Human histone acetyltransferase 1

Target ID:

HAT1

Aliases:

KAT1

Deposition Date:

Thursday 1st March 2007

Families:

Transferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

2P0W

Authors:

H.WU,J.MIN,H.ZENG,P.LOPPNAU,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2P0W

tabs

Structure Details

Histone acetyltransferases (HATs) are a group of enzymes that catalyze the transfer of an acetyl group from acetyl-coenzyme A to the lysine ε-amino groups on the N-terminal tails of histones. HATs play an important role in signal transduction, chromatin remodeling, DNA replication, transcription and repairing. These different functions are correlated with the chromatin structure changes caused by acetylation of histones. The reversible acetylation of specific lysine residues within the N-terminal tails of the core histones is correlated with gene expression activity. Histone acetylation, particularly of histone H4, has also been proposed to play an important role in replication-dependent nucleosome assembly.

Human HAT1 is expressed in the cytoplasm and nucleus of S-phase cells. It acetylates soluble (not nucleosomal) newly synthesized histone H4 at Lys5 and Lys12, and H2A at Lys5. HAT1 has very broad function in many DNA regulatory processes. Cytoplasmic HAT1 acetylates nascent histones H4 before their deposition in replicating chromatin.

Meanwhile, HAT1 from human and yeast have been localized to the nucleus, suggesting a role in transcription or replication-related histone acetylation. In addition, HAT1 has been shown to participate in the nuclear function of telomeric silencing. Abberant HAT activity has been associated with diseases such as cancer. In light of this, it makes HAT1 very attractive targets for the design of specific inhibitors.

We have solved the crystal structure of the human HAT1 protein in complex with acetyl CoA (AcCoA) and histone H4 peptide at 1.9 Å resolution. HAT1 has an elongated, curved structure, and the AcCoA molecule is bound in a cleft on the concave surface of the protein, marking the active site of the enzyme. Histone H4 peptide bound to a channel that runs across the protein, with the Lys12 in close proximity to AcCoA.

Materials and Methods

HAT1

PDB:2P0W

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:4504341
Entry Clone Source:MGC
SGC Clone Accession:HAT1_05:APC038-C3
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPR*GS
Host: E.coli BL21 (DE3) codon plus RIL (Stratagene)

Construct

Prelude:
Sequence:gsKKLAEYKCNTNTAIELKLVRFPEDLENDIRTFFPEYTHQLFGDDETAFGYKGLKILLYYIAGSLSTMFRVEYASKVDENFDCVEADDVEGKIRQIIPPGFCTNTNDFLSLLEKEVDFKPFGTLLHTYSVLSPTGGENFTFQIYKADMTCRGFREYHERLQTFLMWFIETASFIDVDDERWHYFLVFEKYNKDGATLFATVGYMTVYNYYVYPDKTRPRVSQMLILTPFQGQGHGAQLLETVHRYYTEFPTVLDITAEDPSKSYVKLRDFVLVKLCQDLPCFSREKLMQGFNEDMAIEAQQKFKINKQHARRVYEILRLLVTD
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:HAT1 was expressed in E.coli BL21 (DE3) codon plus RIL in 6L Terrific Broth (TB) in the presence of 50 microG/ml of kanamycin. Cell were grown at 37ºC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15degC.

Purification

Procedure
The crude extract was cleared by centrifugation at ~75000 x g for 20 minutes. The clarified lysate was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 20 mM Tris-HCl buffer, pH 8.0, containing 250 mM NaCl and 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl, at flow rate 4 ml/min. Thrombin (Sigma) was added to combined fractions containing HAT1 and incubated overnight at 4oC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 3.8 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation. The cell pellets were frozen in liquid nitrogen and stored at -80ºC. For the purification, 6L of cells were thawed and resuspended in lysis buffer with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through a Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:23.3 mg/ml
Ligand
MassSpec:Expected mass= 37924.22 Da, measured mass = 38095.6689 Da.
Crystallization:Purified HAT1 was complexed with AcCoA (Sigma) and histone H4 peptide (SGRGKGGKGLGKGGAKRHRK) at 1:10 molar ratio of protein: AcCoA/H4 and crystallized using the sitting drop vapor diffusion method by mixing 1 ul of protein solution with 1 ul of the reservoir solution containing 12% PEG 20K, 0.1M MES pH6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: