Principles of ligand binding within a completely buried cavity in HIF2α PAS-B

Article abstract of DOI:10.1021/ja9073062

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment. The C-terminal Per-ARNT-Sim (PAS) domain of HIF2α (HIF2α PAS-B

Full text of DOI:10.1021/ja9073062

Published on Web 11/11/2009
Principles of Ligand Binding within a Completely Buried
Cavity in HIF2r PAS-B
Jason Key,† Thomas H. Scheuermann,† Peter C. Anderson,‡ Valerie Daggett,‡,§ and
Kevin H. Gardner*,†
Departments of Biochemistry and Pharmacology, UniVersity of Texas Southwestern Medical Center,
5323 Harry Hines BouleVard, Dallas, Texas 75390-8816, Biomedical and Health Informatics
Program, UniVersity of Washington, Box 357240, Seattle, Washington 98195-7240, and Department
of Bioengineering, UniVersity of Washington, Box 355013, Seattle, Washington 98195-5013
Received August 28, 2009; E-mail: Kevin.Gardner@utsouthwestern.edu
Abstract: Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the
metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment.
The C-terminal Per-ARNT-Sim (PAS) domain of HIF2R (HIF2R PAS-B) contains a preformed solvent-
inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer.
To better understand how small molecules bind within this domain, we examined the structures and
equilibrium and transition-state thermodynamics of HIF2R PAS-B with several artificial ligands using
isothermal titration calorimetry, NMR exchange spectroscopy, and X-ray crystallography. Rapid association
rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to
permit ligand access, despite the closed conformation observed in the NMR and crystal structures.
Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest
a binding-competent transition state characterized by increased structural disorder. Finally, molecular
dynamics simulations reveal conversion between open and closed conformations of the protein and pathways
of ligand entry into the binding pocket.
Introduction
Both HIFR and ARNT contain an N-terminal bHLH DNA
binding domain and two adjacent PAS domains, referred to as
PAS-A and PAS-B. PAS domains are structural modules found
in proteins from all kingdoms of life that have significant
structural homology despite little conservation of amino acid
sequence. PAS domains often serve as protein-protein interac-
tion components, as in the case of HIFR and ARNT, where
they are needed for assembly of the HIF2 PAS-B heterodimer
via their ꢀ-sheet surfaces.9-11 For many PAS domains, these
protein/protein interactions can be environmentally regulated
by internally bound small molecules or cofactors,12 including
flavin adenine dinucleotide (FAD), flavin mononucleotide
(FMN),13 heme,14 or 4-hydroxycinnamic acid.15 For example,
flavin-binding PAS domains serve as blue-light photoreceptors
A cell’s ability to sense and respond to environmental stimuli
is critical for survival. In metazoans, oxygen concentration is
monitored by the hypoxic response pathway, where hypoxia-
inducible factors (HIFs) regulate numerous genes in response
to oxygen.1 HIF proteins are heterodimeric basic helix-loop-
helix Per-ARNT-Sim (bHLH-PAS) transcription factors2 whose
activity is regulated in an oxygen-dependent manner.3 With
adequate oxygen levels (normoxia), oxygen-dependent hydroxy-
lations of the HIFR subunit block interactions with CBP/p300-
family coactivator proteins4 and also trigger its ubiquitin-
mediated degradation.5 Under low-oxygen conditions (hypoxia),
unmodified HIFR subunits bind the aryl hydrocarbon receptor
nuclear translocator (ARNT, also known as HIFꢀ) protein,
forming the transcriptionally active heterodimer. Thus, cells
translate environmental oxygen concentration into altered gene
expression. HIF proteins are of considerable interest because
of their roles in promoting solid growth6,7 and resistance to
chemotherapy.8
(6) Bos, R.; van der Groep, P.; Greijer, A. E.; Shvarts, A.; Meijer, S.;
Pinedo, H. M.; Semenza, G. L.; van Diest, P. J.; van der Wall, E.
Cancer 2003, 97, 1573.
(7) Zagzag, D.; Zhong, H.; Scalzitti, J. M.; Laughner, E.; Simons, J. W.;
Semenza, G. L. Cancer 2000, 88, 2606.
(8) Sullivan, R.; Pare, G. C.; Frederiksen, L. J.; Semenza, G. L.; Graham,
C. H. Mol. Cancer Ther. 2008, 7, 1961.
† University of Texas Southwestern Medical Center.
(9) Erbel, P. J.; Card, P. B.; Karakuzu, O.; Bruick, R. K.; Gardner, K. H.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15504.
‡ Biomedical and Health Informatics Program, University of Washington.
§ Department of Bioengineering, University of Washington.
(1) Semenza, G. L. Physiology (Bethesda) 2004, 19, 176.
(2) Wang, G. L.; Jiang, B. H.; Rue, E. A.; Semenza, G. L. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 5510.
(10) Scheuermann, T. H.; Tomchick, D. R.; Machius, M.; Guo, Y.; Bruick,
R. K.; Gardner, K. H. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 450.
(11) Yang, J.; Zhang, L.; Erbel, P. J.; Gardner, K. H.; Ding, K.; Garcia,
J. A.; Bruick, R. K. J. Biol. Chem. 2005, 280, 36047.
(12) Denison, M. S.; Nagy, S. R. Annu. ReV. Pharmacol. Toxicol. 2003,
43, 309.
(3) Bruick, R. K.; McKnight, S. L. Science 2001, 294, 1337.
(4) Freedman, S. J.; Sun, Z. Y.; Poy, F.; Kung, A. L.; Livingston, D. M.;
Wagner, G.; Eck, M. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5367.
(5) Kaelin, W. G., Jr. Biochem. Biophys. Res. Commun. 2005, 338, 627.
