Structural Bases of Plexin Signal Transduction

Plexins are a family of transmembrane receptors for the Semaphorin family of repulsive axon guidance molecules. Plexin mediated signal transduction is critical for a variety of cellular processes including regulation of adhesion and actin organization. Aberrant plexin signaling is associated with numerous pathologies. Despite plexin's essential cellular functions, a detailed understanding of the structural basis underlying plexin singling remains elusive. Here, the structural bases for two aspects of plexin signaling are revealed. Plexins relay signals in part by accelerating the GTP hydrolysis reaction catalyzed by the small GTPase Rap. This discovery appears at odds with the known structural features of plexin GAP domains. Plexin GAPs are structurally related to RasGAPs and possess RasGAP catalytic machinery. Conversely, plexins are structurally unrelated to canonical RapGAPs and don't possess their associated catalytic machinery. Here, the structural basis underlying the non-canonical RapGAP activity of plexins is revealed. Plexins induce a unique configuration of Rap's Switch II loop to utilize a non-canonical catalytic residue. Plexins also regulate RhoA activity. The B family plexins recruit two RhoGEFs (PDZ-RhoGEF and Leukemia Associated RhoGEF) by specifically binding to the PDZ domains of these GEFs. Conversely, these PDZ domains bind promiscuously to a variety of ligands. The structural basis by which plexins specifically recognize these two PDZ domains is revealed here. B family plexins interact with the PDZ domain of PDZ-RhoGEF using a secondary interface outside the canonical PDZ binding site. This secondary interface contributes to tight binding and is important for RhoA activation following plexin activation. Secondary interfaces may be a general mechanism utilized by modular protein-protein interaction domains to achieve specificity. Structural and biochemical characterization of plexins can be difficult. Here, two methods for studying plexins are described. These methods were critical to the success of the above studies. First, a method used to generate fusion proteins in-vitro for use in crystallography is described. This method allowed for the successful crystallization of a plexin/Rap complex. Second, a biochemical assay is described for easily measuring Plexin GAP activity in-vitro. This method circumvents the difficulties of many alternative approaches to measuring GAP activity.