Mechanism of actin filament pointed-end capping by tropomodulin

Abstract

Proteins that cap the ends of the actin filament are essential regulators of cytoskeleton dynamics. While several proteins cap the rapidly-growing barbed end, tropomodulin (Tmod) is the only protein known to cap the slowly-growing pointed end. The lack of structural information severely limits our understanding of Tmod’s capping mechanism. We describe crystal structures of actin complexes with the unstructured N-terminal and the leucine-rich repeat C-terminal domains of Tmod. The structures and biochemical analysis of structure-inspired mutants showed that one Tmod molecule interacts with three actin subunits at the pointed end, while also contacting two tropomyosin molecules on each side of the filament. We found Tmod achieves high affinity binding through several discrete low-affinity interactions, which suggests a mechanism for controlled subunit exchange at the pointed end.

Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

“…Similar to the modeled actin-Tmod1 structure (Fig. S2C) (29), actin(A-h) and actin(LRR) of the modeled actin-Lmod2 1-495 structure are expected to locate at subunit n+1 and n, respectively, at the pointed end of actin filament. However, this location leads to a partial overlap of actin(W) with actin subunit n−1 (Fig.…”

“…It binds to the cleft between subdomains 1 and 3 in an opposite direction from the W domain. This binding mode also is seen for other actinbinding proteins including the α-helix at residues 121-139 of gelsolin (29). Located between the LRR domain and the h1 helix is the shortened polyP region in which a stretch of proline residues extends along the surface of the actin(LRR) domain ( Fig.…”

“…Indeed, the actin(LRR)-LRR complex in the modeled actin-Tmod1 structure ( Fig. S2C) (29) was readily superimposable to that in our actin-Lmod2 162-495 (B-GS) structure (Fig. 1C), with rmsds of 1.2 Å and 0.9 Å for actin(LRR) and LRR, respectively (Fig.…”

“…Structural modeling suggested that the Tmod1 A-h and LRR respectively bind to the first (subunit n+1) and second (subunit n) actin protomer at the pointed end of the filament (Fig. S2C) (29). Given that (i) the first ∼340 residues of Lmod2 1-495 share a high (∼45%) sequence identity with human Tmod1; (ii) in the absence of the W domain, Lmod2 behaved just as did Tmod1 (16,20); and (iii) similar to Tmod1, Lmod2 is capable of binding to the pointed end of a preformed actin filament (20), we expect that Lmod2 1-340 binds to subunits n+1 and n in a conformation similar to that of human Tmod1.…”

“…Recently, the crystal structures of actin with human Tmod1 A-h and Tmod1 LRR were determined separately (29). Structural modeling suggested that the Tmod1 A-h and LRR respectively bind to the first (subunit n+1) and second (subunit n) actin protomer at the pointed end of the filament (Fig.…”