Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor

Article (PDF Available)inProceedings of the National Academy of Sciences 100(26):15504-9 · January 2004with21 Reads
DOI: 10.1073/pnas.2533374100 · Source: PubMed
Abstract
Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.

Figures

Structural basis for PAS domain heterodimerization in
the basic helix–loophelix-PAS transcription factor
hypoxia-inducible factor
Paul J. A. Erbel*
, Paul B. Card*
, Ozgur Karakuzu*, Richard K. Bruick*, and Kevin H. Gardner*
†‡
Departments of *Biochemistry and
Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390
Edited by Susan S. Taylor, University of California at San Diego, La Jolla, CA, and approved October 9, 2003 (received for review June 10, 2003)
Biological responses to oxygen availability play important roles in
development, physiological homeostasis, and many disease pro-
cesses. In mammalian cells, this adaptation is mediated in part by
a conserved pathway centered on the hypoxia-inducible factor
(HIF). HIF is a heterodimeric protein complex composed of two
members of the basic helix–loop–helix Per-ARNT-Sim (PAS) (ARNT,
aryl hydrocarbon receptor nuclear translocator) domain family of
transcriptional activators, HIF
and ARNT. Although this complex
involves protein–protein interactions mediated by basic helix–
loop–helix and PAS domains in both proteins, the role played by
the PAS domains is poorly understood. To address this issue, we
have studied the structure and interactions of the C-terminal PAS
domain of human HIF-2
by NMR spectroscopy. We demonstrate
that HIF-2
PAS-B binds the analogous ARNT domain in vitro,
showing that residues involved in this interaction are located on
the solvent-exposed side of the HIF-2
central
-sheet. Mutating
residues at this surface not only disrupts the interaction between
isolated PAS domains in vitro but also interferes with the ability of
full-length HIF to respond to hypoxia in living cells. Extending our
findings to other PAS domains, we find that this
-sheet interface
is widely used for both intra- and intermolecular interactions,
suggesting a basis of specificity and regulation of many types of
PAS-containing signaling proteins.
C
ellular responses to oxygen availability are essential for the
development and homeostasis of mammalian cells, demon-
strated most critically by the link between the cellular adaptation
to reduced tissue oxygenation and disease progression (1, 2). In
mammalian cells, these responses are mediated in part by the
hypoxia-inducible factor (HIF), a heterodimeric transcription
factor composed of HIF
and aryl hydrocarbon receptor nuclear
translocator (ARNT, also known as HIF
) (3). HIF activity is
tightly controlled under normoxic conditions by multiple O
2
-
dependent hydroxylation events of the HIF
subunit, which
coordinately promote the ubiquitin-mediated destruction of this
protein (4) and impair its ability to interact with transcriptional
coactivators (5, 6) (Fig. 1a). These controls are relieved during
hypoxia, allowing HIF to activate the transcription of genes that
facilitate metabolic adaptation to low oxygen levels and increase
local oxygen supply by angiogenesis (7).
All three isoforms of HIF
[HIF-1
,-2
(EPAS1), and -3
]
(8, 9) and ARNT belong to the basic helix–loop–helix (bHLH)–
Per-ARNT-Sim (PAS) family of eukaryotic transcription fac-
tors, which contain bHLH and PAS domains (Fig. 1). The bHLH
domains of these proteins serve as dimerization elements, help-
ing determine the specificity of complex formation while pro-
viding a DNA-binding interface composed of the basic regions
from each monomer (10). PAS domains are widespread com-
ponents of signal transduction proteins, currently identified in
2,000 proteins from organisms in all three kingdoms of life.
These domains, shown to be protein–protein interaction ele-
ments in several systems (11), also appear to contribute to the
dimerization process and thus increase the specificity of bHLH-
PAS transcription factor formation (12, 13). In the case of the
HIF
ARNT complex, coimmunoprecipitation and gel mobil-
ity-shift experiments using truncated forms of HIF
and ARNT
suggest that although the bHLH domains alone are able to
dimerize, the PAS domains are required to build a stable
heterodimer capable of robust DNA binding (14, 15). These data
suggest a model of the complex where the bHLH, PAS-A, and
PAS-B domains of ARNT interact with their counterparts in
HIF
(Fig. 1a). However, most of this model remains speculative
in light of the sparse data describing how PAS domains bind to
each other, or more generally, to any protein partner.
