Computers in Biology and Medicine 149 (2022) 106006

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Computers in Biology and Medicine

journal homepage: www.elsevier.com/locate/compbiomed

An order-to-disorder structural switch regulates HIF-1 transcription

through S247 phosphorylation in the HIF1α PAS-B domain
Chia-Hung Hsu a, Ya-Jyun Chen b, Chia-Ning Yang b, *
a
Department of Internal Medicine, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan
b
Institute of Precision Medicine, National Sun Yat-sen University, Kaohsiung, Taiwan

A B S T R A C T

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular responses to hypoxic stress, is essential for tumor progression. It is a heterodimer
comprising HIF1α and HIF1β, with multiple interfaces among their PAS-A, PAS-B, and bHLH domains. HIF1β is also known as aryl hydrocarbon receptor nuclear
translocator (ARNT). Casein kinase 1δ–dependent phosphorylation of the solvent-front residue S247 on the HIF1α PAS-B domain interrupts HIF1α–ARNT complex
formation and reduces HIF-1 transcription activity. However, S247 is involved in neither HIF1α–ARNT complex formation nor stabilization of the relative orientation
between the HIF1α PAS-A and PAS-B domains. To uncover the underlying allosteric mechanism, we conducted Gaussian accelerated molecular dynamics simulations
and identified two distinct conformations of the pS247-carrying HIF1α PAS-B domain: H291-in and H291-out. The H291-in structure can associate with the HIF1α
PAS-A domain and form a V-shaped pouch to accommodate the ARNT PAS-A domain, but it cannot associate with the ARNT PAS-B domain. By contrast, the H291-out
structure can bind to the ARNT PAS-B domain, but its association with the HIF1α PAS-A domain leads to an unsuitable relative orientation to accommodate the ARNT
PAS-A domain. Both conformations were also collected in parallel simulations of the unphosphorylated PAS-B domain. Both structures manage to associate with the
ARNT PAS-B and HIF1α PAS-A domains; thus, they are adequate for HIF1α–ARNT complex formation. The domain–domain contact pattern in a phosphorylated
variant is shuffled by an order-to-disorder structural switch, triggered by the newly formed K251–pS247 interaction.

1. Introduction
Hypoxia-inducible factors (HIFs) are transcriptional activators that
regulate cellular oxygen sensing and homeostasis. These regulations are
pivotal to tumor growth and progression; therefore, HIFs have become
attractive cancer therapeutic targets [1–10]. To execute their function,
HIFs form HIF1α–HIF1β and HIF2α–HIF1β heterodimers; HIF1β is also
called an aryl hydrocarbon receptor nuclear translocator (ARNT). In this
study, we focused on the HIF1α–ARNT system. In HIF1α, the N-terminal
region comprises a basic helix–loop–helix domain (bHLH), which
functions as part of a DNA reading head. It also has two consecutive
Per-ARNT-Sim (PAS) homology domains—PAS-A and PAS-B—which are
required for dimerization with ARNT, and an oxygen-dependent
degradation domain essential for oxygen-dependent hydroxylation and
degradation. Moreover, the C-terminal region contains an N-terminal
transactivation domain (N-TAD) and a C-TAD, which are separated by
an inhibitory domain. These TADs are responsible for transcriptional
activity under hypoxia. The transcriptional activity of the C-TAD
domain is associated with the binding of the transcriptional coac­
tivators: CREB-binding protein (CBP) and p300. Under normoxic con­
ditions, prolyl hydroxylase (PHD) hydroxylates two proline residues of
HIF1α, and the hydroxylated HIF1α is recognized by the von Hip­
pel–Lindau protein, which then activates the ubiquitin ligase system for
proteasomal degradation. However, PHD is inactive under hypoxic
conditions; therefore, HIF1α is stabilized for dimerization with ARNT.
Both HIF1α and ARNT use their bHLH, PAS-A, and PAS-B domains for
dimerization, as indicated in Fig. 1(A), where two crossed bHLH do­
mains form a reading head to bind to the regulatory hypoxia-response
elements (HREs) in the promoter region of HIF target genes. More­
over, the HIF1α–ARNT heterodimer binds to CBP/p300 to promote the
context-dependent expression of multiple genes that offset cellular
hypoxia [11–14].
HIF1α transcriptional activity is also subject to oxygen-independent
regulation. Kalousi et al. observed that casein kinase 1δ (CK1δ)-depen­
dent serine 247 (S247) phosphorylation on the HIF1α PAS-B domain has
a structural impact, interrupting HIF1α–ARNT dimerization and,
accordingly, abolishing transcriptional activity [15]. The hypoxic
HRE-dependent transcriptional activity of HIF1α was found to be greatly
reduced due to CK1δ overexpression; furthermore, S247 on the HIF1α
PAS-B domain was the target site for modification by CK1δ. However,
CK1δ inhibition—through CK1δ inhibitor PF-670462 [16] or siRNA
knockdown—enhanced HIF1α transcriptional activity under hypoxia.

