User:Valentine J Klimkowski/practice Set7-9
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(HAT1/HAT2 proteopedia page from JF, CG & MB, Spring 2019; VJK edit 191023) |
(HAT1/HAT2 proteopedia page from JF, CG & MB, Spring 2019; VJK edit 191023) |
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proteopedia linkproteopedia linkHistone Acetyltransferase HAT1/HAT2 Complex, Saccharomyces cerevisiae
HistonesHistones are proteins found in the cell nucleus that are the key building blocks of chromatin and are essential for proper DNA packaging and transcription. In the first step of DNA packaging, two copies of the four core histone proteins (H1, H2A, H3, and H4) form an octamer in which DNA directly interacts with and wraps around, forming the nucleosome. 20-24% of residues making up the histone octamer are arginine and lysine, causing a net positive charge, especially at the outer surfaces of the histone core where negatively charged DNA is bound [1] (Figure 1) [2] . After translation, the positively charged residues of the histone core tails are often subject to chemical modifications, such as acetylation or methylation. These modifications can regulate the processes of DNA repair, replication, transcription, and heterochromatin maintenance.
Histone ModificationHistones can be modified in a variety of ways, including: methylation, demethylation, acetylation, deacetylation as well as many others. These modifications all result in either the condensation or relaxation of DNA and consequently turning on or off DNA transcription. Histone acetylation is a histone modification that involves the transfer of an acetyl group from the cofactor Acetyl Coenzyme A (acetyl-CoA) to the ε-amino group of a lysine residue on a histone. This reaction is done by various histone acetyltransferase (HAT) enzymes. The specific histone acetylation modification is an important epigenetic marker. It plays a role in RNA synthesis and there a known correlation between gene activity and histone acetylation. Any misregulation of the HAT enzyme can possibly lead to cancer, cardiovascular disease, and HIV [3].
HAT1 Backgroundwas the first of the enzymes to be identified (in yeast) in the HAT family of enzymes [4]. It is lysine specific for newly synthesized histone 4 (H4). One study showed that the deletion of the HAT gene caused a loss of acetylation on H4K5 and H4K12, leading to the conclusion that HAT1 is the sole enzyme responsible for this evolutionary conserved histone modification.[5] The enzyme is identified as a binding partner for HAT1 to help modulate the substrate specificity of HAT1 [4]. The HAT1/HAT2 complex is highly specific for H4K12 acetylation.
Hat1/Hat2 Complex StructureThe HAT1/HAT2 complex structure was determined by X-ray diffraction at 2.0 A resolution. The complex was crystallized in the presence of coenzyme A (CoA) and a H4 N-terminal peptide (1-48). The complex has four components (HAT1, HAT2, H4 peptide, and CoA) seen with a 1:1:1:1 stoichiometry. While residues 1-48 of the yeast H4 protein were included in the crystallization, only residues 9-46 were well resolved in the refined structure. In the HAT1 subunit of the refined complex, residues 1-4 could not be located. Similarly, in the HAT2 subunit, residues 1-6, residues 87-104, and residues 391-401 were not resolved [4].
HAT1 is not catalytically active until it binds with HAT2 to form the [6]. The HAT1 structure, identified as , includes 317 residues and contains the binding site for acetyl-coenzyme A. HAT2 is identified as , which includes 401 residues in a beta-propeller formation with C7 symmetry. Bound to this complex is the histone protein residues 9-46.