(13) Crosson, S.; Rajagopal, S.; Moffat, K. Biochemistry 2003, 42, 2.
(14) Gilles-Gonzalez, M. A.; Gonzalez, G. J. Inorg. Biochem. 2005, 99, 1.
9
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J. AM. CHEM. SOC. 2009, 131, 17647–17654 17647

A R T I C L E S
Key et al.
by crystal and solution NMR structures. In the MD simulations,
the HIF2R PAS-B protein populates both “open” and “closed”
conformers, with the closed form being preferred. We identified
ligand entry/exit pathways by observing routes taken by solvent
water being transferred to/from the ligand binding pocket. This
transfer takes place primarily in an open conformation of the
protein, although there is a minor route in the closed form. Taken
together, these data provide structural and energetic character-
izations of a protein that is apparently poised to bind natural or
artificial regulatory ligands and cofactors.
Figure 1. Crystal structures of HIF2R PAS-B reveal an internal solvent-
filled cavity that binds artificial ligands. (a) Crystal structure of the PAS B
domain of HIF2R in the apo form, showing the eight bound solvent atoms
within the core of the domain (red spheres). (b) Cutaway of the HIF-2R
surface that reveals the 290 Å3 internal cavity, depicted as a cyan-colored
surface.
Experimental Methods
Sample Preparation. 15N-labeled HIF2R PAS-B was purified
as described elsewhere.10 Briefly, an expression construct encoding
HIF2R PAS-B residues 240-350 was generated through PCR
amplification from cDNA ligated into the pHis6x-GB1 expression
plasmid.20 For ITC studies, protein was expressed in Luria broth,
while NMR studies utilized 15N-labeled protein as produced by
expression in M9 medium containing 15NH4Cl (1 g/L) as the sole
nitrogen source. Protein was purified by nickel chromatography and,
in bacteria and plants16 and assess energy content in bacteria,17
while heme-based PAS domains monitor dissolved gases.18
The sensory roles of many PAS domains, including those
from the HIF proteins, remain an open question, as they have
often been isolated without cofactors. Notably, recent structural
work on the HIF2R PAS-B domain shows that it contains a
290 Å3 water-filled cavity at its core, isolated from bulk solvent10
(Figure 1A,B). Cavities of this size are quite rare in proteins
and suggest a missing cofactor or ligand binding site.10 While
any natural HIF2R ligands remain unknown, we have discovered
a number of artificial ligands that bind within this site with low-
micromolar affinity using an NMR-based small-molecule
screen.19 Several of these compounds modulate the interaction
between HIF2R and ARNT PAS-B domains in vitro, suggesting
a linkage between internal ligand and external protein binding
on opposite faces of the central ꢀ-sheet of the protein, consistent
with PAS domains in other proteins.10,20-23 This raises the
question of how these ligands gain access to the internal cavity
of HIF2R PAS-B, which is completely inaccessible to solvent
in our structures.
To address this point and better understand the nature of HIF/
ligand interactions in general, we determined the crystal
structures of complexes of HIF2R PAS-B with two artificial
ligands bound at this internal site. These structural data
complement isothermal titration calorimetry (ITC) thermody-
namic studies of HIF2R PAS-B with these and three additional
ligands. Using NMR exchange spectroscopy, we determined the
association and dissociation rate constants for each of these
compounds and also, through their temperature dependence, the
transition-state thermodynamics of ligand binding. These pa-
rameters reflect the degree of protein conformational distortion
required to reach a ligand-accessible transition state. Finally,
we combined solution NMR studies and molecular dynamics
(MD) simulations to identify ligand entry/exit pathways to/from
the pocket of HIF2R PAS-B. The NMR-derived results suggest
that HIF2R PAS-B ligands bind rapidly to a partially disordered,
more open conformation of HIF2R PAS-B that must rapidly
interconvert with the ligand-inaccessible conformation illustrated
after cleavage of the N-terminal affinity tag by TEV protease,24
a
second round of nickel chromatography followed by Superdex 75
size-exclusion chromatography. For crystallization, PAS-B* mutants
of the HIF2R PAS-B (R247E) and ARNT PAS-B (E362R) domains
were produced as previously described.10
Crystallization and Structure Determination and Refinement.
Crystals of HIF2R PAS-B* were grown in a high-affinity het-
erodimer complex with the ARNT PAS-B* domain. Liganded
cocrystals were grown by vapor diffusion of 340 µM PAS-B*
heterodimer/440 µM compound solution against 100 mM Bis-Tris
(pH 6.0, 17-25% PEG-33500. Crystals were subsequently treated
with 25% PEG-3350 and 10% PEG-400 as a cryoprotectant prior
to freezing in liquid nitrogen. X-ray diffraction data were collected
at the 19-BM beamline of the Structural Biology Center at the
Advanced Photon Source (Argonne National Laboratory, Argonne,
IL). Data were reduced using HKL200025 and refined using
REFMAC 526 and COOT.27 Data scaling and refinement statistics
are summarized in Table 1. THS-017 cocrystals grew in the C2
space group reported previously10 and were amenable to Fourier
synthesis methods using the apoprotein structure (PDB entry 3F1P)
as an initial model. The THS-020 cocrystals adopted the P21 space
group, and the diffraction data were phased by molecular replace-
ment using the program PHASER.28 The experimental electron
densities for these two structures are shown in Supporting Informa-
tion (SI) Figure 1.
