The WH2 Domain and Actin Nucleation: Necessary but Insufficient

Abstract

Two types of sequences, proline-rich domains (PRDs) and the WASP-homology 2 (WH2) domain, are found in most actin filament nucleation and elongation factors discovered thus far. PRDs serve as a platform for protein-protein interactions, often mediating the binding of profilin-actin. The WH2 domain is an abundant actin monomer-binding motif comprising ∼17 amino acids. It frequently occurs in tandem repeats, and functions in nucleation by recruiting actin subunits to form the polymerization nucleus. It is found in Spire, Cordon Bleu (Cobl), Leiomodin (Lmod), Arp2/3 complex activators (WASP, WHAMM, WAVE, etc.), the bacterial nucleators VopL/VopF and Sca2, and some formins. Yet, it is argued here that the WH2 domain plays only an auxiliary role in nucleation, always synergizing with other domains or proteins for this activity.

Figure 1

Domain diagram of representative members of the four major nucleator subfamilies. The WH2 domain is shown in red and labeled as W. Proline-rich sequences are shown in cyan and labeled as PRD. Other domains specific to each protein include: KIND, kinase noncatalytic C-lobe domain; S, Spire box; FYVE, Fab1/YOTB/Vac1/EEA1 zinc-binding domain; K, basic K-region; SS, signal sequence; NRD, N-terminal repeat domain; CRD, C-terminal repeat domain; AC, autochaperone domain; TMBS1, tropomyosin-binding site 1; ABS2, actin-binding site 2; WH1, WASP-homology 1 domain; B, basic domain; CRIB, Cdc42/Rac interactive binding; C, central region; A, acidic region; GBD, GTPase-binding domain; DID, diaphanous inhibitory domain; DD, dimerization domain; FH2, formin-homology 2 domain; DAD, diaphanousautoregulatory domain. The UniProt accession codes of the proteins shown are: Drosophila melanogaster Spire, Q9U1K1-1; human Cobl, O75128-1; Vibrio parahaemolyticus VopL, Q87GE5; Rickettsia conorii Sca2, Q92JF7; human Lmod2, Q6P5Q4-1; human N-WASP, O00401; human WAVE2, Q9Y6W5-1; mouse WHAMM, Q571B6; Rickettsia conorii RickA, Q92H62; mouse FMNL3, Q6ZPF4; mouse mDia1, O08808; human INF2, Q27J81-1; and Drosophila melanogaster Cappuccino, Q24120.

Figure 2

Sequence and structure of the WH2 domain. (A) Alignments of the WH2 domains and WH2-related sequences of the proteins discussed here. Conservation scores for each amino acid were calculated based on a larger alignment of 100 representative sequences of WH2 domains from different proteins and species (not shown). Ten of the amino acids positions of the WH2 domain are conserved in more than 50% of the sequences (consensus 50%). The UniProt accession codes of the sequences shown are: human WASP, P42768; Drosophila melanogaster Spire, Q9U1K1-1; human Cobl, O75128-1; Vibrio parahaemolyticus VopL, Q87GE5; Rickettsia conorii Sca2, Q92JF7; human Lmod1, P29536; human Lmod2, Q6P5Q4-1; human N-WASP, O00401; human WAVE1, Q92558; human WAVE2, Q9Y6W5-1; human WHAMM, Q8TF30; Rickettsia conorii RickA, Q92H62; Saccharomyces cerevisiae LAS17, Q12446; human WIP, Q8TF74; human MIM, O43312; Dictyostelium discoideum actobindin, Q55DU1; Schizosaccharomyces pombe PAN1, Q10172; human Espin, B1AK53; human INF2, Q27J81-1; mouse mDia1, O08808; mouse FMNL3, Q6ZPF4; and human VASP, P50552. (B) Structure of the WH2 domain of WASP (the founding member of the WH2 domain family) bound to actin (PDB code: 2A3Z) [68]. The actin subdomains are labeled 1 to 4. (C) WH2 domain of human WASP, showing the side chains of the 10 residues that are conserved in more than 50% of the sequences, which most interact with actin.

Figure 3

Hypothetical nucleation mechanisms of tandem WH2 domain-based nucleators. Proteins and domains are colored and labeled according to Figure 1. Note that for this group of nucleators, and specially Sca2 and Cobl, evidence is still lacking concerning the precise mechanisms of nucleation (see Outstanding Questions and main text). For Spire-Cappuccino [2], VopL/VopF [21, 33, 34] and Sca2 [39] similar mechanisms have been proposed, and are supported by structural and biochemical data. The mechanism proposed for Cobl is only supported by cellular data showing strict cooperation between Cobl and dimeric BAR domain proteins [24, 28, 29], and the widespread importance of dimerization (oligomerization) among filament nucleators. Except in the case of formins and Sca2, which function both as nucleation and elongation factors, these proteins are thought to be dissociate after nucleation, which is at least in part due to competition between binding of the WH2 domain and inter-subunit contacts in the filament (Figure S1). Filaments then elongate at barbed end from either monomeric actin or profilin-actin.

Figure 4

Proposed nucleation mechanism of Lmod. Protein domains are colored and labeled according to Figure 1. Lmod contains two actin-binding sites (ABS2 and WH2 domain) and additionally interacts with Tropomyosin (TM) through TMBS1 [41, 43, 44]. ABS2 binds at the interface between three actin subunits of the filament. Lmod dissociates after nucleation, and is replaced at the pointed end by Tmod in muscle sarcomeres. Dissociation is possibly triggered by steric hindrance of the WH2 domain as well as the lack of capping elements present in Tmod, including ABS1, TMBS2, and the N-terminal portion of ABS2, which together contribute to the very high affinity of Tmod for pointed ends.

Figure 5

Proposed nucleation mechanism of NPFs-Arp2/3 complex. Proteins and domains are colored and labeled according to Figure 1. The 7-subunit Arp2/3 complex contains two actin related subunits (Arp2 and Arp3). Interaction with two NPFs [47-50] repositions the Arps into a filament-like conformation and triggers binding on the side of the mother filament. Additionally, NPFs contribute actin subunits to the incipient branch. NPFs dissociated after nucleation, and the branch filament is free to grow from either monomeric actin or profilin-actin.

Figure 6

Proposed nucleation mechanism of formins ± NPFs. Proteins and domains are colored and labeled according to Figure 1. Most formins contain formin-homology 1 (FH1) and 2 (FH2) domains. The FH1 domain is a PRD that mediates the binding of profilin-actin. The FH2 domain is responsible for nucleation and barbed-end binding. For several formins, sequences C-terminal to the FH2 domain enhance nucleation, by either directly recruiting actin monomers [55-58] (top model) or by recruiting other proteins that synergize with the formins during nucleation [16, 18, 59, 60, 63] (bottom model). Formins remain processively bound at the barbed end after nucleation, and accelerate barbed end elongation in a profilin-actin-dependent manner [54].