Human Plexin-D1, ubiquitin-like domain

Target ID:

PLXND1

Deposition Date:

Friday 24th April 2009

Families:

G-Protein Regulators

Structure type:

apo

Download Coordinates:

3H6N

Authors:

Tong Y, Nedyalkova L, Tempel W, MacKenzie F, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Buck M, Park HW

Site:

Toronto

ICM Datapack:

ICM Datapack

3H6N

tabs

Structure Details

The plexin family of transmembrane proteins are receptors for semaphorins, axon growth cone guidance molecules (Nakamura et al., 2000). Plexins were first discovered in axons and developing nervous system (Tamagnone et al., 1999) and later found to play important roles in developmental processes such as cardiovascular development and angiogenesis (Toyofuku et al., 2004), in epithelial cell invasive growth (Giordano et al., 2002), and in the immune system (Wong et al., 2003). The extracellular region of plexins shares significant homology with that of the hepatocyte growth factor receptor and semaphorins. The cytoplasmic region of plexins is also well conserved (Tamagnone et al., 1999) and shows sequence similarity to RASA1, a GTPase activating protein (GAP) for Ras family GTPase (Rohm et al., 2000). The RasGAP homology region of plexins is split into two subdomains with an insertion of ~200 amino acid residues that bind to Rho GTPases(Driessens et al., 2001;Hu et al., 2001;Vikis et al., 2000). Plexins are the first documented example of transmembrane receptors that interact directly with small GTPases.

Plexin D1 (PLXND1) is also found to be a RasGAP for RRAS (Uesugi et al., 2009) and the GAP activity is involved in axon outgrowth of cortical neurons. Binding of RND2 is required for the GAP activity of PLXNB1 for RRAS, representing a novel regulatory mechanism of the RasGAP activity by a Rho GTPase. Plexin D1 (PLXND1) is broadly expressed on tumor vessels and tumor cells in a number of different human tumor types and the expression is strongly correlated with both invasive behavior and metastasis of tumors(Roodink et al., 2008).

Based on the sequence homology with plexin B1, we propose that the ~200 a.a. insertion that separates the RasGAP homology domain should be the region that binds with Rho GTPases and has a ubiquitin-like structure. We solved the crystal structure of the ubiquitin-like domain (the effector domain or Rho GTPases binding domain) of PLXND1. As expected, the domain has an ubiquitin fold that is similar to the RBD domain of PLXNB1 (PDB: 2R2O): a central α-helix is surrounded by a five-strand β-sheet. But while the ubiquitin-like domain of plexin B1 forms a dimer, the ubiquitin-like domain of plexin D1 exists as a monomer.

Materials and Methods

PLXND1

PDB:3H6N

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:CDV PLXND1 (SGC template 16-E4; camR)
Entry Clone Source:Codon Devices Synthesized
SGC Clone Accession:HPC078-G04
Tag:N-terminal tag: mhhhhhhssgrenlyfq*g
Host:BL21(DE3)-V2R-pRARE2

Construct

Prelude:Tag removed

PLXND1:A1553-L1678
Sequence:gAKPRNLNVSFQGCGMDSLSVRAMDTDTLTQVKEKILEAFCKNVPYSQWPRAEDVDLEWFASSTQSYILRDLDDTSVVEDGRKKLNTLAHYKIPEGASLAMSLIDKKDNTLGRVKDLDTEKYFHLVL
Vector:pET28-mhl

Growth

Medium:M9 Medium, SeMet
Antibiotics:Kanamycin 50 µg/mL Chloramphenicol 25 µg/mL
Procedure:LEX bubbling. The target protein was expressed in E.coli using prepacked M9 SeMet growth media kit (Medicilon) following manufacturer\'s instruction. Harvested cells were 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 5 mL 50% flurry of Ni-NTA beads and incubated at 4 degC on rotary shaker for one hour. The mixture was then centrifuged at 2300 rpm for 5 min and the supernant discarded. The beads were then washed with washing buffer containing 30 mM and 75 mM Imidazole, and finally the elution buffer. The flow-trough was collected and further purifed by a Superdex-75 gel filtraton column pre-equilibrated with gel filtration buffer. Fractions were collected and digested with TEV protease. TEV protease was removed from the treated protein sample by adding 100 µL 50% flurry of Ni-NTA beads and the sample was purified with supderdex-75 gel filtration again. Fractions containing the protein were collected and concentrated with Amicon Ultra-15 centrifugal filter (m.w. cut-off 5,000). The purity of the preparation is tested by SDS-PAGE to be around 99%.