Determination of Ligand Binding Affinities. Ligand binding
affinities were determined using a MicroCal VP-ITC calorimeter
(Northampton, MA). Protein was extensively dialyzed against buffer
(25 mM Tris, pH 7.5, 17 mM NaCl, 5 mM ꢀ-mercaptoethanol),
which was used to prepare compound solutions from 50 mM stock
solutions in DMSO-d6. Solutions containing 200-330 µM HIF2R
PAS-B were titrated from the syringe of this instrument into a cell
containing 10-20 µM compound. Compound and protein solutions
all contained 0.02% DMSO, except for KG2-023, which was
supplemented with 5% DMSO to facilitate compound solubility
(minimal effects on thermodynamic parameters were observed in
5% DMSO controls with THS-017 and THS-020). Heats of dilution
were experimentally determined from control titrations of HIF2R
PAS-B into compound-free buffer and were subsequently subtracted
(15) Hoff, W. D.; Dux, P.; Hard, K.; Devreese, B.; Nugteren-Roodzant,
I. M.; Crielaard, W.; Boelens, R.; Kaptein, R.; van Beeumen, J.;
Hellingwerf, K. J. Biochemistry 1994, 33, 13959.
(16) Briggs, W. R. J. Biomed. Sci. 2007, 14, 499.
(17) Taylor, B. L. Mol. Microbiol. 2007, 65, 1415.
(24) Blommel, P. G.; Fox, B. G. Protein Expression Purif. 2007, 55, 53.
(25) Collaborative Computational Project, Number 4. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 1994, 50, 760.
(18) Gilles-Gonzalez, M. A.; Gonzalez, G. J. Biol. Chem. 1993, 268, 16293.
(19) Amezcua, C. A.; Harper, S. M.; Rutter, J.; Gardner, K. H. Structure
2002, 10, 1349.
(26) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 1997, 53, 240.
(20) Harper, S. M.; Neil, L. C.; Gardner, K. H. Science 2003, 301, 1541.
(21) Craven, C. J.; Derix, N. M.; Hendriks, J.; Boelens, R.; Hellingwerf,
K. J.; Kaptein, R. Biochemistry 2000, 39, 14392.
(27) Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2004, 60, 2126.
(22) Moglich, A.; Moffat, K. J. Mol. Biol. 2007, 373, 112.
(23) Gong, W.; Hao, B.; Chan, M. K. Biochemistry 2000, 39, 3955.
(28) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.;
Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658.
9
17648 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Internal Ligand Binding within the Core of HIF2R PAS-B
A R T I C L E S
Table 1. X-ray Crystallography Data Collection and Refinement
µM THS-044. Relaxation rate constants were estimated from
serially collected spectra with time delays of 10, 30, 70, 130, 250,
370, 490, 630, 750, 850, and 1110 ms for R1 and 10, 30, 50, 70,
90, 110, 130, 150, and 190 ms for R2. 15N{1H} nuclear Overhauser
effect (NOE) values were determined from spectra collected with
and without a 3 s saturation period.
Statistics (Molecular Replacement)a
THS-017
THS-020
Data Collection
space group
cell dimensions
a, b, c (Å)
ꢀ (deg)
resolution (Å)
C2
P21
Molecular Dynamics Simulations. All of the computations were
performed using the in lucem molecular mechanics (ilmm) pro-
gram.35 These simulations are part of the Daggett laboratory’s
Dynameomics effort,36-38 whose goal is to create a repository of
molecular dynamics simulations and the corresponding metadata.
The starting model for the simulations was the first conformer
of the NMR structure of the C-terminal PAS domain of HIF2R
(PDB entry 1P97).9 The structure was minimized in vacuo for 1000
steps of steepest-descent minimization. Atomic partial charges and
the potential energy function were taken from Levitt et al.39 The
minimized structures were then solvated in a rectangular box of
flexible three-center (F3C) waters40 with walls located g10 Å from
any protein atom. The solvent density of the box was pre-
equilibrated to 0.997 g/mL, the experimental water density for 298
K and 1 atm pressure.41 The solvent was minimized for 1000 steps.
This solvent minimization was followed by 1 ps of dynamics of
the solvent only, an additional 500 steps of solvent minimization,
and 500 steps of minimization of the entire system. After completion
of this solvation process, the whole system was heated to 298 K.
Three separate 35 ns MD simulations were performed at 298 K
and neutral pH in the microcanonical NVE [constant volume (V),
total energy (E), and number of particles (N)] ensemble. The
protocols and potential energy function have been described
previously.39,42 A force-shifted nonbonded cutoff of 10 Å was
used,39,42,43 and the nonbonded interaction pair list was updated
every three steps. A time step of 2 fs was applied in all of the
simulations, and structures were saved every 1 ps for analysis.
Analysis of MD simulations was performed using ilmm.
73.60, 82.67, 41.10
106.39
40.89, 70.80, 42.36
108.69
20.7 to 1.65
(1.68 to 1.65)
5.1 (46.2)
25.0 (2.1)
105492
26.4 to 1.5
(1.53 to 1.50)
3.7 (23.8)
21.8 (2.5)
101923
Rsym or Rmerge
I/σ(I)
observed reflections
unique reflections
completeness (%)
28092
97.6 (88.5)
34380
94.6 (68.8)
Refinement
22.5 to 1.65
(1.68 to 1.65)
20.2/23.8
resolution (Å)
26.3 to 1.5
(1.53 to 1.50)
19.6/23.4
Rwork/Rfree
no. of atoms
protein
ligand/ion
water
1857
20
182
1933
20
215
B-factors
protein
ligand/ion
water
19.6
30.5
31.4
17.2
18.7
28.1
rms deviations
bond lengths (Å)
bond angles (deg)
0.013
1.48
0.019
1.83
a Values in parentheses are for the highest-resolution shell.
from the corresponding ligand titrations prior to fitting the data to
a single-site binding model (Origin v7.0). Reported equilibrium
thermodynamic parameters are average values derived from three
independent measurements (apart from KG2-023, for which two
measurements were performed).
