Perspective

Physiology meets biophysics: Visualizing the

interaction of hypoxia-inducible factor 1
with p300 and CBP
Gregg L. Semenza*
McKusickNathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287-3914

omplex circulatory and respiratory

systems are established during embryonic development and used in fetal and
postnatal life to ensure oxygen delivery to
every cell in the human body. Within each
cell, the utilization of O2 as a substrate for
biochemical reactions, most notably as an
electron acceptor in the mitochondria, is
also highly regulated. As a result of systemic and cellular physiological mechanisms that control O2 delivery and consumption, O2 concentrations are precisely
maintained within a narrow range that
represents a balance between cellular metabolic requirements and the risk of oxidative damage. Since its identification ten
years ago (1), an exponentially growing
body of experimental data indicates that
the transcription factor hypoxia-inducible
factor 1 (HIF-1) functions as a global
regulator of O2 homeostasis, as it is required for the establishment of the circulatory and respiratory systems as well as
for physiological responses to hypoxia in
prenatal and postnatal life (27).
Altered O2 homeostasis plays a critical
role in the pathophysiology of ischemic
and neoplastic disorders, which are the
most common causes of mortality in the
U.S. population. In ischemic disorders,
HIF-1 activates transcription of genes encoding vascular endothelial growth factor
(VEGF), which promotes angiogenesis;
glucose transporters and glycolytic enzymes, which allow for the production of
ATP in the absence of O2; and survival
factors such as VEGF, erythropoietin
(EPO), and insulin-like growth factor 2,
which inhibit ischemia-induced apoptosis
(reviewed in ref. 8). Thus, therapeutic
strategies designed to increase HIF-1 activity may prevent ischemia or infarction
in patients with advanced atherosclerosis
(9). In cancer, these same pathways are
used to promote tumor cell survival, proliferation, invasion, and metastasis (reviewed in refs. 1012). Increased HIF-1
expression is associated with increased
patient mortality in several types of cancer, and therapeutic strategies designed to

inhibit HIF-1 activity may improve the

survival of such patients (12).
Over the same decade during which the
involvement of HIF-1 in development,
physiology, and pathophysiology has been
established, novel molecular mechanisms
that regulate HIF-1 activity have also been
delineated. Protein purification revealed
that HIF-1 was a heterodimer composed
of HIF-1 and HIF-1 subunits (13),
which dimerize and bind to DNA by
means of basic helixloop helixPAS
(bHLH-PAS) domains (14). Expression of
the 826-aa HIF-1 subunit is tightly regulated by the cellular O2 concentration
(15) by means of ubiquitination and proteasomal degradation (16, 17) that is mediated by binding of the von Hippel
Lindau tumor suppressor protein (VHL)
to HIF-1 (18). This interaction occurs
only when HIF-1 has been hydroxylated
at residues 402 and 564 (1923). O2 is a
rate-limiting substrate for the prolyl hydroxylases that modify HIF-1 (20), thus
providing a molecular mechanism for hypoxia-inducible expression of HIF-1
(Fig. 1).
HIF-1 transactivation domain function is also subject to O2-dependent negative regulation (24, 25). The aminoterminal transactivation domain is
negatively regulated by the recruitment of
histone deacetylases by VHL and by factor
inhibiting HIF-1 (FIH-1), which binds to
both VHL and HIF-1 (26). The binding
of the coactivators CBP (CREB-binding
protein) and p300 to the carboxylterminal transactivation domain (TAD-C;
also known as CAD and CTAD) is critical
for HIF-1 transcriptional activity (27, 28).
HIF-1 TAD-C binds to a domain in p300
and CBP that is known as the cysteine
histidine-rich 1 (CH1) or transcriptional
adapter zinc-binding 1 (TAZ1) domain
(29). CBP and p300 perform both structural and enzymatic functions in mediating the effects of dozens of DNA-binding
transcriptional activators (reviewed in
refs. 30 and 31). First, they function to
physically link these sequence-specific
DNA-binding proteins to the transcription

