Human HIV-1 Rev binding protein

Target ID:

HRB

Aliases:

MGC116938MGC116940RABRIP

Deposition Date:

Friday 19th January 2007

Families:

GTPases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2OLM

Authors:

Y.TONG,W.TEMPEL,L.SHEN,S.DIMOV,R.LANDRY,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2OLM

tabs

Structure Details

HRB (HIV-1 Rev Binding protein), also known as hRIP (human Rev-Interacting Protein), RAB (Rev/Rex Activation domain-Binding protein), is a 60-kDa protein initially identified by yeast two-hybrid screening to interact with Rev protein of HIV-1 and assist in HIV replication [1].
Ablation of HRB activity leads to mislocalization and aberrant accumulation of Rev-directed viral RNAs around nuclear periphery [2].
Based on mouse model, Kang-Decker et al. have found that HRB is associated with proacrosomic vesicles and is required for successful spermatogenesis.
A mutation of Hrb makes male mouse infertile [3].
Full length HRB comprise of an N-terminal region homologous to ArfGAP and a C-terminal region related to nuleoporins. Involvement of Hrb in proacrosomic vesicle fusion implicates a possible involvement of to-be-identified Arf/Rab GTPases regulated by the ArfGAP domain of Hrb.
Since HRB is not required for cell viability, it may be an attractive target for designing new anti-viral drugs.
The high-resolution structure of the ArfGAP domain of HRB is fundamental for such future endeavors.

We solved the structure of the GAP domain of HRB, which has a typical ArfGAP fold (α+β).
The structure features three anti-parallel β-sheets, and a C4-type zinc finger embraced by α helices and a long loop.

Materials and Methods

HRB

PDB:2OLM

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC030592
Entry Clone Source:MGC clone AT27-B10
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with thrombin cleavage site: mgsshhhhhhssglvprgs
Host:E.coli BL21 (DE3) codon plus RIL

Construct

Prelude:
Sequence:gsSAKRKQEEKHLKMLRDMTGLPHNRKCFDCDQRGPTYVNMTVGSFVCTSCSGSLRGLNPPHRVKSISMTTFTQQEIEFLQKHGNEVCKQIWLGLFDDRSSAIPDFRDPQKVKEFLQEKYEKKRWYVPPEQAKVVASVHA
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:The target was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and chloramphenicol at 37 °C. When OD600 was ~3.0, the culture was induced with 1mM IPTG and the temperature was reduced to 15 °C, and the cells were allowed to grow overnight. Cultures were harvested by centrifugation and the cell pellets were flash frozen and stored at -80 °C .

Purification

Procedure
The supernatant was loaded onto 5 mL HiTrap Ni-NTA column (Pharmacia Amersham) equilibrated with the same binding buffer at 4 ºC using a peristaltic pump. The Ni-NTA column was connected to an FPLC instrument and washed with 25 mL of wash buffer. A linear gradient from 50 to 500mM imidazole (in 50 mL) was applied and the protein was eluted at around 175mM imidazole. Collected fractions were digested with thrombin (Sigma, ~ 7U/mg protein) overnight at 4 ºC and then passed through an open column filled with ~ 3mL Ni-NTA resin (Qiagen). The flowthrough was collected and 200 microL PMSF (100mM stock solution) was added to inhibit residual thrombin protease activity. The protein were further purified and desalted using a gel filtration column, Superdex 75 (26/60), which was pre-equilibrated with the binding buffer. Collected fractions were concentrated using an Amicon Ultra centrifugal filter to a final concentration of around 50 mg/mL. Protein concentration was measured using Bradford assay and the purity was greater than 95% based on SDS-PAGE analysis.

Extraction

Procedure
The thawed 1.8 L cell pellets were resuspended in 100 mL of the binding buffer with a protease inhibitor cocktail (0.1 mM benzamidine-HCl, 0.1 mM phenylmethyl sulfonyl fluoride, and 2 microL benzonase [Sigma] ), and 0.5% CHAPS. The cells were lysed by sonication for half a minute. The lysate was centrifuged at 38400 ×g for 60 min and collected the supernatant.
Concentration:20 mg/mL
Ligand
MassSpec:16130.94 D
Crystallization:Crystallization trials were set up using the sitting drop vapor diffusion method. The protein drop was equilibrated against a reservoir solution (1:1 volume ratio) containing 2.27 M (NH4)2SO4, 0.09M BTP, pH 7.0. Crystals reached a size of about 50 microns within two to three days at 20 ºC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Fritz C.C., Zapp M.L., and Green M.R. (1995). A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature 376: 530-533.
Sanchez-Velar N., Udofia E.B., Yu Z., and Zapp M.L. (2004). hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region. Genes Dev. 18: 23-34.
Kang-Decker N., Mantchev G.T., Juneja S.C., McNiven M.A., and van Deursen J.M. (2001). Lack of acrosome formation in Hrb-deficient mice. Science 294: 1531-1533.
Goldberg J. (1999). Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis. Cell 96: 893-902.