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Bromodomain (PCAF)

Introduction

The bromodomain is an evolutionary conserved motif found in chromatin remodeling complexes. It has been identified in over 100 proteins from multiple organisms ranging from unicellular microscopic eukaryotes (e.g., yeast) to humans. The motif is best known for the groundbreaking recent discoveries made to identify it as the only acetyl-lysine binding domain^[1] and to reveal its highly specific ligand selectivity properties^[2]. Due to these novel characteristics, bromodomains are typically found in proteins responsible for modifications in chromatin structure and the regulation of gene expression, such as histone acetyltransferases (HATs), and the ATPase subunits of chromatin remodeling complexes. There are several families of proteins with bromodomains. One such family is the human transcriptional coactivator p300/CBP-associated factor (PCAF) bromodomain.

Structure and Function

Structure

The bromodomain was originally identified as a sequence of roughly 60 amino acid residues that forms 2 alpha helices^[3]. However, it is now known that the bromodomain consist of a highly conserved sequence of approximately 110 amino acids^[4]. The structure of the PCAF bromodomain consists of a bundle (alphaZ, alphaA, alphaB, and alphaC) with a left-handed twist, and a long intervening loop between helices Z and A (ZA loop)^[1]. The ZA loop of the bromodomain has a defined conformation and is packed against the loop between helices B and C (BC loop) to form a . This pocket created by the ZA and BC loops is lined by specific residues (Val 752, Ala 757, Tyr 760, Val 763, Tyr 802 and Tyr 809) that support protein-protein interactions. The ZA loop varies in length between different bromodomains, but almost always contains residues corresponding to Phe 748, Pro 751, Pro 758, Tyr 760 and Pro 767^[1].

Function

Until recently, the function of the bromodomain remained unknown. Its structure and modularity, along with its feature of both N and C termini located together on one end of the protein, suggested that it played a role in protein-protein interactions. It has now been shown that the hydrophobic pocket formed by the loops is the primary binding site for acetyl-lysine proteins, making the bromodomain a functional site for recognition of acetylated lysine residues that play a role in gene regulation via protein-protein interactions^[1]. These interactions have been shown via localization and chemical shift experiments that revealed the specific manner with which the bromodomain hydrophobic cavity binds to acetylated lysine residues.

Once the acetyl-lysine residue makes the initial binding inside the hydrophobic pocket, the ZA and BC loop residues at the entrance of the pocket interact with the amino acids adjacent (+/- 1 or 2) to the already bound acetyl-lysine. Those interactions reinforce binding of the target sequence^[5]. Small structural changes in the conformation of the ZA and BC loops result in exposing other residues that are originally buried within the protein to aid in peptide recognition^[6].

It is also believed that the bromodomain may also play a role in highly specific histone acetylation by tethering transcriptional HATs to specific chromosomal sites^[7] as well as the assembly of multiprotein complexes in transcriptional activation such as the Bromodomain–HIV-1 Tat complex necessary for HIV-1 transcriptional activation^[6].

Mechanism

Acetylation of the lysine and its effects on chromatin remodeling.

The mechanism of protein-protein interaction for the bromodomain of PCAF with target proteins, such as histones^[8] and Tat^[6], begins with the acetylation of lysine residues. The acetylation causes a conformational changes in the histones, which allows for transcriptional machinery to access DNA. The bromodomains of PCAF have three major points of contact that allow for site-specific histone recognition. First, the of the target protein enters a embedded between the ZA and BC loops at the bottom of the protein. The Asn803 residue in the bromodomain forms a hydrogen bond with the amide nitrogen of the acetyl-lysine. Next, residues in the ZA and/or BC loops interact with residues adjacent to the acetyl-lysine, which reinforces the acetyl-lysine binding in the bromodomain. Finally, additional residues in the ZA and BC loops that face opposite to the bromodomain form hydrophobic and/ or electrostatic interaction with the target protein 3 residues away from the acetyl-lysine. This residue clamps on the BC loop together with the acetyl-lysine side chain that is bound inside the hydrophobic pocket of the bromodomain^[8].

The histone acetyltransferase portion of PCAF helps with the transactivation of HIV-1 by acetylating Lys28 of Tat. The acetylated Lys28 of Tat interacts with positive elongation factors, which stimulates elongation of nascent HIV-1 transcripts. Acetylated Lys50 on Tat causes dissociation from TAR RNA and binds to the bromodomain of PCAF^[9].

PCAF Bromodomain-HIV-1 Tat Interaction and Implications

Interaction and Implications

Transcriptional elongation by HIV-1 Tat.

Protein lysine acetylation is a crucial regulatory mechanism in chromatin remodeling and transcription activation for numerous cellular processes. The human immunodeficiency virus type 1 (HIV-1) trans-activator protein (Tat), for example, stimulates transcription of the HIV genome and promotes viral replication in cells. But Tat transactivation activity is dependent on the acetylation of Lys-50 by p300/CBP^[6]. When Tat is acetylated at the Lys-50 residue, Tat dissociates from TAR RNA and binds to the PCAF bromodomain instead. This promotes the formation of a multiprotein complex that is responsible for transcription activation of the HIV genome.

Drug Design

Current anti-HIV drugs target viral proteins such as reverse transcriptase, protease, and integrase^[10]. However, the discovery that Tat transactivation requires Lys-50 acetylation for functional transcription of the viral genome reveals a whole new approach to interfering with virus production. Drugs that target viral proteins have proven inadequate in eradicating the virus because the fast rate of mutations in the target proteins lead to developed drug resistance. Targeting a host cell protein that is necessary for viral reproduction (such as the PCAF bromodomain) as opposed to a viral protein, has the potential to inhibit HIV production much more effectively by disrupting HIV gene expression.

Given the high selectivity of the bromodomain for its target protein, are currently being designed and engineered to block Tat/PCAF association^[11]. In addition, new functions of the bromodomain remain to be discovered with implications for many human diseases such as cancer and Alzheimer's disease, and well as breakthroughs in our knowledge of transcription and gene regulation.

References

1. ↑ ^{1.0 1.1 1.2 1.3} Dhalluin, C. et al (1999) Nature 399, 491 [1]
2. ↑ Zeng, L. (2002) FEBS 513:1, 124 [2]
3. ↑ Haynes, S.R. et al (1992) Nucleic Acids Res. 20, 2603 [3]
4. ↑ Owen, D. J. et al. (2000) EMBO J. 19(22), 6141 [4]
5. ↑ Mujtaba, S. et al (2007) Oncogene 26, 5521 [5]
6. ↑ ^{6.0 6.1 6.2 6.3} Mujtaba, S. et al (2002) Mol. Cell 9, 575 [6]
7. ↑ Brownell, J. et al (1996) Curr. Opin. Genet. Dev. 6, 176 [7]
8. ↑ ^{8.0 8.1} Zeng, L. et al (2008) Structural basis of site-specific histone recognition by the bromodomains of human coactivators PCAF and CBP/p300. Structure 16: 643–652 [8]
9. ↑ Nakatani, Y. (2002) HIV-1 Transcription: Activation Mediated by Acetylation of Tat. Structure 10:443-444 [9]
10. ↑ Garg, R. et al (1999) Chem. Rev. 99, 3525 [10]
11. ↑ Zeng, L. (2005) J. Am. Chem. Soc. 127, 2376 [11]