The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-b4 (Tb4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tb4 (G1-Tb4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tb4:actin complex at 2 Å resolution. The structure reveals that Tb4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tb4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motifcontaining proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

The actin filament-severing functionality of gelsolin resides in its N-terminal three domains (G1-G3). We have determined the structure of this fragment in complex with an actin monomer. The structure reveals the dramatic domain rearrangements that activate G1-G3, which include the replacement of interdomain interactions observed in the inactive, calcium-free protein by new contacts to actin, and by a novel G2-G3 interface. Together, these conformational changes are critical for actin filament severing, and we suggest that their absence leads to the disease Finnish-type familial amyloidosis. Furthermore, we propose that association with actin drives the calcium-independent activation of isolated G1-G3 during apoptosis, and that a similar mechanism operates to activate native gelsolin at micromolar levels of calcium. This is the first structure of a filament-binding protein bound to actin and it sets stringent, high-resolution limitations on the arrangement of actin protomers within the filament.

Participation of actin in cellular processes relies on the dynamics of filament assembly. Filament elongation is fed by monomeric actin in complex with either profilin or a Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2)/beta-thymosin (betaT) domain. WH2/betaT motif repetition (typified by ciboulot) or combination with nonrelated domains (as found in N-WASP) results in proteins that yield their actin to filament elongation. Here, we report the crystal structures of actin bound hybrid proteins, constructed between gelsolin and WH2/betaT domains from ciboulot or N-WASP. We observe the C-terminal half of ciboulot domain 2 bound to actin. In solution, we show that cibolout domains 2 and 3 bind to both G- and F-actin, and that whole ciboulot forms a complex with two actin monomers. In contrast, the analogous portion of N-WASP WH2 domain 2 is detached from actin, indicating that the C-terminal halves of the betaT and WH2 motifs are not functionally analogous.

We present the 2.6 A î resolution crystal structure of a complex formed between G-actin and gelsolin fragment Met25Ĝ ln160 (G1+). The structure di¡ers from those of other gelsolin domain 1 (G1) complexes in that an additional six amino acid residues from the crucial linker region into gelsolin domain 2 (G2) are visible and are attached securely to the surface of actin. The linker segment extends away from G1 up the face of actin in a direction that infers G2 will bind along the same long-pitch helical strand as the actin bound to G1. This is consistent with a mechanism whereby G2 attaches gelsolin to the side of a ¢lament and then directs G1 toward a position where it would disrupt actin^actin contacts. Alignment of the sequence of the structurally important residues within the G1^G2 linker with those of WH2 (WASp homology domain 2) domain protein family members (e.g. WASp (Wiscott^Aldridge syndrome protein) and thymosin L L4) suggests that the opposing activities of ¢lament assembly and disassembly may exploit a common patch on the surface of actin. ß

Gelsolin requires activation to carry out its severing and capping activities on F-actin. Here, we present the structure of the isolated C-terminal half of gelsolin (G4^G6) at 2.0 A î resolution in the presence of Ca 2+ ions. This structure completes a triptych of the states of activation of G4^G6 that illuminates its role in the function of gelsolin. Activated G4^G6 displays an open conformation, with the actin-binding site on G4 fully exposed and all three type-2 Ca 2+ sites occupied. Neither actin nor the type-l Ca 2+ , which normally is sandwiched between actin and G4, is required to achieve this conformation. ß