Comparative studies of the monomeric and filamentous actin-myosin head complexes.
The functional and structural properties of the monomeric and filamentous actin-myosin head (S1) complexes were compared under strictly controlled conditions which avoid the S1-induced polymerization of monomeric actin. Under these conditions, monomeric (G) and filamentous (F) actin were found to activate S1 Mg(2+)-ATPase by 3- and 120-fold, respectively, in the presence of a 5-fold excess of actin over S1. Using the change in fluorescence intensity of pyrene-G-actin induced by S1 binding in the presence of various nucleotide analogues, we discovered that the ternary G-actin-S1-AMPPNP complex could not be formed. Moreover, the association constants of G-actin to S1-ADP or of ADP to the G-actin-S1 complex were at least an order of magnitude lower than in the filamentous state. Such a low affinity between G-actin and the S1-nucleotide intermediates can reasonably explain the lack of ATPase activation by the monomeric complex. Analysis of the G-actin-S1 interface by chemical cross-linking and limited proteolytic experiments showed that, in the monomeric complex, S1 interacted almost exclusively by its positively charged segment 636-642 with the patch of negative residues located on the actin flexible loops 1-7, 20-28, and 90-100. Moreover, the variation in the cross-linking pattern and in the proteolytic susceptibility of S1 segment 636-642 demonstrated that this electrostatic interface was different in the monomeric and the filamentous complexes. Taken together, the results suggested that the G-actin-S1 interaction encompasses only a small fraction of the strong as well as of the weak F-actin-S1 interface. The monomeric complex would in fact resemble more the collision complex which takes place early in the F-actin-S1 interaction.
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Centre de Recherches de Biochimie Macromoléculaire, CNRS-UPR9008, INSERM-U249, Montpellier, France.
Chaussepied
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D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001704:Biopolymers; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D006868:Hydrolysis; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D001704:Biopolymers; D003432:Cross-Linking Reagents; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins