H. sapiens Transcriptional
Coactivator p300/CREB-Binding Protein Associating Factor (PCAF)
Camelia Milnes '15 and Shannon Wright '16
The human p300/CBP-associating factor
(PCAF), named due to its ability to interact with p300/CBP, is a
histone acetyltransferase predominantly known for its ability to
activate transcription.This transcriptional co-activator most
regularly interacts with the transcriptional activator, p53, in order
to acetylate both nucleosomal and free histones (preferentially the H3
subunit) and basal transcription factors such as TFIIF and TFIIE.
Structurally similar to various coenzyme-bound N-acetyltransferases,
the manner in which PCAF binds to and catalyzes the p53 enzyme is an
excellent model in which to better understand structure-function
relationships attributed to various other transcriptional regulators
and histone transferases.
known for its ability to promote transcription of the human
immunodeficiency virus type 1 (HIV-1), the PCAF protein bromodomain
(PCAF BRD), in complex with an acetylated version of the HIV-1
trans-activator, Tat, has recently become highly relevant within the
field of cancer research. PCAF BRD, in complex with Tat-acetylated
Lys50, provides cellular synergy that is essential for the
transcriptional activation of the HIV-1 viral gene promoter.
Exploiting both proteins cooperative mechanism of malignancy could
potentially lead to better therapeutic treatments of HIV-1 and the
AIDS pandemic. We will examine the association of PCAF with the
HIV-1 Tat peptide in the final section of this tutorial in order to
explore this potential target for developing antiretroviral drugs to
inhibit the transcription of HIV-1 and consequently prevent
II. General Structure
The basic structure of the
PCAF-coenzyme A complex contains four distinct domains.
Topologically globular when folded, this protein encloses a histone
acetyltransferase (HAT) core domain associated with the binding of coenzyme A, which is covered, in a sense, with
a pronounced cleft spatially arranged along the top of the binding
cavity. Within this cavity lies a single glutamate
residue , which plays an essential role in terms of
catalysis. N- and C-terminal protein segments flank the protein core
on either side. Each of these terminal protein segments greatly
contribute to histone substrate binding.
PCAF, comprised of a single 832
residue long peptide strand, possesses a
topology. The major domains of this protein include a C-terminal
(bromodomains are found in nearly all nuclear histone
acetyltransferases and function as acetyl-lysine binding domains), a
central histone acetyltransferase domain
, and an N-terminal region
which contains an interaction surface for p300/CBP, other
transcriptional activators, and E1A (an adenoviral oncoprotein able
to recruit and subsequently inhibit PCAF activity via direct binding
to its HAT domain). This N-terminal region contains contributing
substrate-specific side chain determinants required for proper
nucleosomal acetylation. Once you've clicked the following button,
click and drag the protein molecule to rotate it! This will allow
you to more easily visualize the secondary structure of PCAF!
The core protein is formed by two
tertiary structural elements near the center of the protein. The
first element consists of beta-strands 2, 3, and 4 aligned in an
antiparallel configuration directly above helix alpha3. The second
element consists of an adjacent beta5-strand-loop-alpha4-helix.
The N-terminal domain consists of a
beta-strand that interacts via sheet interactions with the
beta2-strand of the core; a helix-turn-helix motif
(alpha1-turn-alpha2) hovers over one side of the protein core.
The C-terminal domain also hovers
above the protein core, opposite of the N-terminal domain, and
consists of a helix-loop-strand (alpha5-loop-beta6). Beta6 interacts
with beta5 of the core domain via parallel sheet interactions.
III. Coenzyme A Binding
Coenzyme A binds, as previously mentioned,
between the two elements of the protein core, oriented in a way as to
have its labile sulfhydryl pointing into the protein cleft. This
ligand is flanked on either side by the beta4-loop-alpha3
segment and the beta5-loop-alpha4
Almost completely buried by the protein cavity, coenzyme A is bound in
a bent configuration between both elements in order to facilitate an
extensive set of protein interactions; PCAF predominantly forms
contacts with the pantetheine arm-pyrophosphate chain of coenzyme A.
All but two of the groups of the 16 membered pantetheine
arm-pyrophosphate chain are contacted by the protein via hydrogen
bonds or van der Waals contacts.
The beta4-loop-alpha3 (residues 580 and 582-587) extensively interacts
via both direct and water-mediated
hydrogen bonds with the pyrophosphate group of the ligand molecule.