Results
Thermodynamics of HIF2r PAS-B Ligand Binding. In-house
NMR-based small-molecule screening efforts identified a num-
ber of artificial ligands for HIF2R PAS-B,10 most of which are
composed of two substituted rings connected by a one- or two-
atom linker. To investigate the structural dependence of the
thermodynamics of interactions between HIF2R PAS-B and
small molecules, we compared calorimetric data collected on
complexes of HIF2R PAS-B with five related bicyclic ligands
(Figure 2). Four of the five compounds used in this study share
a common scaffold consisting of a functionalized benzyl ring
with trifluoromethyl and nitro groups located para and ortho to
the linker, respectively. The fifth compound, KG-721, lacks a
trifluoromethyl group and contains a nitro group para to its
single-atom ether linkage. To determine the affinities and
thermodynamic parameters for binding of these compounds to
NMR Spectroscopy. Experiments were conducted using a 600
MHz Varian Inova spectrometer equipped with a cryogenically
cooled probe and a Z-axis pulsed-field gradient. To determine
association and dissociation rate constants for ligand binding, we
employed a ZZ-exchange-modified 15N/1H heteronuclear single-
quantum correlation (HSQC) experiment, which gives rise to time-
dependent exchange peaks via heteronuclear longitudinal magne-
tization transfer between the bound and unbound states.29 Samples
were prepared for NMR spectroscopy as follows: 133 µM ligand
(from a 50 mM stock solution in DMSO-d6) was added to 266 µM
HIF2R PAS-B in buffer (17 mM NaCl, 50 mM Tris, pH 7.4, 5
mM DTT, 10% D2O). The total DMSO concentration in the
prepared sample was 0.26%. Ligand association and dissociation
rates were extracted from a simultaneous fit of the measured cross-
peak and autopeak intensities to the McConnell equations.29,30 Data
processing and analysis were performed with NMRpipe31 and
NMRviewJ,32 respectively, while curve fitting was performed using
Berkeley Madonna software.33 Data were collected at 25 °C for
determination of binding and dissociation rate constants as well as
at 10, 15, 20, 25, and 30 °C for determination of transition-state
thermodynamic parameters, which were obtained by linear-least-
squares fits to the experimental values of koff and kon by Eyring
analysis.34 Backbone 15N relaxation measurements29 were collected
from 300 µM 15N-HIF2R PAS-B in the presence or absence of 400
(35) Beck, D. A. C., Alonso, D. O. V., Daggett, V. ilmm: in lucem
Molecular Mechanics; University of Washington: Seattle, WA, 2000-
2008.
(36) Beck, D. A.; Jonsson, A. L.; Schaeffer, R. D.; Scott, K. A.; Day, R.;
Toofanny, R. D.; Alonso, D. O.; Daggett, V. Protein Eng., Des. Sel.
2008, 21, 353.
(37) Kehl, C.; Simms, A. M.; Toofanny, R. D.; Daggett, V. Protein Eng.,
Des. Sel. 2008, 21, 379.
(38) Simms, A. M.; Toofanny, R. D.; Kehl, C.; Benson, N. C.; Daggett,
V. Protein Eng., Des. Sel. 2008, 21, 369.
(29) Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol.
NMR 1994, 4, 727.
(30) Iwahara, J.; Clore, G. M. J. Am. Chem. Soc. 2006, 128, 404.
(31) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax,
A. J. Biomol. NMR 1995, 6, 277.
(32) Johnson, B. A. Methods Mol. Biol. 2004, 278, 313.
(33) Macey, R.; Oster, G.; Zahnley, T. Berkeley Madonna User’s Guide,
version 8.0; University of California: Berkeley, CA, 2000.
(34) Eyring, H. J. Chem. Phys. 1935, 3, 107.
(39) Levitt, M.; Hirshberg, M.; Sharon, R.; Daggett, V. Comput. Phys.
Commun. 1995, 91, 215.
(40) Levitt, M.; Hirshberg, M.; Sharon, R.; Laidig, K. E.; Daggett, V. J.
Phys. Chem. B 1997, 101, 5051.
(41) Kell, G. S. Current Contents/Engineering, Technology and Applied
Sciences 1979, 16.
(42) Beck, D. A.; Daggett, V. Methods 2004, 34, 112.
(43) Beck, D. A.; Armen, R. S.; Daggett, V. Biochemistry 2005, 44, 609.
9
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A R T I C L E S
Key et al.
Figure 2. HIF2R PAS-B equilibrium ligand binding thermodynamics. (a) Typical ITC results from compound binding of THS-017 to HIF2R PAS-B. (b)
Compounds evaluated in this study, shown in wireframe with binding and thermodynamic parameters derived from ITC. (c) The ligand binding pocket of
HIF2R PAS-B in the (green) THS-017- and (cyan) THS-020-bound structures. All panels are shown in the same orientation, and the ARNT PAS-B* domain
heterodimer has been omitted for clarity.
the isolated HIF2R PAS-B domain, we used ITC to monitor
the heat evolved upon complex formation. We found that ligand
binding is enthalpically driven with minor entropic penalties
for compounds KG-721, THS-017, and THS-020, while THS-
002 and KG2-023 have favorable entropic contributions to
binding. The enthalpies of KG-721, THS-017, and THS-020
are quite similar, which is notable in light of the reduced
functionality of the KG-721 ligand relative to the other two
compounds. Evidently, the additional functional groups in THS-
017 and THS-020 make enthalpic contributions equivalent to
that of the single p-nitro group in KG-721. THS-002 and KG2-
023 show favorable entropic contributions to binding, both of
which are poorly soluble in aqueous solution and likely draw
an entropic benefit upon binding protein as a result of
desolvation.