* Corresponding author. No. 70 Lien-hai Road, Kaohsiung, 80424, Taiwan.

E-mail address: cnyang@mail.nsysu.edu.tw (C.-N. Yang).

https://doi.org/10.1016/j.compbiomed.2022.106006
Received 24 February 2022; Received in revised form 11 August 2022; Accepted 14 August 2022
Available online 19 August 2022
0010-4825/© 2022 Published by Elsevier Ltd.
C.-H. Hsu et al. Computers in Biology and Medicine 149 (2022) 106006

Moreover, compared with the association of ARNT with wild-type
HIF1α, the association was stronger when S247 of HIF1α was replaced
by alanine and weaker when HIF1α harbored phosphomimetic mutant
S247D. Together, these results indicate that HIF1α S247 phosphoryla­
tion inhibits HIF1α–ARNT heterodimerization, thus impairing tran­
scriptional activity.
The present study demonstrated that structural variance originating
from phosphorylated S247 (pS247) interrupts HIF1α–ARNT dimeriza­
tion. For the HIF1α–ARNT–HRE complex structure retrieved from
PDB:4ZPR, each HIF1α and ARNT has bHLH, PAS-A, and PAS-B do­
mains; these domains are intertwined when associating with HREs
(Fig. 1(A)). Fig. 1(B) presents the secondary structure arrangement of
the HIF1α PAS-B domain, showing the location of S247 on the C-ter­
minal end of the Aβ strand in the domain, with its hydrophilic hydroxyl
group in the solvent front and not in contact with the ARNT PAS-B
domain. Moreover, the HIF1α PAS-B domain (green) is surrounded by
ARNT PAS-A (gray), ARNT PAS-B (gray), and HIF1α PAS-A (blue).
Notably, the coordinates of a short I235–D238 segment (linking the
HIF1α PAS-A and PAS-B domains) and of a rather long V345–P360
segment (connecting the ARNT PAS-A and PAS-B domains) were not
determined due to high mobility.
Because of their strong ability to sample conformational changes,
molecular dynamics simulations are useful for relating protein struc­
tures to their functions. Gaussian accelerated molecular dynamics
(GaMD) offers simultaneous, unconstrained, enhanced conformational
sampling and free energy landscape construction for biomolecules; this
is achieved by considering a harmonic boost potential to smooth the
biomolecular potential energy surface [17–19]. We employed GaMD
simulations to collect conformations of the HIF1α PAS-B domain in the
unphosphorylated wild type (WT) and the pS247-carrying variants to
determine how pS247 modifies the HIF1α PAS-B domain and interferes
with HIF1α–ARNT dimerization. High linkage flexibility, even in the
complex structure, suggests the independent movement of each domain
when in free HIF1α and ARNT. Accordingly, the pS247-induced struc­
tural change can be investigated within the HIF1α PAS-B domain alone;
thus, the following strategy was adopted. First, for each PAS-B of the
unphosphorylated WT and pS247-carrying variants, three independent
simulation runs were performed to cluster conformation ensembles and
extract representative structures. Furthermore, each pS247 representa­
tive conformation was superimposed on a complex structure comprising
the HIF1α PAS-B and ARNT PAS-B domains to produce an initial
PAS-B/PAS-B structure for further binding examination. The process

