Human ADP-ribosylation factor-like 5 isoform 1

Target ID:

ARL5

Aliases:

ARFLP5ARL5

Deposition Date:

Tuesday 16th May 2006

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2H17

Authors:

W.M.RABEH,W.TEMPEL,D.YANIW,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,H.PARK,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Toronto

2H17

tabs

Structure Details

ADP-ribosylation factor-like 5 (ARL5) belongs to the ARF family of GTP-binding proteins. The cellular function of ARL5 is currently unknown. The primary sequence of ARL5 is most similar to that of ADP-ribosylation factor-like 8 (ARL8), which is potentially involved in brain development.

Here, we solved the structure of the complex of ARL5 with GDP. The ARL5-GDP structure is similar to our recent ARL8-GDP structure (PDB code 1YZG) as expected from their sequence similarity. Particularly the N-terminal helix forms hydrophobic interactions with the main body of ARL5. This N-terminal helix binding site of ARL5 is located opposite from the GTP/GDP binding site. Based on the membrane recruitment mechanism of the ARF family proteins, the N-terminal helix will be displaced to interact with membranes when GTP replaces GDP.

Although the ARL5-GTP structure is not known, we hypothesize that the hydrophobic side chains of the N-terminal helix of ARL5 will be responsible for the interactions with membranes. Further studies will be necessary to prove this hypothesis.

Materials and Methods

ARL5:Q15-L176

PDB:2H17

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC001254
Entry Clone Source:MGC AT3-C4
SGC Clone Accession:HPC015-C08, ARL5_40
Tag:mgsshhhhhhssglvpr*gs
Host:E. coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsQEHKVIIVGLDNAGKTTILYQFSMNEVVHTSPTIGSNVEEIVINNTRFLMWDIGGQESLRSSWNTYYTNTEFVIVVVDSTDRERISVTREELYKMLAHEDLRKAGLLIFANKQDVKECMTVAEISQFLKLTSIKDHQWHIQACCALTGEGLCQGLEWMMSRL
Vector:p28a-thrombin-lic

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:We prepared the seeds by inoculating freshly transforming E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After growing overnight, all of the seeds were inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin at 37ºC and grown to an OD600 of 3-4. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.5 mM and grown overnight at 20ºC in SGC LEX bubbling system.

Purification

Procedure
The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4ºC. The Ni-NTA column was washed with 150 mL of the wash buffer (10mM Tris pH7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). Five molar equivalents of GDP, 5 mM TCEP and 5 mM MgCl were added to the purified protein before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 1 mL and the concentration of 10 mg/mL. About 4.5 mg of protein was obtained from 1.8 L of cell culture.

Extraction

Procedure

Concentration:10 mg/mL
Ligand
GDPMassSpec:
Crystallization:Crystallization was setup using sitting drop and 1:1 protein:mother liquor ratio. The crystal used for structure determination was grown in 2.0M sodium formate, 0.1M BTP, pH 7.0 at temperature 291K.

Structure solved by W.M. Rabeh, M&M last updated by ytong at 20080819 according to data in PolyMorph and M&M of other ARL5 structures
NMR Spectroscopy:
Data Collection:
Data Processing: