Opening of the Myosin Nucleotide Triphosphate Binding Domain during the ATPase Cycle

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Abstract
A series of ATP analogs, in which moieties of various sizes have been added to the γ-phosphorus of ATP, bind to the active site of myosin and to the actomyosin complex in myofibrils and in chemically skinned fibers. The affinity of the analogs for the active site shows only a slight dependence on the size of the added moiety. Addition of even our smallest group (CH3) reduced the binding affinity of ATPγ-CH3 for S1 to 40 μM, a factor of 105 less than observed for ATP. Computer molecular docking of ATP-γCH3 into the myosin−ADP·BeF3 crystal structure of Dictyostelium discoideum indicates no steric interference to prevent binding. This suggests that the maintenance of charge at the γ-phosphate is crucial for tight nucleotide binding. Addition of larger groups, (1) an EPR probe (ATP-γSL) or (2) ADP (i.e., P1,P5-diadenosine pentaphosphate, AP5A), reduced the affinity by only approximately a factor of 10 over that of ATP-γCH3. In the crystal structure of S1 complexed with nucleotides, the phosphates are buried within a protein structure called “the phosphate tube”. Both the bulk of the modifying groups and the lack of dependence on the size of the group are incompatible with threading of the phosphates down the Pi-tube, showing that the tube must open. Similar domain movements have been found in other proteins including members of the G-protein superfamily, a family that has structural homologies to myosin.