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

Abstract

Leiomodin (Lmod) is a class of potent tandem-G-actin-binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of highresolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin-Lmod2 162-495 nucleus. The structure contains two actin subunits connected by one Lmod2 162-495 molecule in a non-filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin-binding nucleators.actin nucleation | nemaline myopathy | pointed-end elongation

Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles

“…The interaction is similar to that of other WH2 domains (60,61), with the WH2 domain of Lmod more closely resembling those of Cobl and Las17, in that it presents an extra helical turn in the loop between the N-terminal a-helix and the LKKV motif (62). The authors of the Lmod2-actin structure report an additional interaction with actin, involving the PRD and an adjacent a-helix in the linker between ABS2 and the WH2 domain (59). However, examination of the structure and deposited x-ray data reveals no reliable electron density for the region of Lmod2 between ABS2 and the WH2 domain, calling this claim into question.…”

“…4). These include the 1.5 A resolution crystal structure of ABS2 of Lmod1, two structures of a hybrid Tmod1-Lmod1 ABS2 construct crystallized alone (2.1 A resolution) and in complex with actin (2.4 A resolution) (6), and a 3.0 Å resolution structure of a complex of actin with a fragment of Lmod2 comprising from ABS2 to the C-terminal WH2 domain (59). The structures show that the highly conserved ABS2 of Lmods interacts with actin similarly to that of Tmods (5), with one important distinction; Lmods lack the DBS, which in Tmods is critical for pointed-end capping (Figs.…”

The WH2 Domain and Actin Nucleation: Necessary but Insufficient

“…Nucleation by Lmods is primarily the result of two major adaptations – the loss of pointed-end-capping elements present in Tmods, including ABS1, TMBS2, and the N-terminal portion of ABS2, and sequence variations within the otherwise highly conserved ABS2, allowing this domain in Lmod to recruit two or more actin subunits for nucleation ( Figure 4 ). Thus, similar to the other nucleators described here, the WH2 domain-containing tail of Lmod plays an auxiliary role [43], possibly by helping to recruit the third actin subunit of the polymerization nucleus [45]. ABS2 binds at the interface between three actin subunits in the filament [43, 44], including most likely the subunit bound to the WH2 domain, which explains why this domain plays the most important role in nucleation by Lmod.…”

The cardiomyopathy-associated K15N mutation in tropomyosin alters actin filament pointed end dynamics

“…Therefore, the K15N mutation in Tpm1.1 can be expected to have a greater effect in the Tmod1's binding at the pointed end compared with Lmod2, which has only one Tpm-binding site. Furthermore, Lmod2 contains an extended first actin-binding site compared with Tmod1 [7] and an additional third actin-binding site [17, 27]. Indeed, the K15N mutation in Tpm1.1 seemed to enhance Lmod2's ability to replace Tmod1 bound at the pointed end.…”

“…Lmod shares a similar domain arrangement, but it has only one Tpm-binding site [7, 25]. Additionally, Lmod has a ∼150 amino acid C-terminal extension that contains a proline-rich region and a third actin-binding site, which includes a Wiskott-Aldrich syndrome protein (WASP)-homology 2 (WH2) domain [17, 26, 27]. …”