Human stromal membrane-associated protein 1-like

Target ID:

LOC64744

Aliases:

RP1-228H13.3SMAP1L

Deposition Date:

Friday 13th October 2006

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2IQJ

Authors:

Y.TONG,S.DIMOV,L.SHEN,W.TEMPEL,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

2IQJ

tabs

Structure Details

GTPase Activating Proteins, or the GAPs, are a family of regulatory proteins for GTPases by stimulating their GTPase (hydrolysis) activity and thus alternating signaling events.
Different families of GTPases have corresponding GAPs proteins whose structure and mechanism of function varies quite differently [1].
The Arf family of small GTPases has been implicated in a large number of cellular events, including vesicle biogenesis in intracellular traffic, Golgi morphology, lipid metabolism, endocytosis, exocytosis and cytokinesis.

SMAP1L (stromal membrane-associated protein 1-like), previously coded LOC64744, is a putative ArfGAP protein with 46.2% sequence identity to SMAP1.
The latter is believed to be involved in the haematopoietic progenitor cells to stromal cells interactions[2] and may have a stimulatory effect on stroma-supported erythropoiesis [3].
The full-length SMAP1L features an N-terminal ArfGAP domain and an about 300 a.a. long C-terminal unstructured region rich in prolines, methionines and glutamines.
Whether SMAP1L serves as a GAP protein for Arf or other GTPases has not been determined experimentally so far.

We solved the structure of the GAP domain of SMAP1L (Fig. 1), which has a typical ArfGAP fold (α + β).
The GAP domain of SMAP1L is homo-dimeric in solution as determined by DLS (dynamic light scattering) and in the crystal lattice.
The structure features one single-turn helix, five α-helices, three anti-parallel β-sheets, and a C4-type zinc finger, which stabilizes the whole structure.
Dimerization interface is located on the other side to the zinc finger of the monomers.
Structure alignment of the SMAP1L to the ArfGAP1 in the complex structure of Arf1 and ArfGAP1 [4] shows an RMSD of 2.3 Å for the whole GAP domain (used coordinates from R11 to L128) while the N-terminal 86 residues (R11-Y96) gives an RMSD of 1.3 Å against ArfGAP1, as determined by Dali server [5].
Big conformational change was seen for the C-terminal α-helix in the free state SMAP1L compared with the C-terminal helices of ArfGAP1 in the complex with Arf1.
The putative catalytic Arginine responsible for GAP function of SMAP1L is R56 based on sequence alignment to ArfGAP1 [1,4].

Materials and Methods

SMAP1L (LOC64744)

PDB:2IQJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:MGC clone AU80-G5
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with thrombin cleavage site: mgsshhhhhhssglvprgs
Host:E.coli BL21 (DE3) codon plus RIL

Construct

Prelude:
Sequence:gsMTGKSVKDVDRYQAVLANLLLEEDNKFCADCQSKGPRWASWNIGVFICIRCAGIHRNLGVHISRVKSVNLDQWTQEQIQCMQEMGNGKANRLYEAYLPETFRRPQIDPAVEGFIRDKYEKKKYMDRSLDINA
Vector:pET28a-LIC

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:The target was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and chloramphenicol at 37ºC. 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 before harvesting and flash frozen.

Purification

Procedure
The thawed cell pellets were resuspended in 100 mL of the binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM mercaptoethanol (BME) ) with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl, 0.1 mM phenylmethyl sulfonyl fluoride, and 2uL benzonase [Sigma] ), and 0.5% CHAPS. The cells were further lysed by sonication. The lysate was centrifuged at 16000 rpm for 60 min and the supernatant was loaded onto 5 mL HiTrap Ni-NTA column (Pharmacia Amersham) equilibrated with the same binding buffer at 4 ºC using peristaltic pump. Then the Ni-NTA column was connected to FPLC instrument and washed with 25 mL of the wash buffer (20mM HEPES pH 7.5, 150 mM NaCl, 50 mM imidazole, 1mM BME). A linear gradient of 50mL from 50 to 500mM imidazole concentration was then applied and the protein was eluted at around 175mM imidazole concentration. Collected fraction 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 supplemented with 200?L PMSF (100mM stock solution) to inhibit residual thrombin protease activity. The protein were further purified and desalted using 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

Concentration:30 mg/mL
Ligand
MassSpec:14945.10, expected 15430.63, DNA sequencing confirmed construct design, unclear about the reason of difference.
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 25.0% P3350/0.2M NH4Ac/0.1M Bis-Tris pH6.5. Crystals reached a size of about 50 microns within two to three days.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Scheffzek K. and Ahmadian M.R. (2005). GTPase activating proteins: structural and functional insights 18 years after discovery. Cell Mol. Life Sci. 62: 3014-3038.
Marcos I., Borrego S., Rodriguez de C.S., Galan J.J., and Antinolo G. (2002). Cloning, characterization and chromosome mapping of the human SMAP1 gene. Gene 292: 167-171.
Sato Y., Hong H.N., Yanai N., and Obinata M. (1998). Involvement of stromal membrane-associated protein (SMAP-1) in erythropoietic microenvironment. J. Biochem. (Tokyo) 124: 209-216.
Goldberg J. (1999). Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis. Cell 96: 893-902.
Holm L. and Park J. (2000). DaliLite workbench for protein structure comparison. Bioinformatics. 16: 566-567.