Leiomodin creates a leaky cap at the pointed end of actin-thin filaments

Abstract

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments

“…However, when 19 N‐terminal residues in Tpm1‐1b(1‐19)Zip were replaced with 26 N‐terminal residues in Tpm1‐1b(1‐26)Zip, we detected changes in Tmod NMR spectra. A similar effect was observed when we studied complexes formed between Lmod TpmBS1 and Tpm1.1 model peptides 40,41 . Although we demonstrated that the major Lmod/Tpm interaction occurs within the first 14 residues of Tpm, additional 7 residues included in the model Tpm peptide led to an NMR spectrum change and a modest, two‐fold increase in binding affinity 41 .…”

“…To validate these results, we also predicted the secondary structure of Lmod2‐TpmBS1, a homologous TpmBS of another member of the Tmod family of proteins, leiomodin 2 (Lmod2). We compared the prediction with the secondary structure of Lmod2‐TpmBS1 in complex with the N‐terminus of Tpm1.1, which we have recently determined by NMR 40 (PDB ID 6UT2).…”

“…(a) A “frontal” view of the actin filament with Tmod2‐TpmBS1 shown in green on the foreground and bound to a Tpm protomer (orange), and (b) a “back” view of the actin filament showing Tmod2‐TpmBS2 (cyan) bound to a Tpm protomer on the opposite side from Tmod2‐TpmBS1. The pointed end of the actin filament was modeled as before 40 using an atomic model for the Tpm cable with F‐actin 44 . Actin‐binding sites of Tmod2 actin‐binding site 1 (ABS1) and actin‐binding site 2 (ABS2) were modeled from the crystal structures of the homologous Tmod1 ABS/actin complexes 45 (red and magenta, respectively).…”

“…Very recently, by using NMR and NMR‐guided MDS, we gained a new insight into the mode of interaction of Tpm with TpmBS1 of Lmod (PDB ID 6UT2 40 ). Here, we used this structure as a starting point to create 3D models of complexes of Tpm with Tmod2‐TpmBS1 and with Tmod2‐TpmBS2.…”

“…Both models represent a four‐helix bundle, with a Tpm‐binding site of Tmod forming a α‐helical hairpin positioned over the N‐terminus of the Tpm coiled coil. Similarly to the complex of Tpm with TpmBS1 of Lmod, 40 the complexes of Tpm with Tmod2‐TpmBS1 and with Tmod2‐TpmBS2 mimic the structure of the Tpm head‐to‐tail overlap complex, which was also shown to represent a crisscross arrangement of four α‐helices 29,52,53 . Residues forming the Tmod/Tpm binding interface are conservative between TpmBS1 and TpmBS2 from different Tmod homologs (Figure S1), with some rare exceptions that can potentially be used for isoform‐specific tuning of binding proposed as a basis for tissue‐specific regulation of pointed end capping 18 …”