Localization of the binding interface between leiomodin-2 and α-tropomyosin

Abstract

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7–41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

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

“…However, biochemical and cellular studies have shown that the interaction of Lmods with TM through TMBS1 enhances slightly Lmods' nucleation activity (Fig. 2 F), and is also critical for proper localization in muscle sarcomeres (3,6,40,41,43). On the other hand, the region structurally visualized as ABS1 in Tmod (human Tmod1 residues P58-K99) is only partially conserved in Lmods (Fig.…”

“…Studies using NMR and CD spectroscopy suggest that the intrinsically disordered TMBSs adopt a helical conformation upon binding to the N-terminal $35 aa of the TM coiled coil (32), substituting for end-to-end interactions of TM molecules along the length of the actin filament (34,35). ABS2 is contained within the C-terminal region (residues $160 onward), which has a globular fold consisting mostly of a leucine-rich repeat (LRR) domain (36 very weakly compared to Lmods (3,6,16 (3,6,(40)(41)(42)(43), but lack the conserved TMBS2 of Tmods, with the corresponding region varying widely among Lmod isoforms, and displaying an abundance of negatively charged amino acids (Glu and Asp). Lmod1, in particular, presents a large insertion of >200-aa within this region, which curiously may contain a divergent TMBS, because a fragment of Lmod1 missing the first 30 aa still binds TM (26).…”

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

“…The construct for αTM1a 1-21 Zip was prepared as described previously [9]. The αTM1a 1-21 Zip peptide contained the 21 N-terminal residues of the striated muscle α-Tpm (Tpm1.1) and 18 residues of the leucine zipper domain of the yeast transcription factor GCN4 [28] to stabilize a coiled-coil structure in solution [29].…”

“…The construct for the αTM1a 1-21 Zip[K15N] peptide was prepared by site-directed mutagenesis [9]. The construct for rat striated muscle α-Tpm (Tpm1.1) was a generous gift from Dr. Sarah Hitchcock-DeGregori (Rutgers University, Piscataway, NJ).…”

“…It was previously shown that the K15N mutation in striated muscle a-Tpm (Tpm1.1) decreased its affinity for the Tpm-binding site of Lmod2 and the first Tpm-binding site of Tmod1 [9]. Tpm1.1 is a coiled-coil dimeric protein that binds along thin filaments in cardiac muscle cells (for review see [10]).…”