The HAT1 and HAT2 interface is stabilized by several interactions of various types. Many of these interactions with HAT2 involve residues contained in a HAT1 of residues 200-208. This helix is thought to be important for the heterodimer formation as the deletion of the helix abolishes the in vitro interaction between HAT1 and HAT2. This suggests that there may be another protein involved, such as the N terminus tail of H4, acting as a linker protein interacting with the complex interface to further stabilize the complex interface [4]. This HAT1/HAT2 interface is stabilized by between the two subunits. Three specific interactions at the interface involving hydrogen bonds help stabilize the complex. The side chain atoms of with the main chain nitrogen of Ala202 in HAT1. The side chain of makes hydrogen bonds with Leu288 and Phe205 respectively. The last area of hydrogen bonds between HAT1 and HAT2 is found between . The at the interface of the complex appears to be critical for the complex formation. This core consists of aromatic amino acids from HAT1 and leucine amino acids from HAT2, however it does not form any obvious ring stacking. Once the complex has formed, the histone complex can be catalytically active. The N-terminal segment of H4 that is bound to the HAT1/HAT2 can be divided into . The N-terminal region of H4 is buried within a grove on the surface between HAT1 and HAT2, although H4 mostly interacts with HAT1. The H4 C-terminal helix segment interacts exclusively with HAT2, residing in a groove formed by the LP2 helix, the N-terminal helix and the C-terminus residues. Overall the complex is strongly stabilized by salt bridge interactions between the histone and the heterodimer. Previous studies suggest that H4K12 inserts into the active site of HAT1 to access acetyl-CoA. [6]
Acetyl-CoA (CoFactor) Binding Site The acetyl-CoA HAT1 binding site is parallel to the C-terminal domain of the HAT1 protein. Acetyl-CoA fits structurally into the small binding site due to the kinked pantetheine group giving the molecule a bent confirmation. Once bound, most of the acetyl-CoA molecule is buried in the protein, around 60% (Figure 2). Hydrophobic contacts, hydrogen bonds, and salt bridges help to stabilize the protein-ligand interaction.
The β-methyl of the acetyl group interactions in the hydrophobic pocket formed by the side chain of residues: . The carbonyl oxygen of the acetyl group of the main chain Phe-220 and the sulfur of the acetyl-group . In most HAT1 structures, these interactions keep acetyl-CoA in the correct position of the cofactor active site for the transfer of the acetyl-group. In this HAT1 structure, the sulfur acetyl-CoA atom interaction with Asn 258 is unlikely with the distance of 5.5-5.7 angstroms. [7].
MechanismAfter many structural studies, the total catalytic mechanism for HAT1 remains unclear. In particular, the identity of the general base needed to deprotonate the substrate lysine is uncertain. In a previous study a structural overlay of HAT1 and Gcn5, a better-understood HAT enzyme, found a conserved glutamate residue in the active site of both enzymes. Mutation of this glutamate (equivalent to Glu255 in 2psw.pdb) was shown to greatly decrease the catalytic ability of HAT1, identifying it to be important for catalysis. [4] The crystallized structure of the HAT1/HAT2 complex supports, with the proximity of potentially catalytic residues, a mechanism for histone acetylation involving the following residues and cofactor: .
In this mechanism, first the carboxyl of Glu255 acts to deprotonate lysine 12 of histone 4. (In the featured structure the H4 modified lysine has numbering 14). Surrounding the lysine residue on histone 4 are which interact with the amino end of the lysine to better orient the lone pair electrons for nucleophillic attack. In the second step the lone pair on the lysine attacks the carbonyl carbon of acetyl-CoA (acetyl group not shown in the structure), forming an oxyanion containing tetrahedral transition state. Finally, upon electron reorganization, the C-S scissile bond breaks leaving the H4Lys12 acetylated and Coenzyme A.
InhibitionAlthough HAT1 was the first histone acetyltransferase enzyme discovered, it is difficult to study and is one of the least understood HAT enzymes. While HAT1 has been linked to many disease states, until recently no known enzyme inhibitor had been successfully developed. One exception is the HAT1 inhibitor (H4K12CoA) that is a conjugate of the first 20 residues of the H4 protein, covalently linked to CoA through the lysine 12 side chain. H4K12CoA was found to act as a competitive inhibitor to both the H4 peptide substrate as well as acetyl-CoA. Having this inhibitor now allows for more in depth study of therapeutic targets associated with specific disease states. Additionally, H4K12CoA can be used as tool compound to better elucidate the HAT1 specificity and mechanism regarding epigenetic modification of the H4 histone protein [3]. References
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