To provide insight into this general topic of PAS domain
signaling, particularly its importance in the hypoxia response
pathway, we have studied the structure and interactions of the
C-terminal PAS domain of human HIF-2
(HIF-2
PAS-B) by
NMR spectroscopy. We report that HIF-2
PAS-B adopts a
structure similar to other members of this family, with a central
-sheet flanked on one face by several
-helices. We further
show that HIF-2
PAS-B binds directly to the human ARNT
PAS-B domain in vitro, identifying the interface as a group of
residues located in the central strands of the
-sheet. With
structure-based mutations of this interface in the PAS-B do-
mains of HIF-1
and -2
, we demonstrate that such changes
interfere with the binding of isolated PAS-B domains in vitro but
more importantly disrupt the ability of full-length HIF proteins
to respond to hypoxia in living cells. These observations led us
to compare PAS domains from multiple systems, showing that
the
-sheet interface participates in a wide range of inter- and
intramolecular interactions and suggesting a way that specificity
and regulation may be achieved among these versatile domains.
Materials and Methods
Protein Expression and Purification. DNA-encoding fragments of
human HIF-2
PAS-B (residues 240–350) and ARNT PAS-B
(residues 356470) were subcloned into the pG
1-parallel and
pHis-parallel expression vectors, respectively (16, 17). Esche-
richia coli BL21(DE3) cells transformed with these plasmids
were grown in M9 media containing 1 gliter
15
NH
4
Cl for U-
15
N
samples (supplemented with 3 gliter
13
C
6
glucose for U-
15
N
13
C
labeled samples). These cultures were grown at 37°C to an A
600
of 0.6–1.0, then induced overnight at 20°C by the addition of 0.5
mM isopropyl
-D-thiogalactoside.
The purification of HIF-2
PAS-B has been detailed (18).
NMR samples typically contained 0.9 mM protein in 50 mM Tris
buffer (pH 7.3), 15 mM NaCl, 5 mM DTT, 5 mM NaN
3
and a
protease inhibitor mixture (Sigma) in 90% H
2
O10% D
2
O,
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: HIF, hypoxia-inducible factor; PAS, Per-ARNT-Sim; HIF-2
PAS-B, C-terminal
PAS domain of human HIF-2
; ARNT, aryl hydrocarbon receptor nuclear translocator; bHLH,
basic helix–loop– helix; HSQC, heteronuclear sequential quantum correlation; CHO,
Chinese hamster ovary; HRE, hypoxia responsive element.
Data deposition: The atomic coordinates for the HIF-2
PAS-B domain have been deposited
in the Protein Data Bank (PDB ID 1P97).
To whom correspondence should be addressed. E-mail: kevin.gardner@utsouthwestern.
edu.
© 2003 by The National Academy of Sciences of the USA
15504–15509
PNAS
December 23, 2003
vol. 100
no. 26 www.pnas.orgcgidoi10.1073pnas.2533374100
unless otherwise noted. ARNT PAS-B was expressed and puri-
fied as described in Supporting Methods, which is published as
supporting information on the PNAS web site.
Parallel studies on human HIF-1
PAS-B used a construct
containing residues 238349, chosen by homology with HIF-2
PAS-B. Expression and purification of HIF-1
PAS-B were done
as described for HIF-2
PAS-B.
NMR Spectroscopy. All NMR data were recorded at 30°C with
Varian Inova 500 and 600 MHz spectrometers by using NMRPIPE
for data processing (19) and NMRVIEW for analysis (20). Chem-
ical-shift assignments were made by using standard methods (21)
as detailed in Supporting Methods.
Deuterium exchange reactions were started by resuspending
lyophilized
15
N-labeled HIF-2
PAS-B in 99% D
2
O (uncor-
rected pH 7.3). These samples were then placed into a pre-
warmed magnet (T 30°C), and
15
N
1
H heteronuclear sequen-
tial quantum correlation (HSQC) spectra were sequentially
acquired approximately every 15 min. Observed
2
H exchange
rates were converted into protection factors by using standard
methods (22).