predominantly hydrophobic environment built with Phe244,
Phe254, Phe280, Tyr307, Met309, and Leu319 side chains. This
site within the HIF2R PAS-B cavity contains a single low-
occupancy water molecule in the unliganded crystal structure,
consistent with its hydrophobic composition. As previously
noted, we observe few direct hydrogen bonds or electrostatic
interactions between the protein and the bound ligands, most
of which involve the “A ring” that is common to four of our
five ligands. The ligand/protein interactions consist primarily
of van der Waals contacts, a dipole-π interaction between the
Tyr281 side chain and the ligand aromatic ring, and hydrogen
bonds between the His248 side chain and the ligand nitro and
secondary amine groups. THS-020 has an additional hydrogen
bond between the furan oxygen atom of THS-020 and the
hydroxyl group of Tyr307. The preferred orientation of the
ligands we observe could also help stabilize an intraligand
hydrogen bond between the secondary amine linker and the nitro
group on the A ring. Notably, the scarcity of hydrogen bonds
in the liganded structures starkly contrasts with our structure
of unliganded HIF2R PAS-B, which contains eight solvent-
inaccessible water molecules within the protein core. These
water molecules make up an extensive internal hydrogen-
bonding network10 with a number of the side chains lining the
cavity (SI Figure 2). Thus, a number of protein hydrogen-bond
partners remain unpaired when ligand is bound, leading us to
suggest that much of the energetics favoring ligand binding
arises from other forces (e.g., hydrophobic interactions with the
B ring).
Crystal Structures of the THS-017/ and THS-020/PAS B*
Heterodimer Complexes. To provide a structural context for the
varied thermodynamics we observed, we determined two
additional X-ray crystal structures of HIF2R PAS-B ligand
complexes. A readily crystallized variant of the HIF2R PAS-B
heterodimer (see Experimental Methods), designated as HIF2R
PAS-B*, yielded high-resolution protein/ligand complex struc-
tures with compounds THS-017 (1.65 Å resolution) and THS-
020 (1.5 Å) (Table 1). As previously reported for another
compound, THS-044,10 THS-017 and THS-020 occupy the
internal HIF2R PAS-B* cavity in nearly identical conformations
(Figure 2C). Both ligands within these complexes, like THS-
044, are a minimum of 6.6 Å from bulk solvent and are
completely isolated within the protein core. On the basis of the
three available HIF2R PAS-B/ligand cocomplex structures (those
with THS-017 and THS-020 along with our previously reported
complex with THS-04410) and the chemical similarity of the
compounds evaluated here, we believe that the remaining
bicyclic compounds evaluated in this study likely bind in a
manner analogous to that previously observed.
HIF2r PAS-B Ligand Binding Kinetics Reveals Characteristics
of an Open Conformation. To complement these structural and
thermodynamic data, we determined ligand association and
dissociation rate constants under equilibrium conditions using
NMR ZZ-exchange spectroscopy (Figure 3). These methods
have been used to measure such exchange rates in a range of
macromolecular systems undergoing slow (millisecond-to-
second time scale) dynamic equilibria.29,30,44 The time depen-
dence of the cross- and autopeak intensities from these spectra
provide exchange rates (kex), from which a first-order dissocia-
tion rate constant (koff) can be obtained. The association rate
constant (kon) can then be calculated using the equilibrium
constant derived from ITC measurements. For complexes of
In general, our structural data suggest that HIF2R PAS-B
preorganizes the ligand binding cavity, using shape comple-
mentarity with few specific protein/ligand interactions to guide
binding. This observation is supported by comparisons of the
structures of three protein/ligand complexes and one unliganded
form. Ligand binding is associated with minimal distortion
relative to the water-filled unliganded form, with few side-chain
rearrangements (His248, Met252). Around the variable ring of
the ligands (the “B ring” in SI Figure 2), we observe a
(44) Rubinstenn, G.; Vuister, G. W.; Mulder, F. A.; Dux, P. E.; Boelens,
R.; Hellingwerf, K. J.; Kaptein, R. Nat. Struct. Biol. 1998, 5, 568.
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Internal Ligand Binding within the Core of HIF2R PAS-B
A R T I C L E S
Figure 3. NMR ZZ-exchange data from HIF-2R PAS B and compound.
Shown are fits to NMR peak intensities as a function of exchange mixing
time, and a subset of the corresponding cross-peaks is shown in the inset.
Arrows indicating the autopeak-to-cross-peak directions are shown in the
first inset.
Table 2. Association and Dissociation Rate Constants at 25 °C
no. of probes
(amide cross-peaks/autopeaks)
compound
kon (105 M-1 s-1
)
koff (s-1
)
KG-721
9.2 ( 1.1
4.5 ( 0.4
6.9 ( 0.6
13.9 ( 2.2
13.0 ( 1.0
9.2 ( 1.0
1.6 ( 0.1
2.8 ( 0.6
1.4 ( 0.3
2.5 ( 0.1
18
5
8
6
12
KG2-023
THS-002
THS-017
THS-020
HIF2R PAS-B and these compounds, the ligand dissociation
rate constants are approximately proportional to the affinity
determined by ITC; thus, dissociation is the primary determinant
of binding affinity for these compounds. Despite the completely
buried nature of the HIF2R PAS-B pocket, we detected rapid
ligand association kinetics, with kon values greater than 106 M-1
s-1 for THS-017 and THS-020 at 25 °C (Table 2). Compounds
KG2-023 and THS-002 bind somewhat more slowly, likely
because of solvation effects due to their poor solubility. The
narrow range of association rate constants suggests that the
compounds traverse a similar barrier to binding, which we
interpret as the conformational rearrangement necessary for
HIF2R PAS-B to convert from a “closed” structure into a
binding-competent “open” state. In view of the considerable
size of these compounds relative to the scale of the HIF2R
PAS-B domain,10 it seems apparent that a substantial confor-
mational change is necessary to permit entry. However, the rapid
association rates we observed are more typical of solvent-
accessible ligand binding sites than internal cavities. Thus, the
open conformation of HIF2R PAS-B must either be present in
significant concentration at equilibrium or rapidly interconvert
with the closed native state.