Fig. 1. HIF1α structure in dimerization with ARNT retrieved from PDB:4ZPR. (A) Complex structure of the HIF1α–ARNT heterodimer associated with the HRE
sequence. Dashed lines represent the missing segments connecting the bHLH and PAS-A domains or connecting the PAS-A and PAS-B domains. (B) Arrangement of
the secondary structural elements in the HIF1α PAS-B domain. The residue S247, which is to be phosphorylated, is specified with an all-atom style. (C) Interface
interactions between the HIF1α PAS-A domain (blue) and the PAS-B domain (green). (D) Interface interactions between the HIF1α PAS-B domain (green) and the
ARNT PAS-B domain (gray).

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C.-H. Hsu et al.
was repeated to produce an initial HIF1α PAS-A/PAS-B structure and to
examine the structure’s binding strength. The structure was later used to
determine whether the pocket formed by the HIF1α PAS-A and PAS-B
domains could accommodate the ARNT PAS-A domain, as indicated in
Fig. 1(C). With this work, we intended to determine the structural
impact that results in a weak HIF1α–ARNT association. However, the
whole HIF1α (formed by their bHLH, PAS-A, and PAS-B domains) or the
HIF1α–ARNT complex were not simulated because these structures may
not yield sufficient conformational adjustment that can be attributed to
the phosphate group introduced on S247. For a parallel comparison, all
the aforementioned modeling procedures were also performed for the
WT. In this work, we identified two conformation clusters: H291-in and
H291-out. Without S247 phosphorylation, H291 is interconvertible be­
tween H291-in and H291-out when in PAS-B alone and when associated
with its PAS-A and ARNT PAS-B. By contrast, the H291-in and H291-out
conformations in the pS247 variant restrain PAS-B-binding behavior.
That is, when adopting H291-in, HIF1α PAS-B’s facade for association
with ARNT PAS-B is impaired, whereas that for association with HIF1α
PAS-A is retained; the opposite is observed for H291-out. Altogether,
phosphorylated S247 interferes with the HIF1α–ARNT association.
2. Methods
2.1. System setup
The initial structure of the WT PAS-B domain was extracted from the
crystal structure of the HRE-bound human HIF1α–ARNT complex (PDB
code: 4ZPR) [20]. Because S247 was in the solvent front, S247 was
manually phosphorylated to generate pS247 without spatial conflict.
The two simulated systems were immersed in a cubic TIP3P water box in
which the distance separating any protein atom from the water box wall
was ≥12 Å. To neutralize the protein systems, two and four Na+ ions
were added to the WT and pS247 variants, respectively. Any neigh­
boring TIP3P water molecules within 1.5 Å of the PAS-B domain were
discarded to prevent the formation of biased hydrogen bonds between
PAS-B and TIP3P water molecules prior to modeling. Each PAS-B system
was energy-minimized in three stages, each using 25,000 steps of both
the steepest descent algorithm and conjugate gradient algorithm. In
stage one, PAS-B was restrained to allow reorientation of the added
TIP3P water molecules. In stage two, the PAS-B backbone was restrained
to optimize the interplay between the amino acid side chains and water
molecules. In stage three, the entire solvated system was minimized with
no restraints.
For each HIF1α PAS-B system, three independent GaMD simulations
were performed using the AMBER 18 software package [21]. The stan­
dard protocol covering gradual heating, density, equilibration, and
production procedures in the isothermal isobaric ensemble (P = 1 atm
and T = target temperature) was employed. A minimized solvated sys­
tem served as the initial structure for subsequent GaMD simulations. A
cutoff of 8 Å was applied to treat nonbonding interactions, such as
short-range electrostatics and van der Waals interactions, whereas the
particle mesh Ewald method [22] was used to treat long-range electro­
static interactions. All hydrogen atom bonds were constrained using the
SHAKE algorithm [23,24].
2.2. GaMD-enhanced sampling simulations
GaMD simulations were employed to collect the conformations of the
native and phosphorylated PAS-B domains. With a harmonic boost po­
