Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin

Motifs N2 and N3, also referred to as switch-1 and switch-2 form part of th e active site of molecular motors such as myosins and kinesins. Ln the case of myosin, N3 is thought to act as a gamma-phosphate sensor and moves almo st 6 Angstrom relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the gamma-phosphate. The closed for m seems to be necessary for hydrolysis and is stabilised by the formation o f a salt-bridge between an arginine residue in N2 and a glutamate residue i n 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 sal t-bridge in double-mutant M765-IS eliminates most of the defects observed f or the single mutants. With the exception of a 2,500-fold higher K-M value for ATP, the double-mutant displayed enzymatic and functional properties ve ry similar to those of the wild-type protein. Our results reveal that, inde pendent of its orientation, the salt-bridge is required to support efficien t ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function. (C) 1999 Academic Press.