11570 11572 PNAS September 3, 2002 vol. 99 no. 18

initiation complex that contains RNA

polymerase II and more than 50 associated proteins. Second, they function as
histone acetyltransferases which perform
chromatin remodeling that is required for
gene transcription. Binding of CBP and
p300 to HIF-1 is negatively regulated by
the O2-dependent hydroxylation of asparagine-803 in TAD-C by FIH-1 (32, 33).
Thus, both the stability and transcriptional activity of HIF-1 are negatively
regulated by hydroxylation (Fig. 1). Hydroxylation plays a role similar to that of
other posttranslational modifications
(e.g., phosphorylation) in regulating proteinprotein interactions, but it is unique
in its dependence upon the cellular O2
concentration. These exciting discoveries
have provided an elegant molecular basis
for the O2-dependent regulation of gene
transcription by HIF-1. This understanding has now been advanced to atomic
resolution with the publication of two
recent papers in PNAS describing NMR
spectroscopic analyses of the structural
interaction of HIF-1 TAD-C with the
CH1TAZ1 domain of either p300 (34) or
CBP (35).
In one study (34), the structure of
HIF-1 amino acid residues 786826 in a
complex with p300 residues 323423 was
determined. The p300 CH1 domain consists of four -helices that are folded into
a triangular structure that is stabilized by
the coordination of three Zn2 atoms by
means of a histidine and three cysteine
residues and also by a single hydrophobic
core that is formed by all four helices. The
complex of p300 CH1 and HIF-1 TAD-C
has three remarkable characteristics.
First, the interactions between the two
domains are extensive: the entire TAD-C
is embedded in CH1 with almost half of its
surface buried in the interface. The buried
surface area is more than twice the average for protein complexes in general and
*To whom reprint requests should be addressed at: Johns
Hopkins Hospital, CMSC-1004, 600 North Wolfe Street,
Baltimore, MD 21287-3914. E-mail: gsemenza@jhmi.edu.

www.pnas.orgcgidoi10.1073pnas.192442299

almost three times that of the complex

consisting of the CREB transactivation
domain bound to the KIX domain of CBP.
Second, the tertiary structure of TAD-C
in the complex is determined exclusively
by its interactions with CH1 and, in the
absence of CH1, TAD-C is essentially
unstructured. The proposed induced-fit
model provides a basis for the interaction
of CH1 with a variety of transcriptional
activators (ETS1, HNF4, p53, PIT1,
STAT2, and others) that share no common sequence features, although this will
require experimental verification. Third,
Asn-803 is the single most buried HIF-1
residue in the complex. Although the
chemistry of the asparagine hydroxylation
reaction has not been determined, the
structural data indicate that hydroxylation
in either the pro-R or pro-S position would
be highly disruptive, providing a structural
basis for the regulation of complex formation by O2-dependent hydroxylation.
In the other study published in PNAS
(35), the structure of HIF-1 amino acid
residues 776826 in a complex with the
TAZ1 domain of CBP (residues 323423)
was determined. Although the HIF-1
Semenza

fragment analyzed in this study contained

an additional 10 amino acid residues, it
was also intrinsically disordered in the
unbound state and became structured
upon binding to CBP TAZ1. When isothermal titration calorimetry was used,
the dissociation constant of the complex
between TAD-C and TAZ1 was determined to be 7 nM, indicating a remarkably
high affinity as predicted by the extensive
interactions between these domains.
Whereas binding of HIF-1(786826) to
p300 CH1 induced the formation of two
-helices involving residues 797803 and
816822, the binding of HIF-1(776826)
to CBP TAZ1 induced the formation of
three -helices involving residues 784
788, 796804, and 815824. In the complex with CBP, Asn-803 of TAD-C is
located on an -helix and is buried deep in
the proteinprotein interface (as was observed for the p300 complex), where it
participates in multiple hydrogen-bonding
interactions that are predicted to stabilize
both the helix and the complex.
The absence or presence of hydroxylation of Asn-803 can be viewed as a switch
that turns TAD-C function on or off by

