The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Katalin Ajtai,Susanna P Garamszegi,Shinya Watanabe,Mitsuo Ikebe,Thomas P Burghardt

doi:10.1074/jbc.m310775200

Katalin Ajtai, Susanna P Garamszegi + Show 3 more

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https://doi.org/10.1074/jbc.m310775200

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Abstract

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.

Highlights

The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work
Tryptic Digestion of the C-loop in ␤subfragment 1 (S1)—We showed previously that the tryptic digestion of skeletal S1 and ␤S1 splits the heavy chains at two points, Loops 1 and 2, producing the characteristic 25, 50, and 20-kDa fragments (32, 52) (Fig. 1)
Within unstructured surface loops involved in the rate of substrate release and in regulation of actin activated ATPase (17–19)

Summary

Introduction

The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Limited proteolysis of skeletal S1 cleaves the heavy chain at Loop 1 (aa 202– 217) and Loop 2, producing 25-, 50-, and 20-kDa molecular mass fragments (27). These loops are involved in the regulation of substrate release (Loop 1) and in actin binding and regulation of actin-activated ATPase (Loop 2) (19, 22, 28, 29). A New Functional Structured Surface Loop on Myosin that ␤S1 and skeletal S1 differ in conformation and identify the C-loop as a possible actin binding site. Each mutation caused distinctive changes in actoHMM properties supporting the finding from the proteolysis studies that the C-loop interacts functionally with actin

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