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Abstract

Motifs N2 and N3, also referred to as switch-1 and switch-2, form part of the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a γ-phosphate sensor and moves almost 6 Å relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the γ-phosphate. The closed form seems to be necessary for hydrolysis and is stabilised by the formation of a salt-bridge between an arginine residue in N2 and a glutamate residue in N3. We examined the role of this salt-bridge in Dictyostelium discoideum myosin. Myosin motor domains with mutations E459R or R238E, that block salt-bridge formation, show defects in nucleotide-binding, reduced rates of ATP hydrolysis and a tenfold reduction in actin affinity. Inversion of the salt-bridge in double-mutant M765-IS eliminates most of the defects observed for the single mutants. With the exception of a 2,500-fold higherKM value for ATP, the double-mutant displayed enzymatic and functional properties very similar to those of the wild-type protein. Our results reveal that, independent of its orientation, the salt-bridge is required to support efficient ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function