title: Effect of ATP analogues on the actin-myosin interface.
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 ATPgammaS at 20 degreesC, 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.
authors.affiliation: CRBM du CNRS, IFR24, Montpellier, France.
authors.forename: J
authors.initials: J
authors.lastname: Van Dijk
authors.forename: C
authors.initials: C
authors.lastname: Fernandez
authors.forename: P
authors.initials: P
authors.lastname: Chaussepied
mesh_terms: D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism; D006868:Hydrolysis; D015879:Myosin Subfragments / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012965:Sodium Chloride / Q000494:pharmacology; D013388:Succinimides / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D013388:Succinimides; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D012965:Sodium Chloride; C035982:beryllium fluoride; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D000255:Adenosine Triphosphate; D001608:Beryllium; D005459:Fluorides; D005022:Ethyldimethylaminopropyl Carbodiimide