Mechanochemical consequences of myopathy‐linked mutations in Tpm2.2 on striated muscle contractility

Abstract

AbstractTropomyosin (Tpm) is an actin‐binding protein central to muscle contraction regulation. The Tpm sequence consists of periodic repeats corresponding to seven actin‐binding sites, further divided in two functionally distinct halves. To clarify the importance of the first and second halves of the actin‐binding periods in regulating the interaction of myosin with actin, we introduced hypercontractile mutations D20H, E181K located in the N‐terminal halves of periods 1 and 5 and hypocontractile mutations E41K, N202K located in the C‐terminal halves of periods 1 and 5 of the skeletal muscle Tpm isoform Tpm2.2. Wild‐type and mutant Tpms displayed similar actin‐binding properties, however, as revealed by FRET experiments, the hypercontractile mutations affected the binding geometry and orientation of Tpm2.2 on actin, causing a stimulation of myosin motor performance. Contrary, the hypocontractile mutations led to an inhibition of both, actin activation of the myosin ATPase and motor activity, that was more pronounced than with wild‐type Tpm2.2. Single ATP turnover kinetic experiments indicate that the introduced mutations have opposite effects on product release kinetics. While the hypercontractile Tpm2.2 mutants accelerated product release, the hypocontractile mutants decelerated product release from myosin, thus having either an activating or inhibitory influence on myosin motor performance, which agrees with the muscle disease phenotypes caused by these mutations.