Abstract
Ideas on mechano-chemical transduction by myosin have matured greatly through crystallography and are ripe for testing in vivo. One approach for doing so is termed reversion analysis, in which pairs of compensating mutations are identified. Suppression of one missense mutation by another reveals an interaction at the amino acid level that may be direct or indirect, long-lived or fleeting, but nonetheless physiologically relevant. Reversion analysis thus can test current ideas and, importantly, uncover interactions underlying dynamic or strain-dependent myosin states likely not represented in crystals. We used random mutagenesis to induce suppressors of Caenorhabditis elegans myosin/UNC-54 mutation E524K (=E500 of chicken myosin V), located on helix HQ at the predicted actin-binding region.Worms with E524K alone display disorganized A-bands and have a paralysis that worsens with increasing temperature. Thermodynamically, the heat-sensitivity suggests loss of a salt-bridge. The comparable residue in other myosins forms in the rigor-like and post-rigor crystallographic structures, but not in the pre-powerstroke one, a salt-bridge with a lysine on the relay helix (K460 of myosin V; =K483 of UNC-54). Thus, in the paralyzed worms, electrostatic repulsion between E524K and K483 potentially destabilizes interactions between helix HQ and the relay helix, thereby hindering the powerstroke.Twenty independent lines of suppressed worms were recovered from a screen of 106 mutagenized haploid genomes, and the suppressors mapped to seven residues: near the P-loop, V187I; in the actin-binding domain, E524K to E/T, L547F, A548V, and M579I/L/V; on the SH1-helix, C712Y; and in the converter domain, D724N. Consistent with the crystallographic structures of myosin, the suppressors can be interpreted as diminishing unfavorable interaction between E524K and K483, thus permitting the relay helix to reorganize properly as myosin progresses through the crossbridge cycle.
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