down the onset of resistance. With the existing
benizimidazole inhibitors demonstrating inhibition of the
ATPase activity in the nanomolar range, this is an exciting
possibility toward the development of new drug candidates.
Abbreviations. BI8, benzimidazole number 8; CENP-E,
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AUTHOR INFORMATION
Corresponding Author
f.kozielski@beatson.gla.ac.uk; ddh@andrew.cmu.edu
■
(14) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis,
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Present Address
†School of Chemical and Biotechnology, SASTRA University,
Tirumalaisamudram, Thanjavur 613401, Tamilnadu, India.
Author Contributions
‡These authors contributed equally.
(17) The PyMOL Molecular Graphics System, Version 1.5.0.4,
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Diamond Light Source for access to beamlines I02
(MX6683) and I03 that contributed to the results presented
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comments on ITC and Dr. Marta Dozynkiewicz for her support
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