Structure Determination. Interproton distance constraints were
obtained from 3D
15
N edited NOESY (
m
150 ms),
15
N,
13
C
edited NOESY (
m
100 ms), and 2D NOESY (
m
120 ms)
spectra. Hydrogen bond constraints (1.3 Å d
NH-O
2.5 Å, 2.3
Å d
N-O
3.5 Å) were set for backbone amide protons
protected for 30 min from exchange with D
2
O solvent (30°C,
pH 7.3). Constraints for the
and
dihedral angles were
generated by chemical-shift analyses by using TALOS (23), with
two times the standard deviation of TALOS predictions as the
bounds (minimum 30°). For 19 residues without TALOS
predictions,
dihedral angle constraints were obtained from an
analysis of a 3D HNHA spectrum. Finally, 78
15
N-
1
H residual
dipolar coupling constraints were obtained from a sample
partially aligned in 5% (wtvol) DMPCDHPC ratio of 3:1
(Avanti Polar Lipids) and 5 mM cetyltrimethylammonium bro-
mide at 35°C.
Initial structures were determined without manual assign-
ments by using
ARIA1.2 (24, 25) and subsequently refined with a
mix of automated and manual assignment of NOESY spectra. Of
1,000 structures, the 20 lowest-energy structures were analyzed
with
MOLMOL (26) and PROCHECK-NMR (27).
From this ensemble, the structure closest to the mean was
superimposed against other PAS domains with the
DEEPVIEW
Swiss Protein Data Bank program (28) with the automatic fit
option. The calculated rms deviations ranged between 1.4 and
1.65 Å for HERG (Research Collaboratory for Structural Bio-
informatics Protein Data Bank ID 1BYW), hPASK (1LL8),
RmFixL (1D06), and Phy3 (1G28). The HIF-2
PAS-B structure
was also used to generate a model of the HIF-1
PAS-B
structure (74% sequence identity) by using
MODELLER (29).
HIF
and ARNT PAS-B Titration. Titrations were conducted by the
stepwise addition of natural abundance ARNT PAS-B (up to 800
M) to a sample of 200-
M HIF-2
at 35°C. The peak heights
of HIF-2
PAS-B signals that do not show ARNT-dependent
chemical shift changes (38 residues) were fit to Eq. 1 to obtain
the corresponding K
d
:
I 1 兵⌬I
max
关共A P
T
K
d
共共A P
T
K
d
2
4 A P
T
兲兲
12
2 P
T
兴其, [1]
where I is the observed change in peak height at ARNT
concentration A, I
max
is the change in peak height at saturation,
and P
T
is the total HIF
concentration. Eq. 1 is similar to the
equation used to extract K
d
from chemical-shift changes ob-
served in titrations of complexes undergoing fast exchange (30),
and we apply it here only to sites without chemical-shift changes
(fast exchange) to ensure that the observed peak line widths are
a population-weighted average of the free- and bound-state line
widths (31). The binding of HIF-2
PAS-B mutants to ARNT
PAS-B was assessed by adding 900
M natural abundant ARNT
PAS-B to 250
M HIF
PASBat25°C.
Mutagenesis. Point mutants of full-length HIF-1
and -2
were
created from wild-type DNA and primers including the desired
mutation(s). PAS-B domains containing these mutations were
obtained by PCR amplification of the corresponding full-length
sequence and subcloned into the pG
1-parallel vector. Trans-
formation, protein induction, and purification were performed
as described above.
Transfections. Cells were plated onto 48-well plates (3.5 10
4
cells per well) in 200
l of HyQ DMEF-12 1:1 media (HyClone)
supplemented with 5% FBS 24 h before transfection. Cells were
transfected with 10 ng of each HIF
construct and 20 ng of the
3HRE-tk-luc (HRE, hypoxia-responsive element) luciferase re-
porter construct (8) by using the Lipofectamine PLUS reagent
(Invitrogen). After 3 h, the media were changed and, after an
additional 2 h, cells were incubated for 15 h under normoxic or
hypoxic (1.0% O
2
) conditions. Luciferase activity was measured
as described (32).
Results
Solution Structure of HIF-2
PAS-B. We determined the solution
structure of HIF-2
PAS-B by using standard double- and
triple-resonance NMR experiments conducted on uniformly
15
N
and
15
N
13
C labeled protein samples (Fig. 2). This structure is
Fig. 1. Oxygen-dependent regulation and domain architecture of HIF pro-
teins. (a) HIF regulation is tightly linked to intracellular oxygen levels. Under
normoxic conditions, HIF
is posttranslationally hydroxylated, promoting its
degradation [modication of the oxygen-dependent degradation domain
(ODD)] and interfering with its ability to interact with CBPp300 coactivators
(modication of the transcriptional activation domains NTAD and CTAD).
