Human TBC1 domain family, member 14

Target ID:

TBC1D14

Aliases:

FLJ32400KIAA1322

Deposition Date:

Thursday 26th July 2007

Families:

GTPases

Structure type:

apo

Download Coordinates:

2QQ8

Authors:

Tong Y, Tempel W, Dimov S, Landry R, Arrowsmith CH, Edwards AM, Sundstrom M, Weigelt J, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

2QQ8

tabs

Structure Details

GTPase Activating Proteins, or the GAPs, are regulatory proteins for Ras, Rho, Arf and Rab GTPases by stimulating the nucleotide-hydrolyzing activities of these GTPases. The Ras, Rho, Arf and Rab GTPases have their own GAPs of which the structure and reaction mechanism are different from each other [1]. The Rab GTPases have been implicated in numerous cellular events, including vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion. Most of known Rab-GAPs possesses a TBC domain (Tre-2, Bub2p and Cdc16p) [2]. The TBC1D14 is a 678 a.a. protein that consists of the N-terminal ~ 190 a.a. proline-rich flexible region followed by the TBC domain. The TBC1D14 have no identified Rab GTPases as substrates [3].

We solved the structure of the RabGAP domain of TBC1D14. It features an all-helix fold, similar to the previous RabGAPs, TBC1D22A and Gyp1p. A major conformational difference is seen at helix α4 between TBC1D14 and TBC1D22A. Helix α4 of TBC1D14 is distant from helices α1 and α3 whereas helices α1, α3, and α4 of TBC1D22A form a cluster.

The structure of Gyp1p in complex with Rab33-GDP-AlF3 has generalized that Rab-GAPs use a Arg/Gln dual-finger mechanism for the activation of Rab GTPases. The conformational change surrounding the Arg/Gln dual finger is minimal when bound to the Rab [4]. Interestingly, the conformation of the Arg/Gln dual finger of TBC1D14 is quite different from that of the Gyp1p.

About 60 mammalian RabGAPs and 70 mammlian Rab GTPases are known although the GAP activities of most of these RabGAPs are not determined yet. Furthermore, determinant factors of one particular RabGAP specific for certain Rab GTPases are unclear. The structure of the RabGAP domain of TBC1D14 is the first step to provide insight into these questions.

Materials and Methods

TBC1D14

PDB:2QQ8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AT71-E11
Entry Clone Source:MGC
SGC Clone Accession:HPC057-G06
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgNAVLTWNNEILPNWETMWCSRKVRDLWWQGIPPSVRGKVWSLAIGNELNITHELFDICLARAKERWRSLSTGGSEVENEDAGFSAADREASLELIKLDISRTFPNLCIFQQGGPYHDMLHSILGAYTCYRPDVGYVQGMSFIAAVLILNLDTADAFIAFSNLLNKPCQMAFFRVDHGLMLTYFAAFEVFFEENLPKLFAHFKKNNLTPDIYLIDWIFTLYSKSLPLDLACRIWDVFCRDGEEFLFRTALGILKLFEDILTKMDFIHMAQFLTRLPEDLPAEELFASIATIQMQSRNKKWAQVLTALQKDSREMEKG
Vector:pET28-mhl

Growth

Medium:Terrific BrothFor selenomethionine (SeMet) labeling, prepackaged M9 SeMET growth media kit (Medicilon) was used following manufacturer instructions
Antibiotics:
Procedure:LEX Bubbling

Purification

Procedure
Column 1: 5 mL HiTrap Chelating HP column (GE Healthcare)
Column 1: Superdex 75 (16/60, GE Healthcare)

The lysate was centrifuged at 27,000 x g for 60 min and the supernatant was collected and loaded onto a 5 mL HiTrap Chelating HP column (GE Healthcare) loaded with Ni2+, equilibrated with the same extraction buffer at 4 degC. The HiTrap column was washed with 25 mL purification buffer and the protein was eluted with a linear concentration gradient of imidazole from 30 mM to 500 mM in the HEPES extraction buffer in 50 mL. The fractions containing the target protein were pooled and further purified and desalted using a gel filtration column, Superdex 75 (16/60, GE Healthcare), which was pre-equilibrated with low salt buffer. Collected fractions were concentrated using an Amicon Ultra-15 centrifugal filter (5,000 m.w. cut-off) to a final concentration of 15.1 mg/mL. Protein concentrations were measured using Bradford assay with purity >95% based on SDS-PAGE analysis.

Extraction

Procedure
The thawed cell pellet (from 1.8 L culture) was resuspended in 100 mL of extraction buffer supplemented with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride final concentrations), and 0.5% CHAPS. The cells were lysed by liquid fluidizer.
Concentration:15.1 mg/mL
Ligand
MassSpec:Measured: 38598.31
Expected 38,597.38
SeMet labeled: measured 39073.22
Crystallization:SD12 optimization: 2.0M NaCl, 0.1M Na2HPO4, 0.1M Mes pH 6.5, 0.1M KH2PO4
Buffer 20 mM HEPES pH 8.0, 150 mM NaCl, 5mM DTTSitting drop vaporization
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.
Bernards A. (2003). GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim. Biophys. Acta 1603: 47-82.
Itoh T., Satoh M., Kanno E., and Fukuda M. (2006). Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity. Genes Cells 11: 1023-1037.
Pan X., Eathiraj S., Munson M., and Lambright D.G. (2006). TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442: 303-306.