The Transition State Barrier Exhibits Compensating Enthal-
pic and Entropic Contributions. To better characterize the ligand
binding transition state, we measured the temperature depen-
dence of kon and koff for three HIF2R PAS-B complexes: THS-
002, THS-020, and KG-721. Eyring analyses (Figure 4)
demonstrated a primarily enthalpic barrier to ligand binding
(Table 3). The transition-state enthalpy reflects breaking of
favorable interactions within the protein to reach an accessible,
open transition state. We observed the largest enthalpic penalty
in the transition state with compound THS-020, while binding of
Figure 4. Eyring plots for HIF-2R PAS B ligands KG-721, THS-002, and
THS-020. (a) Temperature dependence of koff. (b) Temperature dependence
of kon.
Table 3. Transition State Thermodynamics Determined Using
NMR ZZ-Exchange Measurements at 10-25°C
∆Hq (kcal/mol) T∆Sq (kcal/mol)a ∆Sq (cal mol-1 K-1
)
∆Gq (kcal/mol)a
Results for kon
KG-721
THS-002 13.5 ( 0.6
THS-020 22.9 ( 0.8
16.8 ( 0.6
7.4 ( 0.2
4.0 ( 0 0.1
13.9 ( 0.4
25.0 ( 0.7
13.4 ( 0.4
46.5 ( 1.4
9.3 ( 0.6
9.5 ( 0.6
9.1 ( 0.9
Results for koff
4.5 ( 0.3
KG-721
THS-002 16.7 ( 0.5
THS-020 23.5 ( 1.1
20.4 ( 1.1
15.0 ( 0.9
0.0 ( 1.8
22.5 ( 1.1
15.9 ( 1.1
16.7 ( 0.5
16.8 ( 1.1
0.0 ( 0.1
6.7 ( 0.3
a At 25 °C.
THS-002 had the lowest transition-state enthalpic barrier. For each
compound, an increase in activation entropy partially compensates
for this distortion of the protein structure. Such compensation
suggests that ligand accommodation entails a transition state with
increased entropy resulting from protein flexibility, release of
ordered solvent, or a combination of these effects.
Molecular Dynamics Simulations Reveal Conversion between
Open and Closed Conformations and Pathways of Ligand Entry.
To identify possible pathways used by the ligands to enter/exit
the cavity within HIF2R PAS-B, we conducted multiple MD
simulations beginning from an NMR-derived structure of apo-
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A R T I C L E S
Key et al.
on the ꢀ-sheet (SI Figure 3B). The movement of FR also
contributes to the open conformation and is correlated with the
changes in the positions of the DR and ER helices. FR moves
away from the protein core, pivoting on its C-terminus in such
a way that the N-terminus of the helix shifts by 20-30°. These
dynamics are the primary factor enabling transport of water
along pathway 1 (between the FR helix and the Gꢀ strand).
The DR and ER helices move 5-8 Å away from the AΒ loop
in the open conformation observed in the MD simulations,
opening the front of the molecule (SI Figure 3). Interestingly,
the C-terminus of the DR helix also extends by two residues in
the open form (SI Figure 3C). The DR and ER helices and
neighboring AB loop are highly dynamic, but the neighboring
Aꢀ-Bꢀ hairpin becomes more ordered when the short helices
move away. The hairpin maintains its contacts with the Iꢀ strand,
while Aꢀ adds residues at its C-terminus and Bꢀ gains structure
and forms hydrogen bonds with Aꢀ. The combined result of
these motions is that a channel is created from the solvent into
the protein core (SI Figure 3D) that serves as the route for water
passage via pathway 2 (Figure 5). The average CR root-mean-
square deviation (rmsd) between the open and closed forms
ranged from 2.2-3.6 Å in the three simulations.
Figure 5. Molecular dynamics suggests routes of ligand entry and egress.
Heat map of HIF2R PAS-B derived from CR rmsd values over the course
of 35 ns MD simulations. Water passage pathways are superimposed on
the crystal structure and labeled with pathway numbers and percentages of
total observed water entry/exit events.
The majority of the water entry and exit events (85%)
occurred while the protein was in the open conformation
(Supplementary Table 1). Transport via pathway 1 was observed
only in the open conformation; in contrast, although most
transport via pathway 2 was in the open conformation, five cases
were observed from the closed state. In these latter cases, the
entry and exit of water via pathway 2 is not controlled solely
by large backbone movements. Instead, the side chain of residue
Met 289 exists as different rotamers, acting as a gate for water
passage. In one rotamer, Met 289 interacts with Met 252 and
Tyr 278, effectively blocking the channel (SI Figure 4), while
an alternate rotamer swings out of the way and permits water
passage (Supplementary Table 1). Our simulations indicate that
pathways 1 and 2 are both sufficiently wide (∼8 Å) in the open
conformation to allow entry of the ligands in this study, all of
which have dimensions of approximately 8 × 12 Å for their
shortest and longest dimensions.