These modications are not made under hypoxic conditions, allowing HIF
to
accumulate and enter the nucleus where it associates with ARNT and binds to
HREs upstream of hypoxia-activated genes. The red box highlights the HIF
and ARNT PAS-B domains. (b) Domain topology of HIF
subunits, including a
bHLH domain, two PAS domains, and C-terminal regulatory domains. A
sequence alignment of the HIF
PAS-B orthologs is shown, with bold letters
indicating the mutated residues described in the text. HIF-2
PAS-B secondary
structure elements are indicated with a gray background.
Erbel et al. PNAS
December 23, 2003
vol. 100
no. 26
15505
BIOPHYSICS
based on 2,500 geometric constraints obtained from measure-
ments of interproton distances, dihedral angles, and
15
N-
1
H
residual dipolar couplings of a partially oriented sample (Table
1). All of these data are well satisfied by the high-precision
ensemble of the 20 lowest-energy structures subsequently used
for further analysis.
HIF-2
PAS-B adopts a typical
PAS domain fold, char-
acterized by several
-helices flanking a five-stranded antipar-
allel
sheet. The similarity of this structure to other PAS
domains is demonstrated by the low-backbone rms deviation
values (1.41.65 Å) of pairwise comparisons between represen-
tative PAS structures and HIF-2
PAS-B. Although several
other PAS domains bind cofactors within their hydrophobic
cores to regulate proteinprotein interactions in response to
various physical stimuli (11), a combination of NMR, mass
spectrometry, and visible spectroscopy shows that HIF-2
PAS-B does not copurify with any such compound (data not
shown). Further, no preformed cavities are present in the protein
core, even at sites occupied by ligands in some other PAS
domains (17, 3336) (Fig. 2b).
Identification of ARNT PAS-B-Binding Surface on HIF-2
PAS-B. The
PAS domains in bHLH-PAS transcription factors are thought to
cooperate with the bHLH domains to facilitate dimerization (12,
13), which implies that the HIF
and ARNT PAS domains bind
to each other (Fig. 1a). To experimentally demonstrate this
interaction, we titrated unlabeled ARNT PAS-B into
15
N-
labeled HIF-2
PAS-B and monitored changes in the HIF-2
15
N
1
H HSQC spectrum (Fig. 3a). Peaks in these spectra showed
both chemical-shift changes and line broadening on addition of
ARNT PAS-B, consistent with binding on the intermediate and
fast exchange time scales. In contrast, we found that HIF-2
PAS-B signals were not affected by the addition of a PAS domain
from PAS kinase, a protein not involved in the hypoxia response
(17) (data not shown), suggesting that the changes observed on
addition of ARNT PAS-B reflect a specific HIF-2
ARNT
interaction.
The ARNT-induced changes in the HIF-2
line widths dem-
onstrate two important effects. First, we observed a general
increase in line width for HIF-2
peaks during the titration,
which we attribute to the slower tumbling of the larger 27-kDa
heterodimeric complex compared with an isolated HIF-2
PAS-B domain. By monitoring this broadening via the decrease
in peak heights as ARNT PAS-B was added, we observed a
titration consistent with a 1:1 binding event with a 30
M K
d
(Fig.
3b). This effect saturated at a 1:3 (HIFARNT) ratio, establish-
ing that it is not caused by nonspecific increases in sample
viscosity or aggregation. Second, we observed that a subset of
residues preferentially broadened on the addition of substoi-
chiometric amounts of ARNT PAS-B. Such differential effects
have been observed in several complexes (17, 37, 38) and arise
from exchange broadening at sites experiencing significant
chemical-shift changes on complex formation. Mapping sites
that exhibit either this differential broadening or significant
ARNT-induced chemical shift changes onto the HIF-2
PAS-B
structure shows that they cluster on the face of the central
-sheet (Fig. 4). This provides a chiefly hydrophobic surface for
ARNT binding that is conserved among the HIF isoforms
(Fig. 1), suggesting that the PAS-B domains of all three interact
similarly with ARNT.
Evidence for the importance of this interface in the HIFARNT
PAS-B dimer was obtained from studies of PAS-B domains con-
taining point mutations. Based on our structure, we altered three
Fig. 2. Solution structure of HIF-2
PAS-B. (a) Superimposition of 20 lowest-
energy structures for HIF-2
PAS-B, calculated as indicated in the text. (b)
Ribbon diagram of the structure closest to the mean of the ensemble shown
in a. Circles indicate the approximate locations of the ligand-binding sites of
several PAS domains (17, 3336).
