Abstract
Srv2/CAP is a conserved actin-binding protein with important roles in driving cellular actin dynamics in diverse animal, fungal, and plant species. However, there have been conflicting reports about whether the activities of Srv2/CAP are conserved, particularly between yeast and mammalian homologs. Yeast Srv2 has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. Here, we dissected the structure and function of mouse CAP1 to better understand its mechanistic relationship to yeast Srv2. Although CAP1 has a shorter N-terminal oligomerization sequence compared with Srv2, we find that the N-terminal-half of CAP1 (N-CAP1) forms hexameric structures with six protrusions, similar to N-Srv2. Further, N-CAP1 autonomously binds to F-actin and decorates the sides and ends of filaments, altering F-actin structure and enhancing cofilin-mediated severing. These activities depend on conserved surface residues on the helical-folded domain. Moreover, N-CAP1 enhances yeast cofilin-mediated severing, and conversely, yeast N-Srv2 enhances human cofilin-mediated severing, highlighting the mechanistic conservation between yeast and mammals. Further, we demonstrate that the C-terminal actin-binding β-sheet domain of CAP1 is sufficient to catalyze nucleotide-exchange of ADP-actin monomers, while in the presence of cofilin this activity additionally requires the WH2 domain. Thus, the structures, activities, and mechanisms of mouse and yeast Srv2/CAP homologs are remarkably well conserved, suggesting that the same activities and mechanisms underlie many of the diverse actin-based functions ascribed to Srv2/CAP homologs in different organisms.
Highlights
Mechanistic and structural conservation between evolutionarily distant Srv2/CAP homologs has remained unclear
N-terminal-half of CAP1 (N-CAP1) is slightly more compact compared with N-terminal-half of yeast Srv2 (N-Srv2), consistent with N-CAP1 having a slightly lower MW. These molecules differ by an acidic N-terminal sequence that mediates Ras-signaling in N-Srv2, and is lacking in N-CAP1 [32, 33]
The overall architecture of the N-Srv2/NCAP1 hexamer is remarkably well conserved between yeast and mice, yet in yeast Srv2 allows the insertion of some residues that introduce new functional capabilities
Summary
Mechanistic and structural conservation between evolutionarily distant Srv2/CAP homologs has remained unclear. Yeast Srv has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. 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] These assays were performed in the absence of cofilin, precluding direct comparison of the two studies. The ability of C-CAP1 to catalyze nucleotide exchange on cofilin-bound ADP-actin monomers requires both its WH2 and -sheet domains These findings indicate that the activities and mechanisms of distantly related Srv2/CAP homologs are highly conserved, and suggest that the diverse members of this protein family may have similar cellular functions in regulating the actin cytoskeleton
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