tential to smooth the potential energy surface and reduce energy bar­
riers, GaMD can enhance conformational sampling [18,25]. The
aforementioned minimized WT and pS247 structures were heated from
0 to 300 K in a 60-ps simulation by applying 10 kcal/(mol⋅Å2) harmonic
position restraints to protein heavy atoms in the constant num­
ber–volume–temperature (NVT) ensemble. Each system was further
re-equilibrated using the constant number–pressure–temperature
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Computers in Biology and Medicine 149 (2022) 106006
ensemble at 1 atm and 300 K for 40 ps with the same restraints as in the
NVT run, followed by a density procedure at 300 K for 100 ps and
constant equilibration at 300 K for 800 ps. A 25-ns conventional mo­
lecular dynamics simulation was performed to collect potential energy
statistics, including the maximum, minimum, average, and standard
deviation. Another GaMD-based 25-ns equilibration was performed
after the boost potential had been applied. For both WT and pS247, three
independent GaMD production simulations were performed in 500 ns
with different randomized initial atomic velocities. Simulation frames
were saved every 1 ps for analysis. In total, 50,000 snapshots were
retrieved from each trajectory, and 150,000 snapshots were used to plot
a free energy landscape against carefully selected coordinates. For each
conformation cluster, 500 conformations centered at low potential en­
ergy were collected for structural analysis.
Furthermore, to examine the ability of the HIF1α PAS-A domain to
associate with the low-energy conformations sampled from GaMD sim­
ulations, we extracted the structures of the HIF1α PAS-A and PAS-B
domains from PDB:4ZPR. and superposed the sampled low-energy
conformations onto PAS-B, and replaced the X-ray-solved PAS-B
domain to generate the initial HIF1α PAS-A/PAS-B structures. To
examine the binding behavior of the HIF1α PAS-B and ARNT PAS-B
domains, the same procedure was used to generate the initial HIF1α
PAS-B/ARNT PAS-B structures. The HIF1α–ARNT complex is formed by
multiple interfaces (Fig. 1(A)). Because the ARNT PAS-A and PAS-B
domains are largely separated, whereas the HIF1α PAS-A and PAS-B
domains are in contact during heterodimerization, the HIF1α PAS-A
and PAS-B domains likely form a V-shape pouch first and consecu­
tively accommodate ARNT PAS-A and PAS-B in any order. Therefore, we
examined the association events between HIF1α PAS-A and PAS-B and
between HIF1α PAS-B and ARNT PAS-B.
3. Results and discussion
3.1. Conformations of the HIF1α PAS-B domain in the WT and pS247
variants
For each of the studied HIF1α PAS-B domains in the WT and pS247
variants, the 150,000 conformations collected from three replicas were
plotted as a free-energy landscape, where the centroid separation dis­
tance H291–D249 was used as the x-axis and H291–E277 was used as
the y-axis. As indicated in Fig. 1(D), the E277 and D249 residues of the
HIF1α PAS-B domain electrostatically interact with the R366’ and N448’
residues of the ARNT PAS-B domain, respectively, whereas H291 and its
nearby H292 and F295, all on the C-terminal portion of Fα, form a hy­
drophobic dent to accommodate the P449’ residue of the ARNT PAS-B
domain. In Fig. 2(A), HIF1α PAS-B domain conformations in the WT
are clustered in two low-energy basins—H291-out and H291-in—with a
similar H291–E277 separation distance of approximately 15 Å. In the
H291-out cluster, the imidazole moiety of H291 moves out of the hy­
drophobic core and moves close to D249, leading to a short H291–D249
separation distance of approximately 8 Å. In the H291-in conformation,
the imidazole ring is slightly embedded inside the hydrophobic core and
shifted away from D249, thereby increasing the H291–D249 separation
distance up to 11 Å. Fig. 2(A) also presents the PAS-B domain structure
(denoted with an asterisk) in the HIF1α–ARNT–HRE complex as deter­
mined through crystallography. The complexed PAS-B structure is be­
tween the two clusters, implying that both H291-in and H291-out
conformations are slightly rearranged after dimerization.
Similarly, Fig. 2(B) presents two clusters, with their representative
structures showing moving out and embedded H291 for the pS247
