Plexin-Bs enhance their GAP activity with a novel activation switch loop generating a cooperative enzyme

Abstract

Plexins receive guidance cues from semaphorin ligands and transmit their signal through the plasma membrane. This family of proteins is unique amongst single-pass transmembrane receptors as their intracellular regions interact directly with several small GTPases, which regulate cytoskeletal dynamics and cell adhesion. Here, we characterize the GTPase Activating Protein (GAP) function of Plexin-B1 and find that a cooperative GAP activity towards the substrate GTPase, Rap1b, is associated with the N-terminal Juxtamembrane region of Plexin-B1. Importantly, we unveil an activation mechanism of Plexin-B1 by identifying a novel functional loop which partially blocks Rap1b entry to the plexin GAP domain. Consistent with the concept of allokairy developed for other systems, Plexin-B activity is increased by an apparent substrate mediated cooperative effect. Simulations and mutagenesis suggest the repositioned JM conformation is stabilized by the new activation switch loop when the active site is occupied, giving rise to faster enzymatic turnover and cooperative behavior. The biological implications, essentially of a threshold behavior for cell migration are discussed..

A putative structural mechanism underlying the antithetic effect of homologous RND1 and RhoD GTPases in mammalian plexin regulation

“…Moreover, our conclusion concerns mostly inter-domain interactions and thus is less likely to be sensitive to the structural details, as the main driving force is the electrostatic interactions between the GTPases and membrane (Figure 3G), rather than any specific residue-residue interactions arising from a specific conformation. Reassuringly, our findings are supported by a recent study on plexin B1, which (Li et al, 2020) identified the functional importance of the buttress segment (or "activation switch loop" as is referred therein) based on analysis of plexin enzymatic turnover, and showed that the segment helps stabilize the dimerization helix when the plexin active site is occupied by Rap.…”

The structural mechanism underlying the antithetic effect of homologous RND1 and RhoD GTPases in plexin regulation

“…Moreover, our conclusion is not sensitive to the fine structures, as the main driving force is the electrostatic interactions from the interface between the GTPases and membrane (Figure 3F), rather than any specific residue-residue interactions arising from a specific conformation. Reassuringly, our findings are supported by a recent study on plexin B1, which (Li et al, 2020) identified the functional importance of the buttress segment (or “activation switch loop” as is referred therein) based on analysis of plexin enzymatic turnover, and showed that the segment helps stabilizing the dimerization helix when the plexin active site is occupied by Rap.…”

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

“…For example, deletion of the plexin A1 sema domain converts the protein from an autoinhibited form to a constitutively active protein (in the absence of ligand) ( 47 ). In principle, it is possible that plexin D1 may undergo an inactive to active state transition without the need for homodimerization ( 7 , 63 , 64 ).…”

“…47 In principle, it is possible that Plexin D1 may undergo an inactive to active state transition without the need for homodimerization. 7,[63][64] The receptors examined here do not form heterotypic interactions in the absence of ligand, except for Plexin A2 and Plexin A4. The class A plexins have conserved residues which may contribute to heterodimerization; however, these interactions had not been reported prior to the present study.…”