EXTENSIVELY METHYLATED MYOSIN SUBFRAGMENT-1 - EXAMINATION OF LOCAL-STRUCTURE, INTERACTIONS WITH NUCLEOTIDES AND ACTIN, AND LIGAND-INDUCED CONFORMATIONAL-CHANGES

The atomic structure of myosin subfragment-1 (S1) has been recently so lved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and n ear-ultraviolet CD spectra, the methylation does not affect the second ary structure of S1 but causes limited changes in its tertiary structu re. The methylation significantly decreases the affinity of S1 for act in in rigor and, to a lesser degree, that of S1 to actin in the presen ce of MgATP gamma S. This modification, like the trinitrophenylation o f Lys-83, accelerates the dissociation of a nucleotide trapped on S1 e ither by phosphate analogs or by cross-linking of the SH1 and SH2 thio ls. Methylation strongly impairs the coupling between the actin- and n ucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a comp lete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication betwe en other sites on S1 including that between the nucleotide-binding sit e and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent pho tocleavage experiments, respectively. The increased rate of thermal de naturation of S1 and the loss of S1 protection by ADP and actin from t his process also indicate flawed communications in methylated S1. It i s concluded that these relatively mild but numerous and important chan ges impair the function of methylated S1.