Human plexin B1 - Rho family GTPase 1 complex

Target ID:

PLXNB1

Aliases:

KIAA0407PLEXIN-B1PLXN5SEP

Deposition Date:

Thursday 27th September 2007

Families:

GTPases

Related Diseases:

Cancer

Structure type:

protein-protein

Download Coordinates:

2REX

Authors:

Tong Y, Tempel W, Shen L, Arrowsmith CH, Edwards AM, Sundstrom M, Weigelt J, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

2REX

tabs

Structure Details

General Description

The plexin family of transmembrane proteins are receptors for axonal growth cone guidance molecule semaphorins (Nakamura et al., 2000). The cytoplasmic region of plexin B1, PLXNB1, is a GTPase activating protein (GAP) for Ras family GTPase RRAS (Oinuma et al., 2004) and the GAP activity plays a role in integrin-mediated actin remodeling and cell collapse (Ito et al., 2006). The RasGAP homology region of PLXNB1 is disrupted by an insertion of ~200 amino acid residues (the effector domain) that bind to Rho GTPases, RAC1, RND1, and RHOD (Vikis et al., 2000;Driessens et al., 2001;Hu et al., 2001). In PLXNB1, its GAP activity for RRAS requires the binding of RND1 GTPase to the effector domain (Oinuma et al., 2004), suggesting that RND1 GTPase regulates the RasGAP activity of PLXNB1. We previously solved the structure of a dimeric form of the effector domain of PLXNB1 (Tong et al., 2007) and proposed that the dimerization of the effector domain is implicated in PLXNB1 function. Here, we solved the structure of the effector domain of PLXNB1 in complex with RND1 GTPase to understand the involvement of RND1 in the regulation of the RasGAP activity of PLXNB1.

Structural features

We solved the structure of the effector domain of PLXNB1 in complex with RND1 GTPase. This is the first complex structure of RND1 GTPase (with its effector). In each asymmetric unit, the effector domain of PLXNB1 forms a dimer, similar to the effector domain of PLXNB1 in the RND1-free state (http://thesgc.com/SGC-WebPages/StructureDescription/2R2O.php). Two RND1 molecules bind to the PLXNB1 dimer toward the opposite sites. Compared to the plexin-free RND1 (PDB code 2CLS), no major conformational change of RND1 is observed upon binding to the effector domain of PLXNB1. In the complex, however, the side chain of Phe47 of RND1 is flipped toward Trp1807 of PLXNB1. Compared to the effector domain of PLXNB1 in the RND1-free state, the conformation of the effector domain of PLXNB1 undergoes significant changes upon binding to RND1. Briefly, the PLXNB1 effector domain moves toward RND1 and there is also conformational adjustment in the central anti-parallel β-sheet. When one molecule of the dimer is fixed and the other molecule is aligned, the movement of the effector domain toward RND1 is obvious. Additionally, the loop, residues Ser1809-Gly1813, rotates ~180 degree from the original position to make room for the interaction of Phe47 of RND1 with Trp1807 of the effector domain. The loop of Ala1755-Gly1766 that was missing in the RND1-free structure of the PLXNB1 effector domain is visible in the complex. The CRIB-like motif LeuValPro (residues 1849-1851) is not involved in RND1 binding (Vikis et al., 2000).

Materials and Methods

PLXNB1 + RND1

PDB:2REX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PLXNB1: Provided by Dr. M.Buck (Case Western Reserve University, Cleveland, Ohio)
RND1: AT22-F2
Entry Clone Source:PLXNB1: Dr. Buck
Rnd1: MGC
SGC Clone Accession:PLXNB1 HPC060-C12
RND1 HPC060-H10
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:
Sequence:PLXNB1:gDVEYRPLTLNALLAVGPGAGEAQGVPVKVLDCDTISQAKEKMLDQLYKGVPLTQRPDPRTLDVEWRSGVAGHLILSDEDVTSEVQGLWRRLNTLQHYKVPDGATVALVPCLTKHVLRENQ
RND1:gRAPQPVVARCKLVLVGDVQCGKTAMLQVLAKDCYPETYVPTVFENYTACLETEEQRVELSLWDTSGSPYYDNVRPLCYSDSDAVLLCFDISRPETVDSALKKWRTEILDYCPSTRVLLIGCKTDLRTDLSTLMELSHQKQAPISYEQGCAIAKQLGAEIYLEGSAFTSEKSIHSIFRTASMLCLNKPSPLPQKSPV
Vector:pET28-mhl

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target proteins were 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 25 μg/mL chloramphenicol at 37 °C. When OD600 reached ~3.0, the temperature of the media was lowered to 15 °C and the culutre was induced with 1mM IPTG. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 °C before use.