determining the ability of the domain to

interact with p300 or CBP. Mutation of
Asn-803 to alanine completely eliminates
the O2-dependent negative regulation of
TAD-C function and interaction with
p300 (33). Taken together with the structural studies, these data suggest that a
small molecule targeted to the -helix
containing Asn-803 might inhibit the interaction of TAD-C with p300 or CBP.
Such an inhibitor would be potentially
useful in the treatment of patients with
cancers in which HIF-1 overexpression is
associated with increased mortality (reviewed in ref. 12).
The pharmaceutical industry has in general avoided transcription factors as molecular targets for drug development and
inhibition of proteinprotein interactions
as a mechanism of drug action. The rationale has been that a nuclear target is more
difficult to hit because the drug must pass
through an additional membrane bilayer.
It is also considered less likely that a small
molecule can specifically disrupt extensive
interactions between two protein domains
as compared with targeting an enzyme for
inhibition by occupying its active site.

PNAS September 3, 2002 vol. 99 no. 18 11571

PERSPECTIVE

Fig. 1. Oxygen-dependent hydroxylation events that regulate HIF-1 protein stability and transcriptional activity. Under normoxic conditions, HIF-1 is
hydroxylated (OH) on proline residues 402 and 564 (P402 and P564) and asparagine residue 803 (N803), which are located within the carboxyl- (TAD-C) and amino(TAD-N) terminal transactivation domains, respectively. bHLH, basic helixloop helix domain. Hydroxylation of P402 and P564 by the prolyl hydroxylases PHD-1,
-2, or -3 (also known as HIF-1 prolyl hydroxylases HPH-1, -2, or -3) is required for the binding of the von HippelLindau tumor suppressor protein (VHL), which
in turn recruits an E3 ubiquitinprotein ligase complex (containing elongin B, elongin C, CUL2, and RBX1) that targets HIF-1 for proteasomal degradation. VHL
also recruits histone deacetylases that repress transactivation. Hydroxylation of N803 by FIH-1 prevents the interaction of TAD-N with the CH1TAZ1 domain of
the coactivators p300 and CBP (CREB-binding protein) which is necessary for transactivation of target genes by HIF-1.

However, the recent identification of

small molecules that inhibit the dimerization of the transcription factors MYC and
MAX in vitro and MYC-induced transformation in cell culture (36) suggests that
the aforementioned historical biases
against targeting transcription factors and
proteinprotein interactions may not have
as compelling a scientific basis in the
present era of high-throughput molecular
screening.
Expression of TAD-C peptides that
block the interaction between HIF-1 and
p300 inhibit tumor xenograft growth in
nude mice (29). However, such peptides
also inhibit the interaction of p300 CH1
with other transcription factors (see
above) and thus the extent to which the
observed effects are attributable to re-

duced interaction of HIF-1 and p300 per

se is unknown. Herein lies a potential
problem with this pharmacologic approach: One would like to inhibit the
interaction of p300 and CBP with HIF-1
specifically, and thus the latter protein is
the preferred target for binding of a small
molecule inhibitor. However, the disordered structure of TAD-C before interaction with p300 or CBP binding may reduce
the likelihood of identifying a small molecule capable of specific high-affinity
binding. Adding to the challenge is the
high affinity of the competing interaction
of p300 or CBP with HIF-1. Nevertheless, the demonstration that hydroxylation
of Asn-803 can disrupt these interactions
suggests that this critical molecular switch
may represent an Achilles heel that can be
targeted therapeutically.

Clinical applications as yet reside in that

fantastic realm where every basic science
discovery is immediately translated into a
powerful disease-conquering therapy, and
such dreams are not disturbed by the
harsh light of empiricism. For now, it is
exciting to appreciate the molecular portraits that are presented in the papers by
Freedman et al. (34) and Dames et al. (35).
These data, along with recently reported
structures of the HIF-1VHL interaction (37, 38), provide us with a remarkable
opportunity to view physiology through
the eyes of the biophysicist.

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11572 www.pnas.orgcgidoi10.1073pnas.192442299

Work in my laboratory is supported by Grants

R01-DK39869, R01-HL55336, and P01-HL65608
from the National Institutes of Health.

Semenza