variant. Fα deformation and Eα downward movement result in the
H291–E277 separation distance (13.5–14 Å) being shorter than that in
the WT (14.5–15 Å). In the H291-in conformation, H291 is shielded by
the M250 aliphatic chain (on Bβ), whereas in the H291-out conforma­
tion, the H291 ring moiety and M250 aliphatic chain are stacked in
parallel for insertion into the hydrophobic core of the HIF1α PAS-B
C.-H. Hsu et al.
Fig. 2. Two-dimensional free energy profiles according to the centroid separation distances of H291–D249 as the x-axis and of H291–E277 as the y-axis; confor­
mations obtained from the GaMD result for the HIF1α PAS-B domain in the (A) WT and (B) pS247 variants. Representative conformations are on the sides.
domain.
We used the dictionary of secondary structures of proteins (DSSP)
method to evaluate Fα (residues D285–F295) intactness. The Fα helical
character is well-preserved in both the H291-in and H291-out clusters in
the WT PAS-B (Fig. 3(A) and 3(B)). In H291-out in the pS247 variant, the
D285–H291 segment exhibits mixed helix and loop/turn characteristics
(Fig. 3(C)). Regarding H291-in in the pS247 variant, the D285–T288
segment has mixed loop/turn and strand characteristics (Fig. 3(D)).
To elucidate the phosphate group–triggered structural change, we
performed a structural analysis of the WT. In Fig. 4(A) for the retrieved
HIF1α PAS-B domain, a buried hydrophobic cavity present above the
β-sheet is maintained by a balanced spatial arrangement of M250 on Aβ-
Bβ; Y276 on Eα; and H291, H292, and F295 on Fα. Moreover, the
K251–R273 and K251–S274 electrostatic interactions stabilize Bβ and
DEβ, further securing Y276. In the pS247 variant, K251 electrostatically
interacts with the phosphate group. The consequence of this K251
movement toward the phosphate group is twofold. One is the rear­
rangement of DEβ and reorientation of the Y276 aromatic ring moiety
(see Fig. 4(B) for a comparison of the WT and pS247 variants). Another
is the repositioning of M250, which also affects the H291 ring moiety
(see Fig. 4(C) for a comparison of the WT and pS247 variants). Overall,
the aforementioned balanced arrangement in the WT, built by M250,
Y276, and H291, is crumpled in the pS247 variant. Several N-terminal
Fα residues sink into the buried cavity, thereby deforming the N-ter­
minal Fα and the shrunk cavity, as estimated using a bio-tool in CAVER
Analyst [26] (see Fig. 4(D) for a comparison of the WT and pS247
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Computers in Biology and Medicine 149 (2022) 106006
variants). Fig. 4(C) also illustrates the relative positions of H291 and
M250 for H291-in and H291-out.
3.2. HIF1α PAS-B binding with ARNT PAS-B
We retrieved HIF1α PAS-B and ARNT PAS-B domains from PDB:4ZPR
as a template structure and replaced the HIF1α PAS-B with the H291-in
and H291-out conformations obtained from the WT and pS247 trajec­
tories as determined using X-ray crystallography. Four initial complex
structures for HIF1α PAS-B were thus generated to associate with the
ARNT PAS-B domain, with each structure submitted for three indepen­
dent GaMD simulation runs. Plotted in Fig. 5(A) and (B) are free-energy
landscapes for ARNT PAS-B binding to HIF1α PAS-B in the WT, where
H291-out and H291-out conformations were used as the initial struc­
tures. These landscapes are plotted against the ARNT Y450′ –HIF1α L248
centroid separation distance (as the y-axis) and H291–D249 centroid
separation distance (as the x-axis). Fig. 1(C) indicates a hydrophobic
interaction between ARNT Y450′ and HIF1α L248 in the complex
structure; thus, their separation indicates an association event between
the two PAS-B domains. Fig. 5(A) and (B) suggest that the H291-in- or
H291-out-carrying WT can associate with the ARNT PAS-B domain.
Notably, the imidazole ring is free to flip in and out regardless of the
initial spatial state of H291, as evidenced by the two basins centered at 8
Å (for H291-out) and 11 Å (for H291-in) along the H291–D249 sepa­
ration distance (Fig. 5(A) and (B)).
Fig. 5(C) presents the landscape plotted for the pS247 variant when
C.-H. Hsu et al. Computers in Biology and Medicine 149 (2022) 106006

