Human HIV-1 Tat interactive protein, 60kDa isoform 2

Target ID:

HTATIP

Aliases:

cPLA2ESA1HTATIP1KAT5PLIPTIPTIP60

Deposition Date:

Friday 9th February 2007

Families:

Transferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

2OU2

Authors:

J.MIN,H.WU,L.DOMBROVSKI,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

2OU2

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. And these different functions are most likely correlated with the chromatin structure changes caused by acetylation of histones.

The histone acetyltransferases can be divided into five families, including the GCN5-related acetyltransferases (GNATs); the MYST (for MOZ, Ybf2/Sas3, Sas2 and Tip60)-related HATs; p300/CBP HATs; the general transcription factor HATs, and the nuclear hormone-related HATs. HAT proteins show very high sequence similarity within families but relatively poor similarity between families. The MYST family of HAT proteins has very diverse biological functions. In yeast, they have been shown to be involved in gene silencing, and cell-cycle regulation, while in human they are involved in leukogenesis.

We have solved the crystal structure of acetyltransferase domain of human HIV-1 Tat interacting protein, 60kDa (HTATIP, TIP60) in complex with acetyl-coA at 2.3 Å resolution. HTATIP belongs to the MYST family of histone acetyl transferases and was originally isolated as an HIV-1 TAT-interactive protein. It has a role in DNA repair, apoptosis and signal transduction. Direct binding of HTATIP to the TAT protein of the human immunodeficiency virus (HIV) is an important feature for efficient TAT transactivation of HIV gene expression.

Materials and Methods

HTATIP

PDB:2OU2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:36287060
Entry Clone Source:MGC
SGC Clone Accession:HTATIP_05:9A11APC005_9
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene).

Construct

Prelude:
Sequence:gsTRMKNIECIELGRHRLKPWYFSPYPQELTTLPVLYLCEFCLKYGRSLKCLQRHLTKCDLRHPPGNEIYRKGTISFFEIDGRKNKSYSQNLCLLAKCFLDHKTLYYDTDPFLFYVMTEYDCKGFHIVGYFSKEKESTEDYNVACILTLPPYQRRGYGKLLIEFSYELSKVEGKTGTPEKPLSDLGLLSYRSYWSQTILEILMGLKSESGERPQITINEISEITSIKKEDVISTLQYLNLINYYKGQYILTLSEDIVDGHERAMLKRLLRIDSKCLHFTPKD
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:The protein was expressed in E.coli BL21 (DE3) codon plus RIL in 2L of Terrific Broth (TB) in the presence of 50 microG/ml of kanamycin. Cell were grown at 37degC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, incubated overnight at 15degC.

Purification

The crude extract was cleared by centrifugation. The lysate was loaded onto 10 ml Chelating Sepharose column (Amersham Biosciences), charged with Ni 2+ . The column was washed with 10 CV of 20 mM HEPES buffer, pH 7.4, containing 0.5 M NaCl , 50 mM imidazole, 5% glycerol and 0.1% CHAPS, and the protein was eluted with elution buffer (20 mM HEPES, pH 7.4, 0.5 M NaCl, 250 mM imidazole, 5% glycerol, 0.1 % CHAPS). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM HEPES buffer, pH 7.4, and 0.5 M NaCl, at flow rate 4 ml/min. The fractions containing HTATIP were pooled and subjected to treatment of thrombin (Sigma) to remove the His-tag. The protein was further purified to homogeneity on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer containing 20 mM Na Citrate pH 6.0 and eluted with linear gradient of NaCl up to 1 M concentration (20CV). Purification yield was 1.2 mg of theprotein per 1L of culture

Extraction

Cells were harvested by centrifugation. The cell pellets were frozen in liquid nitrogen and stored at -80degC. For the purification, the cell paste was thawed and resuspended in 100ml of lysis buffer with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:5 mg/ml
Ligand
MassSpec:The expected mass for HTATIP is 32997.1 Da, measured mass is 33097.2078 Da.
Crystallization:Purified HTATIP was complexed with acetylcoenzyme A (AcCoA, Sigma) at 1:10 molar ratio of protein:AcCoA and crystallized using the hanging drop vapor diffusion method at 20 °C by mixing 2µl of the protein solution with 1 µl of the reservoir solution containing 16% PEG3350, 0.2 M ammonium acetate, 0.1 M Bis-Tris, pH 6.6.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Tang Y, Luo J, Zhang W, Gu W. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis. Mol Cell. 2006 Dec 28;24(6):827-39.
Squatrito M, Gorrini C, Amati B. Tip60 in DNA damage response and growth control: many tricks in one HAT. Trends Cell Biol. 2006 Sep;16(9):433-42. Review.
Col E, Caron C, Chable-Bessia C, Legube G, Gazzeri S, Komatsu Y, Yoshida M, Benkirane M, Trouche D, Khochbin S. HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses. EMBO J. 2005 Jul 20;24(14):2634-45.