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J. Biol. Chem., Vol. 282, Issue 51, 37215-37224, December 21, 2007

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Binding of Rac1, Rnd1, and RhoD to a Novel Rho GTPase Interaction Motif Destabilizes Dimerization of the Plexin-B1 Effector Domain^*

Yufeng Tong¹², Preeti Chugha¹³, Prasanta K. Hota, Rebecca S. Alviani, Mei Li, Wolfram Tempel, Limin Shen, Hee-Won Park^¶, and Matthias Buck^||**4

From the Departments of Physiology and Biophysics, ^||Neuroscience, and ^**Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and Structural Genomics Consortium, ^¶Department of Pharmacology, University of Toronto, Toronto, Ontario M5G 1L5, Canada

Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, β-strands 3 and 4 and a short -helical segment of the plexin domain. The 2.0 Å resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel β-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.

Received for publication, May 8, 2007 , and in revised form, September 20, 2007.

The atomic coordinates and structure factors (code 2r2o) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by the Structural Genomics Consortium, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, Glaxo-SmithKline, Karolinska Institutet, The Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck, the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4.

¹ Both authors contributed equally to this work.

² Postdoctoral fellow of the American Heart Association, Ohio Valley Affiliate 0425678B.

³ Supported by National Institutes of Health Grant T32 HL007653.

⁴ Recipient of American Heart Association Scientist Development Award 0335353N and supported by National Institutes of Health Grants R01GM73071 and K02HL084384 and by a Basil O'Connor award from the March of Dimes Foundation for Birth Defects 5-FY05-117. To whom correspondence should be addressed: Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-8651; Fax: 216-368-3952; E-mail: Matthias.Buck{at}case.edu.