Extraction

Procedure
Frozen cells from 4L M9 medium were thawed and resuspended in 150 mL Binding Buffer with freshly added 0.5% CHAPS and 2mM BME, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 3 &microL benzonase (Sigma Catalog # E1014, 250U/&microL), and lysed using sonication.
Concentration:41.4 mg/mL
Ligand
MassSpec:SeMet: Expected 14498.0 (3 Met, tag cut), measured: 14498.0 (5%), 14574.62(76.62+, ~50%), 14650.6(152.60+, ~40%). The extra mass should come from BME modification.
Crystallization:Crystal used for phasing and refinement was grown using sitting drop vaporization method. The well solution contains 1.39 M NaCitrate, 0.1M Sodium Cocadylate buffer pH 5.2. The protein stock solution was adjusted to contain 15 mM TCEP, supplemented with 1:100 (w/w) Chymotrypsin, and 0.5uL protein solution was mixed with 0.5uL well solution using Mosquito instrument. The plate was stored at room temperature. Crystals appear in the drop in 3 to 5 days. Cryoprotectant used paratone.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

DRIESSENS, M. H., HU, H., NOBES, C. D., SELF, A., JORDENS, I., GOODMAN, C. S., & HALL, A. (2001). Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr.Biol., 11(5), 339-344.
GIORDANO, S., CORSO, S., CONROTTO, P., ARTIGIANI, S., GILESTRO, G., BARBERIS, D., TAMAGNONE, L., & COMOGLIO, P. M. (2002). The semaphorin 4D receptor controls invasive growth by coupling with Met. Nat.Cell Biol., 4(9), 720-724.
HU, H., MARTON, T. F., & GOODMAN, C. S. (2001). Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron, 32(1), 39-51.
NAKAMURA, F., KALB, R. G., & STRITTMATTER, S. M. (2000). Molecular basis of semaphorin-mediated axon guidance. J.Neurobiol., 44(2), 219-229.
ROHM, B., RAHIM, B., KLEIBER, B., HOVATTA, I., & PUSCHEL, A. W. (2000). The semaphorin 3A receptor may directly regulate the activity of small GTPases. FEBS Lett., 486(1), 68-72.
ROODINK, I., KATS, G., VAN, K. L., GRUNBERG, M., MAASS, C., VERRIJP, K., RAATS, J., & LEENDERS, W. (2008). Semaphorin 3E expression correlates inversely with Plexin D1 during tumor progression. Am.J.Pathol., 173(6), 1873-1881.
TAMAGNONE, L., ARTIGIANI, S., CHEN, H., HE, Z., MING, G. I., SONG, H., CHEDOTAL, A., WINBERG, M. L., GOODMAN, C. S., POO, M., TESSIER-LAVIGNE, M., & COMOGLIO, P. M. (1999). Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell, 99(1), 71-80.
TOYOFUKU, T., ZHANG, H., KUMANOGOH, A., TAKEGAHARA, N., SUTO, F., KAMEI, J., AOKI, K., YABUKI, M., HORI, M., FUJISAWA, H., & KIKUTANI, H. (2004). Dual roles of Sema6D in cardiac morphogenesis through region-specific association of its receptor, Plexin-A1, with off-track and vascular endothelial growth factor receptor type 2. Genes Dev., 18(4), 435-447.
UESUGI, K., OINUMA, I., KATOH, H., & NEGISHI, M. (2009). Different requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1. Journal of Biological Chemistry, 284(11), 6743-6751.
VIKIS, H. G., LI, W., HE, Z., & GUAN, K. L. (2000). The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner. Proceedings of the National Academy of Sciences USA, 97(23), 12457-12462.
WONG, A. W., BRICKEY, W. J., TAXMAN, D. J., VAN DEVENTER, H. W., REED, W., GAO, J. X., ZHENG, P., LIU, Y., LI, P., BLUM, J. S., MCKINNON, K. P., & TING, J. P. (2003). CIITA-regulated plexin-A1 affects T-cell-dendritic cell interactions. Nat Immunol., 4(9), 891-898.