Purification

Procedure
The lysates were centrigued at 15,000 rpm for 45 minutes and the supernatants were mixed with 5 mL 50% Ni-NTA beads, and incubated at 4 °C for 1 hours. The supernants were then passed through a gravity column (Poly-Prep, Bio-Rad, Catalog #731-1550) and the beads were washed using 10 mL washing buffer once. The proteins bound to beads were eluted using 8 mL elution buffer twice. The elutants were pooled and added with TEV protease ( 15 μL TEV at 4 mg/mL for every 1 mg target protein) and dialysed against gel filtration buffer at 4 °C overnight. The protein solutions were mixed with 0.5 mL 50% Ni-NTA beads and passed through another gravity column to remove His-tagged TEV protease. The flow-through was collected and loaded onto Supderdex-75 gel filtration column. Eluted fractions were pooled and concentrated using amicon centrifugal filter (m.w. cut-off 10,000 ). For RND1 GTPase, 10 times concentration of GppNHp was added to the protein before concentrating. The purity of the proteins was higher than 95% judged by SDS-PAGE.

Extraction

Procedure
Frozen cells were thawed and suspended in 150 mL the extraction buffer and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 2 μL benzonase (Sigma Catalog # E1014, 250U/μL), and lysed using sonicator at 100W for 5 minutes ( duty cycle: 10\" on, 5\" off)
Concentration:5.0 mg/mL PLXNB1, 8.3 mg/mL RND1, i.e. 1:1 morality ratio
Ligand
GNPMassSpec:Tag-removed Rnd1 21924.61 expected 21924.18
Tag-removed PLXNB1 13281.51, expected 13281.17

Crystallization:PLXNB1 and RND1 were mixed at a molarity ratio of 1:1 before setting up crystallization. Mother liquor using RW-E04 20.0% PEG3350, 0.2M CaCl2. Proteins were in 50 mM Tris, pH 7.5 150 mM NaCl 5 mM DTT, 5 mM MgCl2 buffer. Sitting drop vaporization was set up. Crystals usually appear in two or three days. Most of them appear as stacked plates. Crystals also grow for PLXNB1 and RND1 without tag cleavage.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Nakamura,F., Kalb,R.G., and Strittmatter,S.M. (2000). Molecular basis of semaphorin-mediated axon guidance. J. Neurobiol. 44, 219-229.

2. Oinuma,I., Ishikawa,Y., Katoh,H., and Negishi,M. (2004). The Semaphorin 4D receptor Plexin-B1 is a GTPase activating protein for R-Ras. Science 305, 862-865.

3. Ito,Y., Oinuma,I., Katoh,H., Kaibuchi,K., and Negishi,M. (2006). Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse. EMBO Rep. 7, 704-709.

4. Vikis,H.G., Li,W., He,Z., and Guan,K.L. (2000). The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner. Proc. Natl. Acad. Sci. U. S. A. 97, 12457-12462.

5. Driessens,M.H., Hu,H., Nobes,C.D., Self,A., Jordens,I., Goodman,C.S., and Hall,A. (2001). Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr. Biol. 11, 339-344.

6. Hu,H., Marton,T.F., and Goodman,C.S. (2001). Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron 32, 39-51.

7. Tong,Y.F., Chugha,P., Hota,P.K., Alviani,R.S., Li,M., Tempel,W., Shen,L., Park,H.W., and Buck,M. (2007). Binding of Rac1, Rnd1 and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 Effector domain. J. Biol. Chem. 282, 37215-37224.