Fig. 3. DSSP-based analysis of secondary structural elements (loop/turn, helix, and strand) on HIF1α PAS-B domain’s Fα helix for the (A) H291-out cluster in the WT,
(B) H291-in cluster in the WT, (C) H291-out in the pS247 variant, and (D) H291-in in the pS247 variant.

Fig. 4. Structural details of WT and pS247. (A) The HIF1α PAS-B domain has a cavity (gray). Structure extracted from PDB:4ZPR. (B) Relative orientation of K251
and Y276 in different conformations. (C) Relative orientation of M250 and H291 in different conformations. (D) Comparison of the cavity in different conformations.
WT in H291-out and H291-in maintained a shrunk cavity. In the pS247 variant, having either conformation, the cavity is embedded by the surrounding amino
acid residues.

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C.-H. Hsu et al. Computers in Biology and Medicine 149 (2022) 106006

Fig. 5. Two-dimensional free energy profiles according to the H291–D249 centroid separation distance as the x-axis and the Y450′ –L248 centroid separation distance
as the y-axis; conformations obtained from the GaMD result for the ARNT PAS-B domain associating with the HIF1α PAS-B domain in (A) using the H291-out cluster
in the WT, (B) using the H291-in cluster in the WT, and (C) using the H291-out cluster in the pS247 variant, and (D) using the H291-in cluster in the pS247 variant
with a representative conformation on the side.

using the H291-out conformation as the initial complex structure. The depends on the H291 conformation prior to this association, and only
distance separating the centroids of ARNT Y450′ and HIF1α L248 is H291-out has the opportunity to grasp the ARNT PAS-B domain.
approximately 5 Å, which is identical to that observed in Fig. 5(A) and
(B), suggesting the formation of a complex of two PAS-B domains.
3.3. HIF1α PAS-B binding with HIF1α PAS-A
Notably, the H291–D249 distance indicates that the H291 movement is
restricted in the H291-out conformation and that it is not allowed to flip
HIF1α PAS-A and PAS-B domains that are linked by a flexible
in. As presented in Fig. 5(D), when the initial pS247 variant is in H291-
I235–D238 loop (whose coordinates are not solved in the HIF1α–ARNT
in, the complex structure is not stable since the ARNT Y450′ -HIF1α L248
complex structure) are likely to move independently when not associ­
separation distance is populated between 4.5 and 10 Å. Again, the H291
ated with ARNT. As indicated in Fig. 1(A), the HIF1α PAS-A domain
position is locked in the H291-in confirmation, as indicated by the
(blue) and PAS-B domain (green) form a V-shaped pouch to accommo­
H291–D249 distance centered at 12 Å. To summarize, the association
date the ARNT PAS-A domain. The appropriate opening of the V-shaped
between ARNT PAS-B and HIF1α PAS-B domains in the pS247 variant
pouch is determined using a salt bridge formed by PAS-A’s E143 and

Fig. 6. Two-dimensional free energy profiles according to the Y92–W316 centroid separation distance as x-axis and the E143–K297 centroid separation distance as y-
axis; conformations obtained from the GaMD result for the HIF1α PAS-A domain associating with PAS-B in (A) using the H291-out cluster in the WT, (B) using the
H291-in cluster in the WT, (C) using the H291-out cluster in the pS247 variant, and (D) using the H291-in cluster in the pS247 variant with a representative
conformation on the side.

