EFFECT OF ATP ANALOGS ON THE ACTIN-MYOSIN INTERFACE

The interaction between skeletal myosin subfragment 1 (S1) and filamen tous actin was examined at various intermediate states of the actomyos in ATPase cycle by chemical cross-linking experiments. Reaction of the actin-S1 complex with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimid e and N-hydroxysuccinimide generated products with molecular masses of 165 and 175 kDa, in which S1 loops of residues 626-647 and 567-578 we re 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 b ound to the N termini of two adjacent monomers. In strong-binding comp lexes, i.e., without nucleotide or with ADP, S1 was sequentially cross -linked to one and then to two actin monomers. In the weak-binding com plexes, two types of crosslinking pattern were observed. First, during steady-state hydrolysis of ATP or ATP gamma S at 20 degrees C, the cr oss-linking reaction gave rise to a small amount of unknown 200 kDa pr oduct. Second, in the presence of AMPPNP, ADP.BeFx, ADP.AlF4-, or ADP. VO43- or with S1 internally cross-linked by N,N'-p-phenylenedimaleimid e, only the 265 kDa product was obtained. The presence of 200 mM salt inhibited cross-linking reactions in both weak-and strong-binding stat es, while it dissociated only weak-binding complexes. These results in dicate that, in the weak-binding state populated with the ADP.P-i anal ogues, skeletal S1 interacts predominantly and with an apparent equal affinity with the N termini of two adjacent actin monomers, while thes e ionic contacts are much less significant in stabilizing the rigor ac tin-S1 complexes. They also suggest that the electrostatic actin-S1 in terface is not influenced by the type of ADP.P-i analogue bound to the active site.