HIF2R PAS-B (PDB entry 1P97).9 Despite the significantly
smaller cavity (∼120 Å3) found in this starting structure
compared with the crystal structure, we observed water entering
and exiting the protein core via two primary routes in each of
three independent 35 ns MD trajectories. The most common
entry/exit pathway (pathway 1in Figure 5) is between the FR
helix and the Gꢀ strand, with 50% of solvent entry/exit events
proceeding via this route. The other major pathway (46% of
solvent entry/exit events) is between the short ER helix, the FR
helix and the AB loop (pathway 2 in Figure 5). In pathway 2,
motion in the AB loop and ER relative to FR displaces the
Met252 and Tyr278 side chains, opening a passageway for water
entry and egress. An additional minor pathway (pathway 3)
involved one water molecule entering and another escaping
between Cys257 and Asp259 (between Bꢀ and CR), but this
was only observed early in a single simulation. Consequently,
we focus here on pathways 1 and 2, which were observed
multiple times and in multiple simulations (see Supplementary
Table 1 in the Supporting Information).
To determine whether distinct open and closed protein
conformers were populated in these simulations, we performed
conformational clustering via an all-versus-all comparison of
structures sampled during MD, which was projected into three-
dimensional space via multidimensional scaling (SI Figure 3A).
Two dominant conformations were evident in all three simula-
tions, corresponding to open and closed conformers (SI
Figure 3B). Overall, HIF2R PAS-B spent 57% of the time in
the closed conformer, 41% in the open state, and 2% in neither.
Three transitions from the closed to the open form and two
transitions from the open form back to the closed form were
detected in the combined 105 ns of simulation time. Thus, these
structures were not continuously converting on the MD time
scale. Overall there was 0.05 transition/ns, which unfortunately
falls in a time regime that is longer than the rotational correlation
time of this domain (τc ) 9.1 ns) and difficult to experimentally
examine with NMR relaxation methods.
Discussion
Ligand binding by proteins forms the basis for numerous
biological processes, including the specificity of enzymes,
hormonal control, environmental sensing, and cell-cell recogni-
tion. Biological systems have tuned their respective ligand
affinities for these purposes, utilizing high-affinity interactions
for transport and relatively low affinity ones for rapid response
to stimuli. Thus, understanding the thermodynamics of a protein/
ligand interaction is key to understanding biological function.
Ligand binding also provides an attractive avenue for exogenous
control of biological systems, as exploited by pharmaceuticals
that modulate natural function through specific binding with
molecular targets.
In several cases where proteins bind ligands with high affinity,
evolution has optimized the association rate and achieved the
diffusion limit for ligand binding,45,46 where each molecular
collision between protein and ligand produces a successful
binding event. For the majority of proteins, however, reduced
(45) Miller, D. M., 3rd; Olson, J. S.; Pflugrath, J. W.; Quiocho, F. A. J. Biol.
Chem. 1983, 258, 13665.
The open and closed conformations both contain all of the
secondary structure elements found in the HIF2R PAS-B NMR9
and crystal10 structures but differ mainly in their tertiary packing
of the distorted, one-turn DR and ER helices and the AB loop
(46) Thorsteinsson, M. V.; Bevan, D. R.; Potts, M.; Dou, Y.; Eich, R. F.;
Hargrove, M. S.; Gibson, Q. H.; Olson, J. S. Biochemistry 1999, 38,
2117.
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Internal Ligand Binding within the Core of HIF2R PAS-B
A R T I C L E S
accessibility of a ligand binding site provides a barrier to ligand
binding that is reflected in a reduced value of the ligand
association rate constant relative to this diffusion limit. This
principle is well-illustrated by the work of Scott and colleagues47
on myoglobin, where the bulk of distal-residue side chains
represents a “conformational gate” to diffusional ligand access
that is evident in O2 association rate constants.
values in the MD simulations, suggesting another link between
experiment and simulation indicative of enhanced protein confor-
mational dynamics induced by ligand binding or a more compli-
cated conformational exchange between the two components of
the complex (SI Figure 5 in ref 10). Many of these same sites
show elevated 15N R2/R1 ratios in the apoprotein (SI Figure 6),
suggesting that the motions required for entry exist in the absence
of ligand. Taken together, these observations strongly suggest that
the FR helix has significant conformational heterogeneity, likely
including components of an apo-HIF2R PAS-B conformation that
facilitates ligand binding just as it enables water entry via both
pathways 1 and 2 in the MD simulations.
Given the high HIF2R PAS-B ligand association rate con-
stants, we conclude that the conformational barrier to ligand
entry is small. This was unexpected, considering the inacces-
sibility of the crystallographically determined protein binding
pocket, the size of the bound ligands, and the required expulsion
of eight water molecules from the binding site. Given this low
barrier, one might suppose that a proportion of HIF2R PAS-B
exists in a distinct open conformation in equilibrium with a
closed state in a manner similar to periplasmic carbohydrate
binding proteins, such as maltose binding protein (MBP).48
While MBP and HIF2R PAS-B both exhibit low-micromolar
affinity and rapid ligand association rates for similar-sized
ligands,45 much of our experimental data cannot independently
confirm that HIF2R PAS-B exists to any significant degree in
a discrete open state. A likely cause of this is the rapid time
scale of interconversion between the open and closed states
observed in the MD simulations (21 ns between interconversion
events on average), which we anticipate leading to time
averaging of many NMR parameters that could be different for
the two states. Our data are consistent with this, as we did not
observe distinct chemical shifts for the two conformations
(which would require millisecond or slower interconversion)
or significant 15N relaxation dispersion (sensitive to high-
microsecond to low-millisecond events). Furthermore, our
crystallographic data reveal no substantial differences between
the structures of the free and liganded states, even at the high
resolutions under consideration (1.2 to 1.6 Å), but this is to be
expected if crystallization traps a single, lowest-energy confor-
mation. From these data, unliganded HIF2R PAS-B appears to
be in a single, closed conformation.
