Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins

Abstract

Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1-V6 and G1-G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca 2؉ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca 2؉ , rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca 2؉ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca 2؉ , straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites.Actin is crucial to such processes as cell movement, cell division, and apoptosis, which are regulated by numerous actinbinding proteins, including gelsolin, Arp2/3, and profilin (for review, see Ref. 1). Gelsolin, the most potent actin filamentsevering protein known, can bind to, sever, cap, and nucleate actin filaments in a calcium-, pH-, ATP-, and phospholipid-dependent manner (for review, see Ref.2). Villin, found in microvilli of absorptive epithelium, is a second member of the gelsolin family of actin-binding proteins. In addition to standard gelsolin-type activities, villin is able to bundle actin filaments and is subject to regulation by tyrosine phosphorylation as well as by Ca 2ϩ and phosphatidylinositol 4,5-bisphosphate (for review, see Ref.3). Many comparisons have been made between gelsolin and villin. The two share 50% amino acid sequence identity and show similar proteolytic cleavage patterns (4). Both contain six similarly folded domains, but villin possesses a seventh domain at its C terminus, the headpiece (HP) 2 domain, which folds into a compact structure that introduces a second F-actin-binding site into the protein. Recent studies indicate that villin uses the HP F-actin-binding sites to achieve bundling (5). In an environment devoid of free Ca 2ϩ

Gelsolin: The tail of a molecular gymnast

“…Calcium binding to type‐2 sites induces small‐scale local structural changes such as the exact positioning of the long helix with respect to the β‐sheet, and the straightening of the long helices of domains 3 and 6 (Fig. B) [Wang et al, ]. The long helix acts as a calcium sensor that transmits local changes at the calcium‐binding site to other areas of the parent domain to trigger large‐scale rearrangements of domain positions relative to each other, while maintaining the structural integrity of the individual domains [Choe et al, ; Kazmirski et al, ; Burtnick et al, ].…”

Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

“…We cloned human profilin 1 into the pSY5 vector, a modified version of pET-21d(+), and expressed it with an 8×His tag 39 . We purified the protein with a HisTrap column followed by gel filtration with a Superdex 75 HiLoad 16/60 column (GE Healthcare).…”

Gelsolin amyloidosis: genetics, biochemistry, pathology and possible strategies for therapeutic intervention

“…These sites have the highest affinity for calcium. Calcium binding at these sites breaks the G2–G6 interface and also straightens the C-terminal helical latch in G6 (Figure 2B) (Choe et al, 2002, Wang et al, 2009). A series of conformational rearrangements then occurs: A continuous β-sheet between G1 and G3 is ruptured (cf.…”