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H. sapiens Transcriptional Coactivator p300/CREB-Binding Protein Associating Factor (PCAF)


Camelia Milnes '15 and Shannon Wright '16

Contents:

I. Introduction

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.

Also 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 infection.


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 beta-alpha-alpha-beta-beta-beta-alpha-beta-alpha-alpha-beta topology. The major domains of this protein include a C-terminal bromodomain (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 segment. 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.

Beta5-loop-alpha4 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 A.


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 catalysis.

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 general base. 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.


VI. References

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 1: 277-296.

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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