The WASP family is defined by a conserved C-terminal VCA domain named for its three sub-elements, the Verprolin homology (also called WASP homology 2), Central hydrophobic and Acidic regions. The VCA domain strongly stimulates actin nucleation by Arp2/3 complex (1
). The three elements have distinct functions during this process. The C-terminal A region contributes substantial binding affinity toward Arp2/3 complex (9
). There is also a connection between the A region and activity; different WASP family VCAs have quite different maximal activities, which correlate with A region sequence (11
). The C region also contributes both to binding affinity and to an affinity-independent aspect of activation (10
), as well as to autoinhibitory regulation (see below). The C region appears to contact Arp2/3 complex using the hydrophobic face of an amphipathic helix (13
The V region binds to G-actin and delivers it to the Arp2 and/or Arp3 subunits of Arp2/3 complex (which both are homologous to actin), creating a pseudo-trimer/tetramer nucleus for filament growth (7
). A recent SAXS study suggests that a high-affinity site for the VCA:actin complex is positioned to deliver actin to the Arp2 subunit (18
); it is also possible that a second VCA binding to a different site (see below) could deliver actin to Arp3. V region peptides form an amphipathic helix that inserts into the hydrophobic groove between actin subunits 1 and 3, followed by an extended segment that traverses up the actin face toward the nucleotide binding cleft () (16
) (see also RSCB database entry PDB#2vcp). As detailed in recent reviews, V/WH2 repeats are also found in many other proteins, and can act to either sequester actin monomer or, when in tandem arrays, nucleate filaments directly (20
An important caveat to this straightforward characterization of the V, C and A functions is that nearly all experiments to date do not take into account the recent discovery of a second VCA binding site on Arp2/3 complex (22
). This finding requires new experiments (and reconsideration of previous data) to analyze these functions in the context of the individual sites. In general, the VCAs could have quite different actions at the two sites, perhaps even promoting temporally distinct steps in the nucleation process. Understanding both the thermodynamic and kinetic aspects of Arp2/3 activation through the two VCA binding sites represents an important avenue for future research.
N-terminal elements of WASP family proteins control localization, association with ligands and biochemical activity of the VCA. These elements are divergent among different family members, affording the proteins distinct molecular details of regulation. However as argued below, the physical regulatory principles are likely similar across the family. These principles have emerged from studies of the archetypal members, WASP and N-WASP.
The N-terminal Ena/VASP homology 1 (EVH1) domain of WASP and N-WASP () binds to a conserved proline motif in members of the WIP family (23
). This interaction stabilizes and localizes WASP and N-WASP in cells (23
). Structural studies show that the WIP proline motif contacts the canonical EVH1 ligand binding site through a type II polyproline helix () (26
). The flanking sequences extend linearly around the EVH1 domain in opposite directions, wrapping over half way around the domain. Mutations that disrupt this interface decrease WIP binding, leading in vivo to proteolytic degradation of WASP (25
) and many forms of WAS (27
). WIP binding also negatively regulates the N-WASP VCA (28
The EVH1 domain is followed by a hydrophilic, low-complexity sequence, without an ascribed function for either WASP or N-WASP. Following this is a sequence of basic residues known as the basic (B) region (). In both proteins the B region stabilizes autoinhibitory interactions (see below) (30
). In N-WASP it is clear that this element also binds PIP2
and mediates many PIP2
effects on activity (31
) (see below). The element probably plays a similar role in WASP, but additional regions appear necessary for the functional interaction (35
). As elaborated below, the B region also plays an important role in selective binding of the WASP activator, Cdc42 (36
After the B region, WASP and N-WASP contain an ~85 residue element termed the GTPase binding domain (GBD, ). The first 20 residues of this element contain a Cdc42-Rac Interactive Binding (CRIB) motif (37
), which mediates nucleotide-dependent binding of numerous effectors to the Cdc42 and Rac GTPases. The GBD in WASP and N-WASP binds the VCA intramolecularly and plays a central role in controlling activity toward Arp2/3 complex (38
Between the GBD and VCA, WASP and N-WASP contain a 100–125 residue proline rich domain (PRD, ). The PRD contains approximately six canonical binding sites for SH3 domains, and three profilin binding sites (40
). This segment mediates interactions with SH3 domains of a large and diverse range of ligands. As WIP also contains a PRD, WASP:WIP complexes will have even more SH3 binding sites.
The five functional elements of WASP allow binding to multiple ligands simultaneously. A central concept developed in the following sections is that the ligands can act through two distinct regulatory mechanisms to control the biochemical activity of WASP toward Arp2/3 complex: allostery, which controls accessibility of the VCA to Arp2/3 complex, and oligomerization, which controls how strongly the exposed VCA can stimulate Arp2/3 complex. When multiple ligands engage one or both of these mechanisms simultaneously, they act cooperatively to control WASP activity, thus integrating multiple signals.