Table 1. Statistics for HIF-2
PAS-B solution structure
determination
List of constraints
NOE distance restraints
Unambiguous 2,767
Ambiguous 496
Hydrogen bond restraints 60
Dihedral angle restraints 96
15
N-
1
H residual dipolar couplings 78
Stereospecic assignments
(Val
, Leu
)
12
Structural analysis
Mean rms deviation from
experimental restraints
NOE, Å 0.022 0.002
Dihedral angles, deg 1.04 0.16
Average number of:
NOE violations 0.5 Å 0
NOE violations 0.3 Å 1.9 1.2
Dihedral violations 5° 1.6 1.1
Mean rms from idealized covalent
geometry
Bonds, Å 0.0045
Angles, deg. 0.65
Impropers, deg. 1.69
Geometric analysis of residues
691 and 98112
rms deviation to mean 0.53 0.07 Å (backbone)
1.08 0.10 Å (all heavy)
Ramachandran analysis (
PROCHECK) 81.0% most-favored
16.4% additionally allowed
1.6% generously allowed
1.0% disfavored
15506
www.pnas.orgcgidoi10.1073pnas.2533374100 Erbel et al.
residues with solvent-exposed side chains within the H
and I
strands (Q322E, M338E, and Y342T) (Fig. 4b).
15
N
1
H HSQC
spectra of this triple mutant (trHIF-2
PAS-B) retain the chemical-
shift dispersion and general pattern of the wild-type protein,
confirming that the protein structure remains intact (Fig. 5a). The
ARNT-binding capability of this mutant was assessed by comparing
15
N
1
H HSQC spectra before and after addition of unlabeled
ARNT PAS-B. As demonstrated by the minimal ARNT-induced
changes in peak locations and intensities, the interaction of the
triple mutant HIF-2
PAS-B with ARNT has been very signifi-
cantly weakened. These data establish that subtle changes on the
surface of the H
and I
strands of HIF-2
PAS-B can disrupt the
HIFARNT PAS-B interaction.
Comparison of HIF-1
and -2
PAS-B. Sequence alignments of
HIF-1
and -2
indicate that the PAS-B domains of these
proteins are extremely similar (74% identity; Fig. 1b). Never-
theless, the HIF-1
homolog of our HIF-2
PAS-B construct
Fig. 3. Characterization of the HIF-2
ARNT PAS-B-binding interaction. (a)
Titration of unlabeled ARNT PAS-B (black, 0
M; light blue, 200
M; blue, 600
M; red, 800
M ARNT) into a 200
M
15
N-labeled HIF-2
PAS-B solution.
Arrow shows direction of peak shifts with increasing amounts of ARNT.
Residues with peak broadening beyond detection during the titration are
indicated with
*
.(b) Normalized peak heights of HIF-2
PAS-B (38 resonances)
plotted against increasing amounts of ARNT PAS-B. The concentration depen-
dence of the observed reduction in peak heights can be t to a 1:1 binding
event with a K
d
of 30
M (dotted line).
Fig. 4. Identication of the ARNT PAS-B-binding site on the surface of HIF-2
PAS-B. (a) Chemical-shift changes from
15
N
1
H HSQC spectra of HIF-2
PAS-B in
the presence of 800
M ARNT are plotted as a function of residue number. The red line indicates chemical-shift changes 0.16 ppm. Residues with peak
broadening beyond detection are shown as an arbitrary chemical-shift change of 0.5 ppm. Secondary structure elements are indicated with a gray background.
(b) Surface representations of HIF-2
PAS-B showing the location of the ARNT PAS-B-binding site. Colors indicate residues with large chemical-shift changes (0.4
ppm) or broadening beyond detection (red), residues with a signicant chemical-shift changes (0.16 ppm) (orange), and the site of the mutated residues that
disrupt the HIFARNT PAS-B interactions (yellow). Figs. 4 and 6 were made with
PYMOL (www.pymol.org).
Fig. 5. Point mutations in the HIF
PAS-B central
-sheet disrupt the binding
of ARNT PAS-B. (a) Superimposed
15
N
1
H HSQC spectra of 250
M
15
N labeled
HIF-2
PAS-B (Left) or triple mutant (Q322EM338EY342T) (Right). Spectra in
the presence of 900
M unlabeled ARNT PAS-B are shown with red contours;
those without ARNT are shown in black contours. Similar data for HIF-1
PAS-B
are provided in Supporting Methods.(b) PAS-B domain interaction is impor-
tant to form a biologically active HIFARNT complex. A construct expressing a
luciferase reporter under the control of an HRE promoter was transfected into
Ka-13 (columns 15) or CHO (column 6) cells along with various HIF
con-
structs. Values represent the average luciferase activity of three samples, with
bars indicating standard error. Luciferase expression was induced by cotrans-
fection of HIF-1
(column 2) or HIF-2
(column 4), particularly under hypoxic
conditions. Cotransfection of trHIF-1
(column 3) or trHIF-2
(column 5),
full-length HIF
proteins containing the three PAS-B mutations, shows a
signicant drop in luciferase activity compared with wild-type HIF
.
