Molecular Mechanism of Actin Filament Nucleation by Leiomodin (Lmod)

Abstract

Leiomodin (Lmod) is related to tropomodulin (Tmod), and the two proteins have similar localization in striated muscle, i.e. near the M-line, in association with the pointed end of the actin filaments. However, in vitro the two proteins have fundamentally different activities: Lmod is a powerful actin filament nucleator, whereas Tmod caps the pointed end. Originally, it was assumed that the different domain organizations of the two proteins explained their different activities. Thus, Tmod has two tropomyosin (TM)- and two actin-binding sites, organized as: TMBS1-ABS1-TMBS2-ABS2. Lmod is longer, featuring a C-terminal extension that contains a Pro-rich region and a WH2 domain, which constitutes a third actin-binding site. The presence of the WH2 was considered to be the major feature distinguishing Lmod from Tmod. Here we show that this is not the case. Among the main findings are: 1) Lmod not only lacks TMBS2, but also ABS1, such that the entire region N-terminal to ABS2 has very little effect on nucleation, 2) The C-terminal extension of Lmod has also a limited effect on nucleation, and adding it to Tmod produces a very modest increase in nucleation, 3) Despite being relatively well conserved, the major feature distinguishing Lmod from Tmod is ABS2, consistent mostly of a Leu-rich repeat domain. Structural analysis shows that ABS2 can bind up to 3 actin subunits, and subtle differences between Lmod and Tmod dictate the affinities of their interactions with actin, and thus their roles in nucleation vs. capping. Understanding these differences allowed us to engineer an ABS2 Tmod-Lmod hybrid with nucleation activity equal to that of full-length Lmod.