Abstract
Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.
Highlights
Dissecting the molecular mechanism of muscle myosin function in vivo has proved difficult
Ile508 can be cross-linked to Arg759 in Dictyostelium non-muscle myosin II when they are each substituted by cysteine [23]
We previously demonstrated that converter mutation R759E disrupts biochemical, biophysical, mechanical, and ultrastructural properties of Drosophila muscle myosin and indirect flight muscles [20, 21]
Summary
Dissecting the molecular mechanism of muscle myosin function in vivo has proved difficult. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile508, Asn509, and Asp511) in communicating with converter domain residue Arg759. The details of myosin motor function have been examined through kinetics studies, comparison of crystal structures, molecular dynamics, and mutational analyses (for a review, see Ref. 1) It has been shown, for instance, that the relay domain of the molecule rearranges during the mechanochemical cycle, acting as a signaling pathway that connects the active site, the actin binding region, and the converter domain [2,3,4]. We previously showed that a mutation in converter residue Arg759 results in significantly reduced myosin ATPase activity and in vitro actin sliding velocity and eliminates the enhanced tryptophan fluorescence indicative of relay loop reconfiguration and lever arm swing [20]. Defining the molecular basis of relay-converter domain interaction in muscle provides insight into the mechanism of the myosin motor and its role in myofibril contraction and stability
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