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C.-H. Hsu et al.
PAS-B’s K297 and can be monitored in accordance with the separation
distance between PAS-A’s Y92 and PAS-B’s W316, as depicted in Fig. 1
(C). Both Y92 and W316 exhibit nonnegligible hydrophobic interactions
with the ARNT PAS-A domain. To elucidate how pS247 causes a struc­
tural change to weaken HIF1α–ARNT complex formation, we also
examined the interaction between HIF1α PAS-A and PAS-B domains.
The structure of these domains recovered from PDB:4ZPR was used as a
template structure where the HIF1α PAS-B determined through X-ray
crystallography was replaced by the H291-in and H291-out conforma­
tions from WT and pS247 trajectories; this resulted in four initial HIF1α
PAS-A bindings with the HIF1α PAS-B domain without the flexible
linkage. Fig. 6(A) and (B) show free energy landscapes obtained when
using the PAS-B domain with H291-out and H291-in, respectively,
plotted against the K297–E143 and Y92–W316 center-of-mass separa­
tion distances for the aforementioned reasons. The narrow K297–E143
span (8.5–9.5 Å) means that the salt bridge must be formed in a precise
orientation. By contrast, the rather wide Y92–W316 span (21–33 Å)
denotes the staunch K297–E143 salt bridge that enables open-to-closed
movement of two PAS domains until the ARNT PAS-A domain is
accessed, indicated by an asterisk sign in Fig. 6(A), which displays the
corresponding separation distances in the HIF1α–ARNT complex
structure.
Fig. 6(C) illustrates the association between PAS-A and PAS-B do­
mains when the pS247 H291-out conformation is used as the initial
structure. H291 stays out in all three replica trajectories. Owing to the
extensive Fα distortion, as indicated in Fig. 3(C), the misplaced E143
extensively disrupts the interface between the HIF1α PAS-A and PAS-B
domains, resulting in disintegrated clusters. In a cluster with
K297–E143 span of 8.5–9.5 Å and Y92–W316 span of 33–43 Å, the
separation between Y92 and W316 is too large to undergo induced-fit
when the ARNT PAS-A domain is encountered. Regarding H291-in
(Fig. 6(D)), although the population is diffusing along the y-axis, a
cluster with a K297–E143 span of 8.5–9.5 Å and a Y92–W316 span of
22–31 Å is likely for HIF1α PAS-B to first associate with the HIF1α PAS-A
domain and subsequently bind to the ARNT PAS-A domain. Meanwhile,
the slightly broad K297–E142 separation in Fig. 6(D) is attributable to
Fα distortion. In summary, our simulation results suggest that only the
H291-in conformation of the pS247 variant has the opportunity to
associate with HIF1α PAS-A and then accommodate the ARNT PAS-A
domain.
4. Conclusion
Enhanced conformational sampling through GaMD revealed two
structures in terms of the H291-in and H291-out conformations for the
HIF1α PAS-B domain. The unphosphorylated WT structure maintains a
flexible hydrophobic facade held by structured Fα, inflexible Eα, and Aβ-
Bβ loop to enable the interconversion of H291 between the H291-in and
H291-out positions without the whole conformation being disturbed.
Moreover, K251 points toward the solvent and occasionally electro­
statically interacts with S274 near Eα and D249 on the tip of the Aβ-Bβ
loop. Upon S247 phosphorylation, K251 is driven away toward the
phosphate group. Such a keystone displacement consecutively de­
stabilizes Eα and the Aβ-Bβ loop and disrupts the N-terminal Fα, ulti­
mately deforming the hydrophobic facade stabilized by M250, Y276,
and H291 in the WT.
Many inhibitors bind to an internal water-filled cavity of the HIF2α
PAS-B domain to interrupt the interaction between the HIF2α PAS-B and
ARNT PAS-B domains for therapeutic purposes [27–33]. HIF2α PAS-B
domain-bound inhibitors cause conformational changes in M252,
Y278, and H293 (M250, Y276, and H291 in HIF1α PAS-B) to interfere
with HIF2α–ARNT dimerization [27,30,31]. This is consistent with our
finding that these three residues, when spatially rearranged, reshape the
interaction pattern between the HIF1α PAS-B and ARNT PAS-B domains.
Along with research on drug development in relation to HIFs, mo­
lecular modeling studies on HIFs have been conducted to identify the
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Computers in Biology and Medicine 149 (2022) 106006
structural impact of accommodating an artificial ligand in the PAS-B
domain [34,35]. For instance, Masetti’s group evaluated the
ligand-bound and ligand-unbound states of the HIF2α PAS-B domain and
a complex formed by the HIF2α PAS-B and ARNT PAS-B domains, where
the considered ligand was a prototypical disrupting ligand [34]. Chong
