Mapping the functional surface of domain 2 in the gelsolin superfamily

The crystal structure of the F-actin binding domain 2 of severin, the gelso lin homologue from Dictyostelium discoideum, has been determined by multipl e isomorphous replacement and refined to 1.75 Angstrom resolution. The stru cture reveals an alpha-helix-beta-sheet sandwich similar to the domains of gelsolin and villin, and contains two cation-binding sites, as observed in other domain 1 and domain 2 homologues. Comparison of the structures of sev eral gelsolin family domains has identified residues that may mediate F-act in binding in gelsolin domain 2 homologues. To assess the involvement of th ese residues in F-actin binding, three mutants of human gelsolin domain 2 w ere assayed for F-actin binding activity and thermodynamic stability. Two o f the mutants, RRV168AAA and RLK210AAA, demonstrated a lowered affinity for F-actin, indicating a role for those residues in filament binding. Using b oth structural and biochemical data, we have constructed a model of the gel solin domain 1-domain 2-F-actin complex. This model highlights a number of interactions that may serve as positive and negative determinants of filame nt end- and side-binding.