The only side chain hydrogen bond to the pyrophosphate oxygen of the
coenzyme is assigned to Thr587. Aliphatic
residues 574-576 containing a Cys-Ala-Val
sequence and Gln581 on the beta4-strand
make numerous van der Waals contacts with the entirety of the
pantetheine arm length. The backbone residues of Cys574 and Val576
also form hydrogen bonds with the pantetheine arm. This region has
amazing sequence-specificity: single point mutations of essentially
all residues within the beta4-loop-alpha3 that contact coenzyme A
drastically reduce HAT activity of PCAF in vitro.
residues (Ala613, Tyr616, and Phe617)
predominantly interact with the beta-mercaptoethylamine segment of
the pantetheine arm (through hydrophobic interactions) in order to
intimately orient the reactive sulfhydryl atom into the protein
cleft to expediate acetyl transfer. Tyr616 also makes van der Waals
contacts with the pantetheine arm closer to the pyrophosphate group.
Residues in the alpha4-helix make van der
Waals interactions with the adenosine base present within coenzyme
Residues Gln525 and Leu526
in the N-terminal segment make van der Waals with the pantetheine arm
and may play an important role in substrate-specific binding and/or
IV. Histone Acetyltransferase (HAT) Domain
Slightly deviating from the overall globular topology of the
protein, the PCAF-coenzyme A complex has a striking pronounced cleft
placed above the protein core, which is flanked by the N- and
C-terminal protein segments on opposite sides of the cavity. Chains
of Glu570 and Asp610, and the backbone carbonyls of Ile571, Val572,
and Tyr608, form an acidic patch at the base of the cleft,
establishing an attractive site onto which the basic lysine
substrate can favorably bind.
Made of somewhat flexible loops (alpha1-alpha2 and alpha5-beta6), the
parameters of this cleft are about 10x10x20 angstroms--a perfectly
sized patch to accommodate a protein strand including a reactive
lysine side chain!--and is made of somewhat flexible loops to
accommodate protein that possesses a reactive lysine side-chain.
The junction between the cleft and the
coenzyme A-binding site plays an important role in substrate
binding and/or catalysis. Catalysis proceeds through the formation
of a ternary protein-cofactor-substrate complex, followed by the
transfer of an acetyl group to the substrate via direct
nucleophilic attack of acetyl-coenzyme A-dependent
transferases. This mechanism requires the presence of a protein
side chain to serve as a general base for substrate proton
extraction to facilitate acyl addition. Glu570
in the beta4-strand and Asp610 in the
loop between the beta5-strand and the alpha4-helix of the protein
core are in close enough proximity to the cleft to serve as a
Glu570 strength is greatly
diminished when mutated, so this residue is likely most important
in the catalysis process. Further, this residue is in an ideal
environment to play a catalytic role, as it is also surrounded by
several hydrophobic residues which raise the pKa of the glutamate
side chain and thus facilitate proton extractions from the lysine
substrate. Depending on the location of the substrate, protein
extraction may proceed directly through the carboxylate of Glu570, or through the mediation of a water
molecule. Hydrogen bond donors must be present in order to
stabilize the tetrahedral reaction intermediate. These hydrogen
bond donors come from the backbone amino group of Cys574
and the backbone amino groups of the substrate.
V. HIV-1 Tat Peptide Binding
The Tat peptide binds tightly
to the PCAF bromodomain, but only upon
acetylation of Lis50. The specificity of
this binding depends on interactions with residues flanking AcK50.
The Tat peptide interacts with four left-handed alpha helices of
PCAF (residues 723-830). The side chain of acetyl-lysine
fits into the hydrophobic cavity of the protein and participates in
extensive interactions with residues F748,
V752, Y760, I764, Y802, and Y809.
Changes in loop conformation are necessary to expose critical
residues F748, V752, and I764.
The residues flanking AcK50 (G48,
R49, R53) also interact with the protein and are
responsible for the specificity of this binding. Y47
and E54 side chains interact with V763 and E756
respectively. Mutations of any of these protein residues results in
a major reduction or complete loss in Tat-binding.
Clements, Adrienne, Jeannie R. Rojas, Raymond
C. Trieval, Lian Wang, and Ronen Marmorstein. 1999. Crystal
structure of the histone acetyltransferase domain of the human
PCAF transcriptional regulator bound to coenzyme A. The EMBO
Journal 18(13): 3521-3532.
Mujtaba, Shiraz, Yan He, Lei Zeng, Amjad
Farooq, Justin E. Carlson, Melanie Ott, Eric Verdin, and
Ming-Ming Zhou. 2002. Structural Basis of the Lysine-Acetylated
HIV-1 Tat Recognition by PCAF Bromodomain. Molecular
Cell 9: 575-586.
Quy, Vo Cam, Sergio Pantano, Giulia
Rossetti, Mauro Giacca, and Paolo Carloni. 2012. HIV-1 Tat
Binding to PCAF Bromodomain: Structural Determinants from
Computational Methods. Biology
Wang, Qiang, Ruirui Wang, Baiqun Zhang,
Shuai Zhang, Yongtang Zheng, and Zhiyong Wang. 2013. Small
organic molecules targeting PCAF bromodomain as potent
inhibitors of HIV-1 replication. Med.
Chem. Commun. 4: 737-740.
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