SUMMARY Vav proteins are guanine nucleotide exchange factors (GEFs) for Rho family GTPases. They control processes including T cell activation, phagocytosis and migration of normal and transformed cells. We report the structure and biophysical and cellular analyses of the five-domain autoinhibitory element of Vav1. The catalytic Dbl homology (DH) domain of Vav1 is controlled by two energetically coupled processes. The DH active site is directly, but weakly, inhibited by a helix from the adjacent Acidic domain. This core interaction is strengthened 10-fold by contacts of the calponin homology (CH) domain with the Acidic, pleckstrin homology and DH domains. This construction enables efficient, stepwise relief of autoinhibition: initial phosphorylation events disrupt the modulatory CH contacts, facilitating phosphorylation of the inhibitory helix and consequent GEF activation. Our findings illustrate how the opposing requirements of strong suppression of activity and rapid kinetics of activation can be achieved in multi-domain systems.

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected by bacteria into eukaryotic host cells. VopL contains three N-terminal WiskottAldrich Homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own, and requires the VCD for activity. The VCD forms a Ushaped dimer in the crystal, which is stabilized by a terminal coiled-coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model where VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.Actin cytoskeletal dynamics are important in numerous cellular processes, including migration, division and maintenance of morphology [1][2][3] . These functions all require rapid assembly of new actin filaments de novo from actin monomers. However, actin assembly is intrinsically slow, due to kinetic and thermodynamic barriers to forming the actin dimers and trimers needed to initiate filament growth 4 . Cells have therefore developed specialized factors to stabilize these actin nuclei and thus catalyze filament assembly 4 . Three classes of so-called actin nucleation factors have been identified: Arp2/3 complex, formin proteins and WASP Homology domain 2 (WH2)-based nucleators [5][6][7][8][9][10][11] . Arp2/3 complex binds to the side of an existing filament and initiates growth of a new filament from its actin related protein 2 (Arp2) and Arp3 subunits 12,13 . Alignment of the actin homologs Arp2 and Arp3 to resemble Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript the first two monomers at the base of an actin filament is believed to be a key aspect of Arp2/3-mediated nucleation 14 . Formin proteins nucleate linear filaments through a conserved formin homology 2 (FH2) domain, which is also thought to organize three actin monomers into a structure that resembles the base of an actin filament 15,16 . After nucleation, formins remain associated with the growing filament barbed end as new monomers are added 5 . Several mechanisms have been proposed to account for this processive activity [15][16][17][18] . The WH2-based actin nucleators have been identified most recently, and include Spire, Corbon blue (Cobl) and Leiomodin (Lmod) [7][8][9]11 . These proteins all contain a series of tandem WH2 motifs, each capable of binding an actin monomer. These proteins are also thought to act through organization of multiple actin monomers into a stable structure...