Human full-length centaurin alpha-1 in complex with the FHA domain of KIF13B

Target ID:

CENTA1

Deposition Date:

Friday 19th December 2008

Families:

G-Protein Regulators

Related Diseases:

Signalling

Structure type:

protein-protein

Download Coordinates:

3FM8

Authors:

Shen L, Tong Y, Tempel W, MacKenzie F, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H.

Site:

Toronto

3FM8

tabs

Structure Details

Centaurin alpha1, CENTA1, is an Arf6 GAP (Venkateswarlu et al., 2004) containing two tandem PH domains. It was discovered as a PIP3 binding molecule (Donie & Reiser, 1991) and later found to be an adaptor protein for KIF13B to carry PIP3 vesicle to the developing axon tips (Venkateswarlu et al., 2005).

KIF13B is a member of kinesin-3 family and features an FHA domain after the motor domain. FHA (Forkhead-Associated) domains are the only known phosphor-threonine binding domain that was discovered. The FHA domain was found to be responsible for CENTA1 binding.

We solved the complex structure of the full length centaurin alpha1 bound with the FHA domain of KIF13B. The FHA domain was found to interacts mainly the first PH domain of centaurin alpha1 but also ArfGAP domain.

Materials and Methods

CENTA1+KIF13B

PDB:3FM8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:CENTA1: BC033747
KIF13B: BC005977
Entry Clone Source:CENTA1: MGC AT53-B3
KIF13B: OpenBiosystem CloneID:4103715
SGC Clone Accession:CENTA1: HPC060-A04
KIF13B: HPC079-B11
Tag:Both have N-terminal tag: mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:Tag not removed
CENTA1:M1-P374
KIF13B:G440-P545
Sequence:CENTA1:
mhhhhhhssgrenlyfqgMAKERRRAVLELLQRPGNARCADCGAPDPDWASYTLGVFICLSCSGIHRNIPQVSKVKSVRLDAWEEAQVEFMASHGNDAARARFESKVPSFYYRPTPSDCQLLREQWIRAKYERQEFIYPEKQEPYSAGYREGFLWKRGRDNGQFLSRKFVLTEREGALKYFNRNDAKEPKAVMKIEHLNATFQPAKIGHPHGLQVTYLKDNSTRNIFIYHEDGKEIVDWFNALRAARFHYLQVAFPGASDADLVPKLSRNYLKEGYMEKTGPKQTEGFRKRWFTMDDRRLMYFKDPLDAFARGEVFIGSKESGYTVLHGFPPSTQGHHWPHGITIVTPDRKFLFACETESDQREWVAAFQKAVDRPMLPQEYAVEAHFKHKP
KIF13B:
mhhhhhhssgrenlyfqgGIKVGDDKCFLVNLNADPALNELLVYYLKEHTLIGSANSQDIQLCGMGILPEHCIIDITSEGQVMLTPQKNTRTFVNGSSVSSPIQLHHGDRILWGNNHFFRLNLP
Vector:pET28-mhl (GI:134105571)

Growth

Medium:Terrific Broth
Antibiotics:Kanamycin 50 µg/mL Chloramphenicol 25 µg/mL
Procedure:LEX Bubbling. The target protein was expressed in E. Coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into 2L Terrific Broth medium in the presence of 50 µg/mL kanamycin and 50 µg/mL chloramphenicol at 37 degC. When OD600 reached ~3.0, the temperature of the medium was lowered to 18 degC and the culture was induced with 1 mM IPTG. The cells were allowed to grow overnight before they were harvested and flash frozen in liquid nitrogen and stored at -80 degC.

Purification

Procedure
The lysate was centrigued at 15,000 rpm for 45 minutes and the supernatants were mixed with 1.5 mL 50% Ni-NTA beads, and incubated at 4 degC for 1.5 hours. The supernatant was then passed through a gravity column (Poly-Prep, Bio-Rad, Catalog #731-1550) and the beads were washed using 10 mL washing buffer twice. The protein bound to beads were eluted using 10 mL elution buffer twice. The flow-through was collected and loaded onto Supderdex-200 gel filtration column. Eluted fractions were pooled and concentrated using amicon centrifugal filter (m.w. cut-off 10,000 for CENTA1, cut-off 5,000 for KIF13B-FHA). The purity of the proteins was higher than 95% judged by SDS-PAGE. The complex was formed by mixing purified CENTA1 and KIF13B at 1:1 ratio and run through a Superdex-75 column. The fractions were collected and concentrated using Amicon centrifugal filter and then used to setup crystallization.

Extraction

Procedure
Frozen cells were thawed and resuspended in 80 mL extraction buffer with freshly added 1mM PMSF/Benzomidine, 5U/ml of Benzonase (Sigma Catalog # E1014, 250U/&microL), 0.5% CHAPS, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and lysed using microfludizer(17000 psi).
Concentration:28.2 mg/mL (complex concentration using Bradford measurement)
Ligand
ZnMassSpec:CENTA1: Native: 45619.63, expected 45618.66
KIF13b: Native: 13951.10, expected 13950.80
Crystallization:Buffer for the protein is gel filtration buffer.
Crystal used for data collection was grown in Optimized SGC-B12 condition:Well solution: 1.20 M Li2SO4, 0.1M Sodium Citrate pH 6.0 0.5M (NH4)2SO4Protein complex was added 5% MPD and then mixed with well solution at 1uL:1uL ratio, and setup as hanging drop in a 24-well optimization plate. Crystals grown to a size of 60micron in about 3-4 weeks. Cryo used 1.6M Li2SO4 and 0.5M (NH4)2SO4.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

DONIE, F. & REISER, G. (1991). Purification of a high-affinity inositol 1,3,4,5-tetrakisphosphate receptor from brain. Biochem.J., 275 ( Pt 2), 453-457.
VENKATESWARLU, K., BRANDOM, K. G., & LAWRENCE, J. L. (2004). Centaurin-alpha1 is an in vivo phosphatidylinositol 3,4,5-trisphosphate-dependent GTPase-activating protein for ARF6 that is involved in actin cytoskeleton organization. Journal of Biological Chemistry, 279(8), 6205-6208.
VENKATESWARLU, K., HANADA, T., & CHISHTI, A. H. (2005). Centaurin-alpha1 interacts directly with kinesin motor protein KIF13B. J.Cell Sci., 118(Pt 11), 2471-2484.