Erbel et al. PNAS
December 23, 2003
vol. 100
no. 26
15507
BIOPHYSICS
was too poorly behaved in solution to be amenable to structure
determination. However, by combining the high sequence iden-
tity between these PAS-B domains and the HIF-2
PAS-B
solution structure, we generated a homology model of HIF-1
PAS-B (29). This guided our mutation of three solvent-exposed
residues in the HIF-1
PAS-B
-sheet (Q320EV336EY340T)
at sites analogous to those changed in HIF-2
PAS-B. Interest-
ingly, this HIF-1
PAS-B triple mutant was significantly better
behaved in solution than the wild-type domain. To determine
whether HIF-1
PAS-B bound ARNT PAS-B in a similar
fashion as HIF-2
PAS-B, we recorded
15
N
1
H HSQC of
wild-type and triple-mutant HIF-1
PAS-B in the presence and
absence of ARNT PAS-B (Fig. 7, which is published as support-
ing information on the PNAS web site). In the case of wild-type
HIF-1
PAS-B, the combination of specific peak shifting and
line broadening on the addition of ARNT PAS-B indicated
binding. In contrast, this interaction was disrupted in the triple
mutant (Fig. 7), demonstrating that the interface identified in
HIF-2
is as crucial for the ARNT-binding function of HIF-1
.
Functional Importance of PAS-B in Full-Length HIF
. Our demonstra-
tion of specific interactions between the isolated HIF
and
ARNT PAS-B domains suggested that they function as dimer-
ization elements in the HIF heterodimer. To address this ques-
tion in living cells with full-length HIF
, we used a luciferase-
based assay that uses Chinese hamster ovary (CHO) cells lacking
functional HIF
(CHO-Ka13) (39). CHO-Ka13 cells were tran-
siently transfected with full-length HIF
constructs under the
control of the HIF-1 promoter (40) and encoding either wild-
type or a mutant sequence containing the three PAS-B domain
mutations. Low levels of DNA were transfected to roughly match
the HIF activity in wild-type CHO cells. The abilities of these
HIF
constructs to activate transcription were determined by
measuring the expression of luciferase under the control of a
minimal HRE promoter (8).
After expressing either wild-type HIF-1
or -2
, the hypoxia
response of the transfected CHO-Ka13 cells mimicked that of
wild-type CHO cells, confirming that HIF
activity can be
rescued by expression from these plasmids (Fig. 5b). Cells
transfected with HIF-2
constructs either lacking the entire
PAS-B domain (240350) or containing a mutation known to
unfold PAS-B (C339P; unfolded as established by NMR) showed
no significant luciferase activity under any conditions (data not
shown). Although these results suggest that HIF
PAS-B plays
a crucial role in forming a biologically active HIF
ARNT
heterodimer, the question remained whether the subtler PAS-B
modifications that disrupted in vitro binding would have a similar
effect. Therefore, we transfected CHO-Ka13 cells with full-
length HIF-1
and -2
constructs containing the three PAS-B
mutations (trHIF
). For both mutant proteins, the HRE-driven
luciferase response is decreased compared with that observed in
cells containing vectors expressing wild-type HIF
. In the case
of trHIF-1
, the response is virtually eliminated (Fig. 5b, column
3), whereas a less pronounced but significant reduction is
observed for trHIF-2
(Fig. 5b, column 5). These results are
surprising in light of the three apparently redundant sets of
proteinprotein interactions implied by the model of the HIF
heterodimer (Fig. 1a). Our data show that the subtle disruption
of the PAS-B interaction within the full length HIF
protein is
sufficient to prevent the formation of the HIF transcription
factor, thereby abolishing the hypoxia response.
