(D-139) Evidence of a fourth actin-binding site on leiomodin-2

Introduction:: Leiomodin (Lmod) and tropomodulin (Tmod), actin-binding proteins found in striated muscle cells, regulate the length of actin thin filaments in the sarcomere¹. According to the sliding filament theory, the length of the actin filament is crucial for generating proper force for muscle contraction. Lmod2 and Tmod1, the cardiac isoforms, compete for binding at the pointed end of actin thin filaments to regulate their length¹. Tmod1 is localized at the pointed end and functions as a cap that prevents polymerization of the filament. Lmod2 is an actin nucleator and serves as a loose cap that allows for polymerization of the filament to continue¹. Lmod2 is found at the pointed end as well as along the length of the filament and has been shown to bundle actin filaments³. Lmod2 and Tmod1 are homologous in several regions, however, Lmod2 extends ~150 amino acid residues beyond the C-terminus of Tmod1². In this C-terminal extension exists an additional actin-binding WH2 domain that gives Lmod2 the ability to nucleate actin³. Previous cosedimentation experiments have shown that the C-terminal extension of Lmod2 is required for bundling of filaments^1,4, however, because the WH2 domain binds the barbed end of actin⁵, we suggest that there is an additional actin-binding site that gives Lmod2 it’s bundling ability.

To investigate the role of the C-terminal extension of Lmod2 and test whether the WH2 domain is responsible for bundling filaments, we performed cosedimentation assays using two truncated versions of Lmod2. Lmod2[1-524], missing the WH2 domain, and Lmod2[1-384], missing the entire C-terminal extension.


Materials and Methods:: Prior to cosedimentation experiments monomeric, or G-actin, was purified as in⁶. 3 µM G-actin was polymerized at room temperature for 1 hour in concentrated 20X polymerization buffer. Varying concentrations (1-5 µM) of Lmod2[1-524] or Lmod2[1-384], with or without 2 mM CaCl₂ were added and incubated at room temperature for 10 minutes. Final composition of the polymerization buffer in the mixtures was: 100 mM KCl, 2 mM MgCl₂, 2 mM EGTA, 1 mM DTT, 0.1 mM ATP, 25 mM imidazole, pH 8.0. The F-actin and Lmod mixtures were centrifuged for 30 minutes at 4,000 x g and 4°C. The supernatants were then centrifuged for 30 minutes at 213,483 x g and 4°C. The pellets from both centrifugation steps and the supernatants were analyzed using SDS-PAGE. Cosedimentation experiments were also repeated using full length Lmod2 and two other actin-binding proteins, tropomyosin (Tpm) and troponin (Tn), to investigate Lmod2’s bundling ability under physiological conditions.

Results, Conclusions, and Discussions:: Results and Discussion:

If bundling has occurred, at 4,000 x g bundles would sediment and individual filaments would remain in the supernatant. Analysis of pellets using SDS-PAGE revealed that Lmod2[1-524] maintains the ability to bundle actin filaments while Lmod2[1-384] does not. This result confirms that not the WH2 domain is responsible for Lmod2’s bundling ability, but rather an additional actin-binding domain in the C-terminal extension.



Conclusions:

In this study we demonstrated the presence of a fourth actin-binding domain between ABS2 and the WH2 domain of Lmod2 for the first time. The results of this study call for further investigation into the exact location and function of the fourth actin-binding site.


Acknowledgements (Optional): : These studies were supported by a grant from an NIH R01 GM120137. ML was supported by an NIH training grant T32 GM0008336. We thank Dr. Carol Gregorio’s lab at the University of Arizona for the preparation of Lmod2 and Lmod2 mutants.


References (Optional): : 1. Tolkatchev et. al (2022). FEBS J, 289(20):6119–6131. 2. Chereau et. al (2008). Science, 320(5873): 239-243. 3. Ly et. al (2019). Molecular Biology of the Cell, 30(2):268-281. 4. Skwarek-Maruszewska et. al (2010). Molecular Biology of the Cell, 21(19):3352-3361. 5. Fowler VM, Dominguez R. (2017). Biophys J, 112(9):1742-1760 6. Smith et. al (2022). Protein Science, 31(7):e4358.