The β-myosin G256E mutation impairs relaxation by altering transducer stiffness.

Abstract

Associations between mutations in sarcomeric proteins and familial cardiomyopathies are myriad. However, detailed structural and functional studies are required to describe the mechanistic relationship between a mutation and its pathology. To accelerate such studies, we utilize a combined computational and experimental platform to probe how mutations to b-myosin lead to familial cardiomyopathies. Here, we report results on the HCN-associated MYH7 mutation G256E, which is located in the myosin transducer. Experimentally, the specific force generation of mutant G256E myofibrils was ∼2x greater than isogenic control myofibrils. The activation rate was similarly increased. The initial slow phase relaxation kinetics of G256E myosin were significantly reduced relative to isogenic controls, indicating a slower cross-bridge detachment rate. Using stopped flow kinetics, the ATP binding rate was reduced in G256E relative to WT. Molecular dynamics simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations modeled the rigor (nucleotide free) to post-rigor (ATP-bound) structural transition along multiple reaction coordinates. The transition required that G256E myosins surmount a greater energetic barrier that describes the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Based on our combined computational and experimental results, we hypothesize that G256E leads to a hypercontractile and delayed relaxation phenotype via modulation of the transducer structure. This alters communication between the nucleotide binding pocket and the actin binding surface during steps in the chemo-mechanical cycle that involve transducer motion during nucleotide release-induced strong actin binding and ATP binding-induced release from actin. Other effects on myosin structure and its activation during contraction are being investigated.