Study of Hcm Causing β-Cardiac Myosin Mutations Located at Different Structurally Significant Regions of the Myosin-Head

Abstract

β-cardiac myosin dwells in an equilibrium between an actin-interacting ‘on’ state and an inactive folded-back ‘off’ state. During actin-interaction, myosin hydrolyzes ATP and undergoes conformational changes that move the thin filament with respect to the thick filament, resulting in cardiac contraction. ∼35% of heritable hypertrophic cardiomyopathy has been linked to β-cardiac myosin mutations. Although the myosin motor is highly allosteric, different regions of the motor serve distinct functions. As myosin heads interact with the actin, an ATP molecule gets hydrolyzed at the ATP-binding site (p-loop), and the converter domain and relay helix bring about required cyclic-conformational changes. Throughout the whole process the 7-stranded β-sheet at the core of myosin-head structure provides required rigidity to transfer force. Residues in the mesa and converter regions in the myosin-head, and residues in the proximal part of myosin-tail control the equilibrium between the ‘on’ and ‘off’ states. We plan to investigate how pathological β-cardiac myosin mutations — Y115H (β-sheet region), R169G (mesa region/near p-loop), E497D (relay helix), E536D (actin interacting/mesa region), and I702N (near converter domain) — alter function by performing ensemble and single molecule measurements. We prepared two new two-headed human β-cardiac constructs having different length of heptad units in the tail region for our test. One of the constructs has fifteen heptad repeats (15-hep) and is designed to support head-tail interaction in the “off” state, while the other construct has just eight heptads and is predicted to lack the head-tail interaction sites. Preliminary experiments show that the 15-hep, but not the 8-hep, construct showed a significant population in the super relaxed state by a single ATP turnover assay, consistent with only the 15-hep forming the off-state. Both construct show similar single molecule force measurements, as expected.