A central role for the WH2 domain of Srv2/CAP in recharging actin monomers to drive actin turnover in vitro and in vivo

Abstract

Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assemblycompetent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K d ~1.5μM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.

Structure and Mechanism of Mouse Cyclase-associated Protein (CAP1) in Regulating Actin Dynamics

“…Further, C-CAP1-98 (which mutates a key actin-binding surface on the WH2 domain) almost completely abolished the activity. These results demonstrate that the WH2 domain of CAP1 is critical for actin monomer recycling specifically in the presence of cofilin, similar to what has been observed for yeast Srv2 (23). Thus, the mechanisms used by yeast and mammalian CAP homologs for catalyzing actin monomer recycling appear to be highly conserved.…”

“…However, earlier studies on yeast Srv2 showed that mutating the central pair of Lys residues in its LKKV motif had little effect on function either in vitro or in vivo (18,23). Instead, mutating both of the flanking hydrophobic residues (Leu and Val) was required to disrupt WH2 function in vitro and in vivo (23).…”

“…For yeast Srv2, these two functions were assigned to separate halves of the protein that function in a largely autonomous manner (22). The N-terminal-half mediates enhanced severing, which depends on its ability to form large hexameric structures (20), while the C-terminal-half mediates actin monomer recycling, which depends on actin-binding surfaces located in both its WH2 and ␤-sheet domains (18,23). One question that has remained open is how the hexameric Srv2/CAP structures enhance cofilin-mediated severing of filaments.…”

“…This activity requires conserved surfaces on both the WH2 and ␤-sheet domains (18,20,23,31). Thus, neither domain is sufficient for this function, and instead the two actin-binding domains work in concert to displace cofilin and stimulate nucleotide exchange on G-actin.…”

“…There is also some disagreement about the mechanism by which the C-terminal-half of Srv2/CAP catalyzes actin monomer recycling. Studies on yeast Srv2 show that the ability to catalyze nucleotide exchange on cofilin-bound ADP-actin monomers requires both the WH2 and ␤-sheet domains (23). In contrast, recent studies on mouse CAP1 reported that the ␤-sheet domain alone was sufficient to catalyze nucleotide exchange on ADP-actin monomers, and have called into question the role of the WH2 domain in C-CAP1 functions (24).…”