Discussion
Despite the intense interest in hypoxia signaling, previous un-
derstanding of the roles of various proteinprotein interactions
in the HIF heterodimer remained unclear. Much of the data
underlying prior work in this area were based on deletions
generated at a time when the lack of structural data hampered
the design of constructs cleanly corresponding to PAS domain
boundaries. Unfortunately, this approach has complicated the
interpretation of these findings with respect to the functional
importance of individual PAS domains. In this context, our
results provide the structural insight to clarify how PAS-B
domains contribute to the stability of HIF and other bHLH-PAS
transcription factor complexes. We demonstrate that the PAS-B
domains of HIF
and ARNT directly associate in vitro, using a
group of predominantly hydrophobic residues located on the
solvent-exposed face of the HIF
␣␤
-sheet. Critically, mutations
that disrupt the interaction between isolated PAS-B domains in
vitro also interfere with the ability of the full-length heterodimer
to activate transcription in living cells. These results establish the
importance of the
-sheet interaction surface of HIF
PAS-B
domains within the complexes that are central to the hypoxia
response pathway.
More broadly, the
-sheet interface we identified appears to
be important for a wide array of inter- and intramolecular
interactions within several PAS domains (Fig. 6). Evidence of
this commonality is provided by PAS domains from two blue-
light photoreceptors, phototropin1 (AsLOV2) (36) and photo-
active yellow protein (33). Both of these domains are flanked by
additional
-helical elements that fold back on the PAS domain
itself, associating with the same
-sheet region used by HIF
to
bind ARNT (Fig. 6). An additional example is provided by the
human ether-a-go-go-related gene (HERG) potassium channel,
which contains a PAS domain that controls the poststimulation
deactivation kinetics of this channel, possibly by interacting with
Fig. 6. Versatility of protein interactions involving PAS domain
-sheets. HIF2
is shown in the same orientation as Fig. 4b and colored by residues experiencing
signicant
15
N
1
H chemical shifts on complex formation (red) and those used to generate the complex-disrupting trHIF-2
(blue). Phototropin (AsLOV2) (36) and
photoactive yellow protein (33) highlight the
-helices external to the PAS core (magenta) and any atoms located within 5 Å of those helices (pink). HERG (42)
shows functionally important, solvent-exposed residues (dark blue) and residues present in a surface hydrophobic patch suggested to be important for channel
function (light blue) (42).
15508
www.pnas.orgcgidoi10.1073pnas.2533374100 Erbel et al.
the S4-S5 linker (41). Point mutations in the PAS domain that
impair channel function have been identified by both alanine
scanning mutagenesis (42) and sequencing of HERG genes from
individuals affected by long QT syndrome (41), an inherited
cardiac disorder associated with defects in HERG and other ion
channels. Biochemical studies of channels containing one of
several conservative mutations in solvent-exposed residues
(F29L, F29A, N33T, Y43A, and R56Q; Fig. 6) demonstrate that
the region analogous to the HIFARNT interface plays an
important role in regulating HERG channel kinetics. In sum-
mary, these comparisons indicate that the exposed face of the
central
-sheet in PAS domains is well suited for making many
functionally important types of associations.
This comparison also suggests a route that might be exploited
to regulate the formation of the HIF
ARNT complex by using
small organic compounds. Although neither a natural ligand nor
a ligand-binding cavity has been identified for the HIF
PAS-B
domains, this should not be interpreted to mean this domain
cannot bind any small compounds. Indeed, it has been demon-
strated that certain artificial chemicals can specifically bind into
the well packed hydrophobic core of a PAS domain from PAS
kinase (Fig. 2b) (17), likely producing significant conformational
changes therein. Parallel studies of two PAS domains with
natural cofactors, AsLOV2 and photoactive yellow protein, have
shown that relatively small structural changes in these internally
bound compounds are sufficient to displace
-helices bound
onto the same
-sheet interface we identified in HIF
(36, 43).
Combining these observations and an earlier model proposed for
PAS domain signaling (44) raises the possibility that binding
compounds in the HIF
PAS-B core could generate structural
changes that disrupt the interactions needed to form the HIF
heterodimer, analogous to the HIF
mutations demonstrated
here. Given the central role of HIF in the hypoxia response
and the importance of this pathway in cancer progression,
this approach may serve as the basis for a novel therapeutic
strategy (1, 2).
We thank M. Rhima for technical assistance; P. Ratcliffe (Oxford
University, Oxford) for the gift of the Ka13 cell line; and D. Russell, J.
