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 the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a gamma-phosphate sensor and moves almost 6 A relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the gamma-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 higher KMvalue 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.
Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, Heidelberg, D-69120, Germany.
M
M
Furch
S
S
Fujita-Becker
M A
MA
Geeves
K C
KC
Holmes
D J
DJ
Manstein
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004023:Dictyostelium / Q000737:chemistry*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins