Biophysical Society Thematic Meeting | Canterbury 2023

Towards a More Perfect Union: Multi-Scale Models of Muscle and Their Experimental Validation

Poster Abstracts

5-POS Board 5 HYPERTROPHIC CARDIOMYOPATHY MUTATIONS IN CARDIAC MYOSIN BINDING PROTEIN C N-TERMINAL DOMAINS CAUSE LOCAL AND ALLOSTERIC EFFECTS ON PROTEIN MOBILITY AND INCREASE ACTIN BINDING Rhye-Samuel Kanassatega 1 ; Fiona L Wong 1 ; Thomas A Bunch 1 ; L. Michel Espinoza Fonseca 2 ; Brett A Colson 1 ; 1 University of Arizona, Cellular & Molecular Medicine, Tucson, AZ, USA 2 University of Michigan, Internal Medicine, Ann Arbor, MI, USA Mutations in the gene encoding cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). HCM affects more than 1 in 500 individuals and is a leading cause of death in young individuals. However, the mechanisms leading to cardiac dysfunction in HCM remain unclear and therapies are limited. We have used molecular dynamics (MD) simulations and biochemical and biophysical approaches to gain insight into the molecular mechanisms by which mutations in cMyBP-C cause HCM disease. cMyBP-C N terminal domains C0 through C2 (C0-C2) are considered to be the “business end” of the molecule as they contain regions for binding actin and myosin and phosphorylation, which are important for cMyBP-C’s critical roles in normal cardiac function. We selected three HCM causing mutations in C0-C2 expected to be pathogenic according to the SHaRe Registry and NIH ClinVar databases to investigate: P161S and Y237S in the hydrophobic core of domain C1 and surface-exposed P371R in domain C2. Using a fluorescence lifetime-based actin-binding assay, we determined that all 3 HCM mutations increased in vitro binding affinity for actin in both unphosphorylated and phosphorylated C0-C2. We also found that the root mean square fluctuation (RMSF) values used to measure the in silico mobility of the protein in the trajectories (a total of 4 ms each) at either 25 or >50 o C induced moderate local structural changes in P161S and P371R and allosteric structural changes in Y237S as compared to immunoglobulin-like wild type C1 or C2 domain structures. In addition, mutations reduced in vitro protein folding stability of C0-C2, C1, and C2 relative to wild type, as expected, using differential scanning calorimetry (DSC) and protein solubility assays. These in vitro and in silico results suggest that structural changes in mutant cMyBP-C domains can cause altered myofilament binding, and thereby lead to dysfunction and HCM disease.

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