Garcia, and S. McKnight for comments on the manuscript. This work was
supported by grants from the National Institutes of Health (CA90601 to
K.H.G. and CA95471 to K.H.G. and R.K.B.), the University of Texas
Southwestern Endowed Scholars Program (to K.H.G. and R.K.B.), and
the Robert A. Welch Foundation (I-1424 to K.H.G.). P.B.C. was
supported by a National Institutes of Health Training Grant GM08297
to the University of Texas Southwestern Graduate Program in Molecular
Biophysics. R.K.B. is supported by a Career Award in the Biomedical
Sciences from the Burroughs Wellcome Fund.
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Erbel et al. PNAS
December 23, 2003
vol. 100
no. 26
15509
BIOPHYSICS
    • "dimer model (Fig 9B), do not affect dimerization confirms the applicability of the CLOCK:BMAL1 dimerization mode for AhR:ARNT dimerization model. The topologically equivalent position of AhR:I374 was previously investigated in the homologous dimer HIF2α:ARNT (mutation HIF2α:M338E in [37]). In that study, the mutation maps on the dimerization interface involving the β-sheets of both the HIF2α an ARNT partners and the observed negative effect was due to dimerization disruption, since activation of HIF2α is not dependent on ligand binding. "
    [Show abstract] [Hide abstract] ABSTRACT: The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.
    Full-text · Article · Jun 2016
    • "The complete mRNA sequences of the porcine AhR and ARNT which were obtained in our previous experiments ([20]; Sadowska et al., unpublished) were used to construct reliable spatial models of AhR-LBD and ARNT-LBD in the pig by means of homology modeling. To perform this analysis, the crystalline protein structures of the PAS domains of human Hif-2􃙐 [10] and ARNT [5] were used in the present study as modeling templates. The aim of the current study was to predict the structure of the porcine AhR-and ARNT-LBD by means of homology modeling. "
    [Show abstract] [Hide abstract] ABSTRACT: The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and natural chemicals, including toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the present study, homology models of the porcine AhR-ligand binding domain (LBD) and the porcine aryl hydrocarbon receptor nuclear translocator-ligand binding domain (ARNT-LBD) were created on the basis of structures of closely related respective proteins i.e., human Hif-2α and ARNT. Molecular docking of TCDD to the porcine AhR-LBD model revealed high binding affinity (-8.8 kcal/mol) between TCDD and the receptor. Moreover, formation of the TCDD/AhR-LBD complex was confirmed experimentally with the use of electrophoretic mobility shift assay (EMSA). It was found that TCDD (10 nM, 2 h of incubation) not only bound to the AhR in the porcine granulosa cells but also activated the receptor. The current study provides a framework for examining the key events involved in the ligand-dependent activation of the AhR.
    Full-text · Article · May 2016
    • "The bHLH-PAS TFs are well known in vertebrate TFs in which two PAS domains are present, contrary to the stramenopile sequences that have only one PAS [9, 76]. In vertebrates, the PAS domains are involved in the dimerization of PAS domains containing TFs, such as the Hypoxia Inducible Factor [77, 78]. The presence of bHLH and PAS domains in the same sequence in both vertebrates and stramenopiles may be an example of convergent evolution, which suggests that this fusion occurred in a parallel fashion in different lineages. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Studying transcription factors, which are some of the key players in gene expression, is of outstanding interest for the investigation of the evolutionary history of organisms through lineage-specific features. In this study we performed the first genome-wide TF identification and comparison between haptophytes and other algal lineages. Results: For TF identification and classification, we created a comprehensive pipeline using a combination of BLAST, HMMER and InterProScan software. The accuracy evaluation of the pipeline shows its applicability for every alga, plant and cyanobacterium, with very good PPV and sensitivity. This pipeline allowed us to identify and classified the transcription factor complement of the three haptophytes Tisochrysis lutea, Emiliania huxleyi and Pavlova sp.; the two stramenopiles Phaeodactylum tricornutum and Nannochloropsis gaditana; the chlorophyte Chlamydomonas reinhardtii and the rhodophyte Porphyridium purpureum. By using T. lutea and Porphyridium purpureum, this work extends the variety of species included in such comparative studies, allowing the detection and detailed study of lineage-specific features, such as the presence of TF families specific to the green lineage in Porphyridium purpureum, haptophytes and stramenopiles. Our comprehensive pipeline also allowed us to identify fungal and cyanobacterial TF families in the algal nuclear genomes. Conclusions: This study provides examples illustrating the complex evolutionary history of algae, some of which support the involvement of a green alga in haptophyte and stramenopile evolution.
    Full-text · Article · Apr 2016
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