Chaussepied, P., Van dijk, J. & Fernandez, C.
CRBM du CNRS, B.P.5051, 34090 Montpellier Cedex, FRANCE. France
The electrostatic components of the Acto-myosin interface were proposed to be predominant during the early steps of the formation of the acto-myosin complex and when the nucleotide analogues ATP and ADP.Pi were present in the myosin active site, i.e. when the acto-myosin complex was in the so-called weak binding state (see for review Geeves & Conibear, 1995, Biophys.J. 68, 194s). These interactions take place between two positively charged loop structures of the myosin head (residues G635-G643 and K567-A575 of the skeletal myosin isoform) and the negatively charged residues located on the surface of actin subdomain 1 (residues E1-E4,D24-D25,E93, and E99-E100) (Shroder et al., 1993, Nature 364, 171). We took benefit of the high reactivity of these residues to the chemical cross-linking reagents to study their contact during the ATP hydrolysis cycle using various nucleotide analogues. The pattern of the cross-linking reaction induced by 1-ethyl-3(3-(dimethylamino)-propyl(-carbodiimide was sensitive to the nucleotide present in the acto-myosin head complex. In the absence of nucleotide or in the presence of ADP, four cross-linked products of apparent MWs of 165, 175, 200 and 265kDa were obtained while with the analogues ADP.BeFx, ADP.AlF4 and ADP.VO4 the 265kDa band was almost exclusively produced. When ATP was added to the acto-myosin head complex the major cross-linked product was the 200kDa band. Time course analysis of these cross-linking reactions together with the estimation of the amount of complex formed in all the conditions revealed that the differences observed were more likely due to changes in the acto-myosin interface. Moreover identical results were obtained at low and at 90mM ionic strength. The 165 and 175kDa band are composed of one myosin head cross-linked to the stretch of residues 1-4 of one actin monomer and the 265kDa band contain one S1 bound to the N-terminus of two adjacent actin monomers (Bonafe & Chaussepied, 1995, Biophys.J. 68, 35s). Only the nature of the 200kDa product is still of unidentified though it was found to be composed of one actin bound to one myosin head (Combeau et al., 1992, J.Biol.Chem. 267, 14038). These results revealed that in the presence of the ADP.Pi analogues, the myosin head interacts with an apparent equal affinity with the N-terminus segment of two actin monomers while in the rigor like states (with ADP or no nucleotide present) the myosin head interacts mainly with the N-terminus of one actin monomer. Based on this conclusion and together with the data already published, we will propose a new model for the formation of the different acto-myosin contacts during the mechano-chemical cycle.