Human ADP-Ribosylation Factor-Like 10B

Target ID:

ARL10B

Aliases:

ARL10BFLJ45195GIE2

Deposition Date:

Friday 15th April 2005

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

1ZD9

Authors:

J.CHOE,A.ATANASSOVA,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,A.BOCHKAREV,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

1ZD9

tabs

Structure Details

ADP-ribosylation factor-like 10B (ARL10B) is a member of the ARF GTPase family
currently with unknown function. The structure of a truncated mutant of ARL10B (ARL10B-N17)
that lacks the N-terminal 17 amino acids has been solved at 1.7Ã… resolution.

The structures of ARF family members in GDP and GTP conformations
have suggested that the GDP-GTP exchange induces global conformational changes
for effecter recognition and membrane binding;
thus the bound nucleotide at the active site determines the differences of ARF proteins
between the GDP and the GTP conformations.
However, the ARL10B-N17 structure resembles the ARF structures in the GTP conformation
although it is still bound to GDP.

This finding suggests that the N-terminal truncation,
which mimics the interactions of the N-terminal helix with phospholipid membrane,
triggers the conformational switch of ARL10B from the GDP conformation to the GTP conformation.
Therefore, the GDP-GTP conformational switch of ARL10B occurs by the N-terminal helix interactions
with phospholipid membrane rather than by the GDP-GTP nucleotide exchange at the active site.

Although guanine nucleotide exchange factors (GEFs) of ARL10B are unknown,
other ARF GEFs stimulate GDP dissociation with an invariant glutamate.
The ARF1-GEF structures have suggested that the GDP conformation of ARF1 is recognized by the GEF.
On the basis of the structure of GDP-bound ARL10B-N17,
which is similar to the GTP conformation;
however, we hypothesize GEF recognition mechanism of ARL10B
whereby membrane-recruited ARL10B-GDP adapts a GTP conformation before forming a complex with GEF.
Consequently, GDP is dissociated from the ARL10B-GEF complex.

Materials and Methods

ARL10B

PDB:1ZD9

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AT20-E2
Entry Clone Source:MGC
SGC Clone Accession:HPC003-F10
Tag:mgsshhhhhhssglvpr*gs
Host:E. coli BL21 (DE3)

Construct

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

Growth

Medium:
Antibiotics:
Procedure:We prepared a seed culture by inoculating freshly transformed E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After overnight, the seed culture was 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 4.63. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.5 mM and grown overnight at 20ºC in a 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 pH7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The eluate was dialyzed overnight against a buffer containing 10 mM Tris pH7.5, 0.5 M NaCl, 5% glycerol. The protein concentration was estimated based on the extinction coefficient of the protein, 22430 at 280 nm. Five molar equivalents of GDP, 5 mM TCEP and 5 mM MgCl2 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 30 mg/ml. About 55 mg of protein was obtained from 1.8 L of cell culture.

Extraction

Procedure
Cultures were centrifuged and the cell pellets were suspended in 100 ml of the binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole) with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride) and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was used for subsequent steps of purification.
Concentration:30 mg/mL
Ligand
MassSpec:
Crystallization:Purified His-tagged ARL10B was crystallized using the sitting drop vapor diffusion method. Crystals grew in one day when the protein (30 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 30 % PEG 4000, 0.1 M Tris, pH 8.5 and 0.2 M LiSO₄.
NMR Spectroscopy:
Data Collection:
Data Processing: