Sandbox Reserved 774

This Sandbox is Reserved from Sep 25, 2013, through Mar 31, 2014 for use in the course "BCH455/555 Proteins and Molecular Mechanisms" taught by Michael B. Goshe at the North Carolina State University. This reservation includes Sandbox Reserved 299, Sandbox Reserved 300 and Sandbox Reserved 760 through Sandbox Reserved 779.

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spinqualitylabelsall models 3D Model of Hpa2. Clicking links in the test will highlighted : chains A,B,C,D, β-sheets, main chain, side chain, and residues 100-104. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Histone Acetyltransferase Hpa2Histone Acetyltransferase Hpa2

Histone Acetyltransferase Hpa2 is a member of the GNAT (Gcn5-related N-acetyltransferases) super-family of enzymes that are found spread out across nature and use acyl-CoA's to acylate their cognate substrates. GNAT is a catalytic subunit of ADA and SAGA histone acetyltransferase complexes. Hpa2 is found in the organism Saccharomyces Cerevisiae, which is more commonly known as Baker's Yeast. It was also discovered in other organisms, such as Pelagibacterium halotolerans B2 - a marine halotolerant bacterium in the East China Sea. In vitro, Hpa2 serves to acetylate histone H3 'Lys-4' and 'Lys-14' and histone H4 'Lys-5' and 'Lys-12.' The acetylation of the e-amino group of lysines on the histone N terminal tails and core regions cause changes in the chromatin structure and dynamics, which often times leads to transcriptional activation. In solution, Hpa2 forms a dimer, and upon binding with AcCoA forms a tetramer. It is classified as a transferase.

StructureStructure

Has a four chain structure (, , , ) with 2.4 Å resolution, and 2.9 Å resolution with a co-factor (acetyl-CoA). Core fold features include four conserved sequence motifs of the GNAT family, and comprises a central, highly curved five stranded surrounded on both sides by helical segments.

Each monomer has a similar and compact α-β structure. The structure's core contains a central mixed five-stranded sheet structure from sheets β1 to β5. Strands β1 to β4, however, are organized in an anti-parallel arrangement while β4 and β5 are parallel, but only at their amino-terminal ends. At the other end of the parallel β4 and β5 strands, they are spread apart because of a β bulge in strand β4 caused by residue N74 of strand β3 as well as N91 and D92 of β4. The central sheet is accompanied on each side by two α-helices. Helices α1 and α2 are on one side of the sheet with α1 lying nearly flat against and perpendicular to the other direction of the strands, while helices α3 and α4 are on the opposite side of the sheet with helix α3 cupped within the curved face of the sheet. The method used to determine the structure was X-ray crystallography. Sedimentation and crystal structure analysis clearly shows that Hpa2 is dimeric in solution and tetramerizes in the unit crystal. The crystal structure of the oligomer reveals that two Hpa2 dimers are held together by interaction between the bound acetyl-CoA molecules. The average B-factor value of the is 23.9 with a 25.4 . The R-factor is 0.19.

Figure 1. Sequence of Hpa2.

Co-factorCo-factor

Most of the hydrogen bonding between Hpa2 and its co-factor AcCoA are highly conserved and occur via the main-chain groups, not the side-chains. This explains the uniformity of the binds with the co-factors, despite the low degree of sequence conservation. The conserved main-chain contacts seen in a segment of Motif A forms a loop before and around the first turn of the helix in Motif A. This is found in residues . Most of the residues in this loop contribute to a series of hydrogen bonds between the α and β phosphate oxygen atoms via main-chain groups. This includes a conserved solvent molecule interaction. This loop was not located within Hpa2 in the absence of its co-factor, indicating that this region is formed upon binding. It appears that this area of interactions is a vital determinant for the binding of AcCoA. The pantetheine arm of CoA interacts with the backbone amide and carbonyl groups of β4, and is wedged between the spread apart areas on the strands of β4 and β5. The loop connecting β4 and α3 provides five amide hydrogen bonds to the pyrophosphate group of the bound CoA.

