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

Plexins are semaphorin receptors that play essential roles in mammalian neuronal axon guidance and in many other important mammalian biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism, and is modulated by small GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations we showed that RND1 reinforces the plexin dimerization interface whereas RhoD destabilizes it due to their differential interaction with the cell membrane. Upon binding plexin at the Rho-GTPase binding domain (RBD), RND1 and RhoD interact differently with the inner leaflet of the cell membrane, and exert opposite effects on the dimerization interface via an allosteric network involving the RBD, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively charged membrane interface.

1H, 13C, and 15N NMR chemical shift assignment of the complex formed by the first EPEC EspF repeat and N-WASP GTPase binding domain

LEE-encoded effector EspF (EspF) is an effector protein part of enteropathogenic Escherichia coli’s (EPEC’s) arsenal for intestinal infection. This intrinsically disordered protein contains three highly conserved repeats which together compose over half of the protein’s complete amino acid sequence. EPEC uses EspF to hijack host proteins in order to promote infection. In the attack EspF is translocated, together with other effector proteins, to host cell via type III secretion system. Inside host EspF stimulates actin polymerization by interacting with Neural Wiskott-Aldrich syndrome protein (N-WASP), a regulator in actin polymerization machinery. It is presumed that EspF acts by disrupting the autoinhibitory state of N-WASP GTPase binding domain. In this NMR spectroscopy study, we report the 1H, 13C, and 15N resonance assignments for the complex formed by the first 47-residue repeat of EspF and N-WASP GTPase binding domain. These near-complete resonance assignments provide the basis for further studies which aim to characterize structure, interactions, and dynamics between these two proteins in solution.

Allosteric N-WASP activation by an inter-SH3 domain linker in Nck

Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott–Aldrich-syndrome protein (N-WASP) and the Arp2/3 complex. An inactive conformation of N-WASP is stabilized by intramolecular contacts between the GTPase binding domain (GBD) and the C helix of the verprolin-homology, connector-helix, acidic motif (VCA) segment. Multiple SH3 domain-containing adapter proteins can bind and possibly activate N-WASP, but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2/3 complex. Here, we have used purified components to reconstitute a signaling cascade driven by membrane-localized Src homology 3 (SH3) adapters and N-WASP, resulting in the assembly of dynamic actin networks. Among six SH3 adapters tested, Nck was the most potent activator of N-WASP–driven actin assembly. We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains. This linker sequence, reminiscent of bacterial virulence factors, directly engages the N-WASP GBD and competes with VCA binding. Our results suggest that animals, like pathogenic bacteria, have evolved peptide motifs that allosterically activate N-WASP, leading to localized actin nucleation on cellular membranes.

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin–based motility

Vaccinia virus dissemination relies on the N-WASP–ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP–ARP2/3 and Rac1–FHOD1 pathways.