However, the combination of other NMR methods sensitive
to motions on different time scales, ligand association rates,
and MD simulations suggest that the protein also populates a
more open conformation. Furthermore, the combination of NMR
and MD provides clues to the mechanism of ligand entry into
the core of HIF2R PAS-B. NMR-based 2H exchange measure-
ments are particularly useful in this context. For much of the
central core of HIF2R PAS-B, binding of the THS-044 ligand
significantly (by a factor of >100) stabilizes many of the amides
near the ligand, particularly in the ER helix and the central Aꢀ,
Hꢀ, and Iꢀ strands of the ꢀ-sheet10 (shown schematically in SI
Figure 5). This is consistent with a ligand-dependent shift of
an open/closed conformational equilibrium in the apoprotein
being shifted toward a closed conformation (although it is likely
that a component of this stabilization comes from the expulsion
of water from the core of the domain). Moreover, sites with
high rmsd values in the MD simulations show no significant
2H exchange protection in the apoprotein (Figure 5, SI Figure 5).
This is particularly conspicuous for the long FR helix,10 despite
the fact that this section has backbone chemical shift and 1H-1H
NOE characteristics of helical secondary structure.9 Notably,
complex formation with THS-044 significantly broadens the NMR
signals from many AB-loop, ER, and FR sites with high rmsd
The existence of an open, binding-competent conformation
of HIF2R PAS-B is also supported by the enthalpy-entropy
compensation observed in our transition-state thermodynamics
data. Enthalpy-entropy compensation of this nature is charac-
teristic of a loss of protein structure49 in the transition state that
permits ligand access to the binding site. Mulder and co-workers
have characterized a similar interconversion between a sterically
inaccessible closed state and a partially unstructured open state
of the T4 lysozyme L99A mutant50 that also binds small ligands
51
with an association rate of 106 M-1 s-1
.
The L99A mutation
in T4 lysozyme creates a 150 Å3 cavity (significantly smaller
than the 290 Å3 HIF2R PAS-B cavity) that binds small
substituted benzene compounds.52 Though HIF2R PAS-B
ligands are substantially larger and were observed in a natural
protein rather than a point-mutant variant, the principle here
appears to be the same: a conformational equilibrium exists
between a highly populated ligand-inaccessible ground state and
a binding-competent state characterized by increased dynamics
and altered structure.
A notable difference between T4 lysozyme and HIF2R PAS-B
is the time and spatial scale of conformational motions necessary
to permit access to the ligand binding site. T4 lysozyme requires
increased millisecond-time-scale dynamics within the ER, FR,
and IR helices and their adjacent loops that permit ligand
access.50 The HIF2R PAS-B dynamics appear to be more rapid
than these motions, suggesting that ligand entry requires
nanosecond-time-scale motions, likely side-chain rotamerization,
loop displacement, and helical motions, as observed in the MD
simulations. While the experimental studies cannot flesh out
the details of the PAS-B conformational substates, these states
were both detectable and characterizable by MD. Future studies
will involve use of the MD-generated models to devise specific
experiments to check the predictions.
We close by asserting that a ligand-accessible state of HIF2R
PAS-B exists in equilibrium with the native state, although this
open state is characterized by increased dynamics and altered
structure. As such, the open state conformation is better
described by the open state model of L99A T4 lysozyme than
the discrete open structure of MBP. Efforts to quantify the
relative populations of native and open conformations of HIF2R
PAS-B are challenging because of the rapid exchange involved,
but they have important implications for both the binding of a
natural ligand and the development of targeted ligands aimed
at disrupting the functionally important HIF2R PAS-B/ARNT/
PAS-B interaction within the intact HIF2 transcription factor.
(49) Dunitz, J. D. Chem. Biol. 1995, 2, 709.
(50) Mulder, F. A.; Mittermaier, A.; Hon, B.; Dahlquist, F. W.; Kay, L. E.
Nat. Struct. Biol. 2001, 8, 932.
(51) Feher, V. A.; Baldwin, E. P.; Dahlquist, F. W. Nat. Struct. Biol. 1996,
3, 516.
(47) Scott, E. E.; Gibson, Q. H.; Olson, J. S. J. Biol. Chem. 2001, 276,
5177.
(52) Eriksson, A. E.; Baase, W. A.; Zhang, X. J.; Heinz, D. W.; Blaber,
M.; Baldwin, E. P.; Matthews, B. W. Science 1992, 255, 178.
(48) Tang, C.; Schwieters, C. D.; Clore, G. M. Nature 2007, 449, 1078.
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A R T I C L E S
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Acknowledgment. We acknowledge Paul Erbel and Rick Bruick
for ongoing discussions through various aspects of this work, Doug
Frantz and the UT Southwestern Medicinal Chemistry Facility for
synthesis of the THS series compounds used in this study, and
Carlos Amezcua for development of the initial small-compound
library and assistance with NMR spectroscopy. This work was
supported by grants from the NIH (P01 CA95471 to K.H.G., R01
GM50789 to V.D., and T15 LM07442 to P.C.A.) and the American
Cancer Society (High Plains Division-North Texas Postdoctoral
Fellowship PF-06-270-01-GMC to T.H.S.).
NMR exchange data, conformational changes revealed in MD
simulations, deuterium exchange protection factors mapped onto
the structure of HIF2R PAS-B, 15N relaxation measurements
from HIF2R PAS-B in the absence or presence of an artificial
ligand, the McConnell equations used to fit ZZ-exchange data,
and a table of water entry/exit events. This material is available
free of charge via the Internet at http://pubs.acs.org. The
coordinates and structure factors of the THS-017/PAS B* and
THS-020/PAS B* heterodimer complexes have been deposited
with the Protein Data Bank as entries 3H7W and 3H82,
respectively.
Supporting Information Available: Experimental electron
density maps of ligand-bound structures, protein/ligand interac-
tions within the core of HIF2R PAS-B, details of fitting the
JA9073062
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