and Anderson characterized the structural stabilization attributed to
eight ordered water molecules in the druggable pocket of the HIF2α
PAS-B domain [35]. Our group previously conducted a series of classical
molecular dynamics simulations on the HIF-2α PAS-A and PAS-B do­
mains of the WT and of three mutants carrying R171A and/or V192D for
the HIF-2α–ARNT dimer destabilization [36]. For both R171 and V192
in the interface of the HIF-2α PAS-A and PAS-B domains, mutations alter
the relative domain–domain orientation and accordingly damage the
V-shaped pouch that can hold the ARNT PAS-A domain.
Through S247 phosphorylation, HIF1α PAS-B experiences an order-
to-disorder structural switch, which leads to an inability to dimerize
with ARNT, thus interrupting the transcription activity. Such an order-
to-disorder structural switch was also observed for the regulation of
FoxM1 transcription factor, whose transcription activity is inhibited by
the association of its transactivation domain (TAD) to its negative reg­
ulatory domain (NRD). Upon phosphorylation of TAD’s S715, TAD
dissociates from NRD and turns into a disordered conformation, which is
able to bind to its transcriptional co-activator CBP [37].
Fig. 7 summarizes the structural impact of pS247. In the unphos­
phorylated WT, there is a minor structural difference between the H291-
in and H291-out conformations, and H291 is interconvertible between
the two states when alone and upon associating with HIF1α PAS-A and
ARNT PAS-B. However, in the pS247 variant, the H291-in and H291-out
conformations rigidly restrict PAS-B-binding behavior. When in H291-
in, the facade for association with ARNT PAS-B is impaired, whereas
that for association with HIF1α PAS-A is retained; the opposite is
observed in H291-out. Taken together, this allosteric mechanism ex­
plains the diminished HIF1α–ARNT heterodimerization.
In this study, we provide structural clues to link a local structural
variance caused by the phosphorylated Ser247 toward the interface
between the HIF1α PAS-B and ARNT PAS-B domains and the interface
shared by the HIF1α PAS-A, HIF1α PAS-B, and ARNT PAS-A domains.
Because all the concluding remarks were based on molecular modeling,
further analysis of protein–protein association events among the PAS-A
and PAS-B domains of HIF1α and ARNT are required to confirm our
results. Because HIF1α-ARNT is a heterodimer with six interfaces [20],
as indicated in Fig. 1(A), our model of the propagated structural disorder
suggests that HIF1α PAS-B plays a critical role in heterodimer formation.
However, these six interfaces certainly impede propagation of the
structural influence from the phosphorylated site as if we had used the
intertwined HIF1α-ARNT complex in the beginning for structural eval­
uation. The fact that the segment connecting the HIF1α PAS-A and
PAS-B domains was too flexible to be solved [20] suggests that these two
PAS domains tend to move independently when not associated with
ARNT. On the basis of these facts, we characterized conformational
features of the phosphorylated and unphosphorylated HIF1α PAS-B
alone, instead of the entire HIF1α-ARNT complex, to shed light on the
structural change that occurs prior to the binding event, and we further
surmised its possible binding behaviors with other domains. Such a
bottom-up strategy should be useful for studies on protein–protein
recognition.
CRediT authorship contribution statement
Chia-Hung Hsu: Project discussion and administration; Conceptual­
ization. Ya-Jyun Chen: Data curation and validation. Chia-Ning Yang:
Draft writing, reviewing, and editing; Investigation; Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
C.-H. Hsu et al.
Fig. 7. Summary of the difference caused by pS247. In the WT, the H291-in and H291-out conformations are observed and interconvertible upon association with the
ARNT PAS-B and HIF 1α PAS-A domains. In the pS247 variant, the H291-in and H291-out conformations are not interconvertible. Moreover, H291-in conformation
cannot associate with the ARNT PAS-B domain but can appropriately associate with the HIF 1α PAS-A domain and further accommodate the ARNT PAS-A domain. By
contrast, H291-out conformation can appropriately bind to the ARNT PAS-B domain but not to the HIF 1α PAS-A domain.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
The authors thank Zuoying Branch of Kaohsiung Armed Forces
General Hospital, Kaohsiung, Taiwan (grant number: ZBH 107-06), for
the financial support.
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