Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1

Recent crystallographic studies have suggested structural differences between the complexes of S1 . Mg . ADP with the phosphate analogs aluminium fluoride (AlF4-), vanadate (VO43-) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404-5417]. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707-Cys697 (SH1-SH2) cross-linking, were used to compare the S1 . ADP . BeFx, S1 . ADP . AlF4- and S1 . ADP . VO43- complexes with specific states of the myosin-ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethyl has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20 degrees C lead to the conclusion that S1 . ADP . BeFx is similar to S1* . ATP, while S1 . ADP . AlF4- and S1 . ADP . VO43- are more similar to S1** . ADP . P-i. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1-N-6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (by N,N'-p-phenylene dimaleimide) S1 . ADP[C2H2] . AlF4- and S1 . ADP[C2H2] . BeFx. Our results suggest that the conformations of the S1 . ADP . AlF4-, S1 . ADP . VO43- and S1 . ADP . BeFx complexes in the Cys707-Cys697 region are distinct from each other, with the former two at least partially resembling the S1** . ADP . P-i state, while the latter is similar to the prehydrolyzed S1* . ATP state.