Active SiteActive Site

The region around the has potential consequences for substrate binding. For Hpa2, there exists a pocket adjacent to the active site which is sealed off on two sides by the second Hpa2 monomer in each dimer. This pocket restricts the conformation of a polypeptide backbone in the vicinity of the active site. Therefore, Hpa2 is able to distinguish between potential substrate, whereby the only lysine side-chains that may enter the active site are those with surrounding polypeptides that can adopt a conformation with the ability to fit through the pocket.

spinqualitylabelsall models Hpa2 + AcCoA, Clicking links in the test will highlighted : co-factor, backbone, β-strands, active site, α-helices. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Secondary StructureSecondary Structure

Most of the secondary structure elements of the monomer contribute residues involved in dimer contacts. A large part of the interface is formed by two projections from the core part of the monomer structure. The first projection is formed by the C-terminal end of strand β3, turn β3-β4, and the N-terminal end of strand β4, while the second is formed by strand β7. Together with strands β5 and β6 they form a barrel-like structure containing ten strands in which the component strands of the barrel are locked together. Most importantly, strand β7 from each monomer interacts between strands β5 and β6 of the opposite monomer, which also extends the central sheet structure by two strands. Also, the two projections interact with residues from helices α1 and α2, turn α1 α2, turn α2 β2, and helices α3 and α4 of the opposite monomer. There are eight , four , and ten turns. Thirty-three percent of the secondary structure is helical (5 helices and 50 residues), while thirty-one percent consists of β-sheets (6 strands and 47 residues).

MechanismMechanism

Protein surfaces near the active site are characterized by a positive electrostatic potential. In each structure there are several multi-chain carbonyl groups without hydrogen bonding partners in the active site. These could act in a proton transfer pathway by helping locate water molecules. Around the acetyl group, there exists a hydrophobic pocket which would stabilize the neutral charge while the substrate is bound to the enzyme once the amino group is deprotonated.

Kinetic MechanismKinetic Mechanism

The binding of substrates and release of products can be random, fully ordered, or a combination of both. It operates on a Bi-Bi mechanism. A study that employed product inhibitors CoA and acetylated (Lys14Ac) H3 peptide and dead-end inhibitor desulfo-CoA in order to determine the order of substrate binding has yielded results consistent with a fully ordered Bi-Bi kinetic mechanism where AcCoA is the first substrate to bind, and CoA is the last product that is released. It is also important to note that the transcriptional co-activator GCN5 from yeast (yGCN5) is a histone acetyltransferase that is essential for the activation of target genes. Bi-substrate kinetic analysis using acetyl-coenzyme A and an H3 histone synthetic peptide indicated that both substrates must bind to form a ternary complex before catalysis. Product inhibition studies revealed that the product CoA was a competitive inhibitor as opposed to AcCoA. Desulfo-CoA, a dead end inhibitor, also demonstrated simple competitive inhibition versus AcCoA. Acetylated (Lys14Ac) H3 peptide displayed noncompetitive inhibition against both H3 peptide and AcCoA.

Chemical MechanismChemical Mechanism

Post-translational modification of histones is linked to numerous cellular processes. These include transcriptional regulation, DNA damage repair, and DNA replication. One common histone modification, N-e-lysine acetylation, is controlled by the opposing actions of histone acetyltransferases (HATs) and deacetylases (HDACs). After the formation of a ternary complex of acetyl-CoA, histone and enzyme, an active site base deprotonates lysin, which allows for direct attack of the N-e-lysine on the carbonyl carbon of acetyl-CoA. Additionally, without a histone acceptor, slow rates of enzyme auto-acetylation (7 x 10-4 s-1, or ~2500-fold slower than histone acetylation; kcat = 1.6 s-1) and of CoA formation (0.0021 s-1) were not consistent with a kinetically competent acetyl-enzyme intermediate. The figure below illustrates the direct attack mechanism for acetyl transfer by Esa1. After binding acetyl-CoA and peptide substrate to form a ternary structure, glutamate 338 from Esa1 deprotonates the e-amine of lysine inside the substrate. After Lysine attacks the carbonyl carbon of the acetyl moiety of acetyl-CoA, it forms a tetrahedral intermediate, which in turn collapses and forms CoA-SH as well as acetylated product.

ImplicationsImplications

The analysis of a loss-of-function mutant of the Hpa2 gene suggests that the Hpa2 affects bacterial proliferation in host plants and a hypersensitive response in nonhost plants. As this is the first of such enzyme activity identified in the Hrp protein family, it is speculated that the Hpa2 contributes to the assembly of the TTSS by enlarging gaps in the peptidoglycan meshwork of bacterial cell walls. Members of the GNAT family have been found in E. Coli, a common organism with some strands that cause sickness to humans. Three other GNAT's have been attributed to acetylating three ribosomal proteins: S5, S18, and L12. Hpa2 also plays a role in governing gene expression through its effects on chromatin structure and assembly. Additionally, it affects gene regulation, antibiotic resistance, and hormonal regulation of circadian rhythms. It was also found that in head and neck carcinoma patients, Hpa2 expression was noticeably elevated, which correlated with prolonged time to disease recurrence, as well as inversely correlated with tumor cell spreading within lymph nodes. This would make it appear that Hpa2 restrains tumor metastasis, most likely by constricting heparanese enzymatic activity, granting a favorable outcome to the head and neck cancer patients.