Effect of ATP Analogues on the Actin−Myosin Interface

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Abstract
The interaction between skeletal myosin subfragment 1 (S1) and filamentous actin was examined at various intermediate states of the actomyosin ATPase cycle by chemical cross-linking experiments. Reaction of the actin−S1 complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide generated products with molecular masses of 165 and 175 kDa, in which S1 loops of residues 626−647 and 567−578 were cross-linked independently to the N-terminal segment of residues 1−12 of one actin monomer, and of 265 kDa, in which the two loops were bound to the N termini of two adjacent monomers. In strong-binding complexes, i.e., without nucleotide or with ADP, S1 was sequentially cross-linked to one and then to two actin monomers. In the weak-binding complexes, two types of cross-linking pattern were observed. First, during steady-state hydrolysis of ATP or ATPγS at 20 °C, the cross-linking reaction gave rise to a small amount of unknown 200 kDa product. Second, in the presence of AMPPNP, ADP·BeFx, ADP·AlF4-, or ADP·VO43- or with S1 internally cross-linked by N,N‘-p-phenylenedimaleimide, only the 265 kDa product was obtained. The presence of 200 mM salt inhibited cross-linking reactions in both weak- and strong-binding states, while it dissociated only weak-binding complexes. These results indicate that, in the weak-binding state populated with the ADP·Pi analogues, skeletal S1 interacts predominantly and with an apparent equal affinity with the N termini of two adjacent actin monomers, while these ionic contacts are much less significant in stabilizing the rigor actin−S1 complexes. They also suggest that the electrostatic actin−S1 interface is not influenced by the type of ADP·Pi analogue bound to the active site.