Probing the Conformational States of the SH1−SH2 Helix in Myosin: A Cross-Linking Approach

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Abstract
Previous biochemical studies have shown that the SH1 (Cys707) and SH2 (Cys697) groups on rabbit skeletal myosin subfragment 1 (S1) can be cross-linked by using reagents of different cross-linking lengths. In the presence of nucleotide, this cross-linking is accelerated. In the crystal structure of S1, the SH1 and SH2 residues are located on an α-helix, 19 Å apart. Thus, the cross-linking results could be indicative of helix melting or increased flexibility in the presence of nucleotides. Nucleotide-induced changes in this region were examined in this study by monitoring the cross-linking of SH1 and SH2 on S1 with dimaleimide reagents of spans ranging from 5 to 15 Å. A method was devised to directly measure the kinetic effects of nucleotides on the rates of cross-linking reactions. The slow and reagent-insensitive rates of the SH1−SH2 cross-linking in the absence of nucleotides reveal that the equipartitioning of the SH1−SH2 helix among states with different SH1−SH2 separations occurs infrequently. In the presence of MgADP, MgATP, and MgATPγS, the rates of SH1 and SH2 cross-linking were increased ∼2−7-fold for the shortest reagent (5−8 Å). Rate accelerations were much greater for the longer reagents (9−15 Å): 40−50-fold for MgADP, 25−40-fold for MgATP, and 80−270-fold for MgATPγS. To account for any nucleotide-dependent differences in the reactivities of the reagents toward SH2, the rates of monofunctional SH2 modification on SH1-labeled S1 were also measured for each reagent. These experiments showed that the nucleotide-induced increases in the rates of SH2 modification were similar for all of the reagents. Thus, the changes observed in the cross-linking rates are due not only to the type of nucleotide bound in the active site but also to the span of the cross-linking reagent. These findings are interpreted in terms of nucleotide-induced shifts in the equilibria among conformational states of the SH1−SH2 helix.