Nitao Lisa K, Loo Rachel R Ogorzalek, O'Neall-Hennessey Elizabeth, Loo Joseph A, Szent-Györgyi Andrew G, Reisler Emil
Department of Chemistry and Biochemistry and the Molecular Biology, Institute University of California, Los Angeles, California 90095, USA.
Biochemistry. 2003 Jul 1;42(25):7663-74. doi: 10.1021/bi027312u.
Atomic structures of scallop myosin subfragment 1(S1) with the bound MgADP, MgAMPPNP, and MgADP.BeF(x) provide crystallographic evidence for a destabilization of the helix containing reactive thiols SH1 (Cys703) and SH2 (Cys693). A destabilization of this helix was not observed in previous structures of S1 (from chicken skeletal, Dictyostelium discoideum, and smooth muscle myosins), including complexes for which solution experiments indicated such a destabilization. In this study, the factors that influence the SH1-SH2 helix in scallop S1 were examined using monofunctional and bifunctional thiol reagents. The rate of monofunctional labeling of scallop S1 was increased in the presence of MgADP and MgATPgammaS but was inhibited by MgADP.V(i) and actin. The resulting changes in ATPase activities of S1 were symptomatic of SH2 and not SH1 modification, which was confirmed by mass spectrometry analysis. With bifunctional reagents of various lengths, cross-linking did not occur on a short time scale in the absence of nucleotides. In the presence of MgADP, cross-linking was greatly enhanced for all of the reagents. These reactions, as well as the formation of a disulfide bond between SH1 and SH2, were much faster in scallop S1.ADP than in rabbit skeletal S1.ADP and were rate-limited by the initial attachment of the reagent to scallop S1. The cross-linking sites were mapped by mass spectrometry to SH1 and SH2. These results reveal isoform-specific differences in the conformation and dynamics of the SH1-SH2 helix, providing a possible explanation for destabilization of this helix in some scallop S1 but not in other S1 isoform structures.
结合MgADP、MgAMPPNP和MgADP·BeF(x)的扇贝肌球蛋白亚片段1（S1）的原子结构为包含反应性巯基SH1（半胱氨酸703）和SH2（半胱氨酸693）的螺旋结构不稳定提供了晶体学证据。在先前的S1结构（来自鸡骨骼肌、盘基网柄菌和平滑肌肌球蛋白）中未观察到该螺旋结构的不稳定，包括溶液实验表明存在这种不稳定的复合物。在本研究中，使用单功能和双功能巯基试剂研究了影响扇贝S1中SH1 - SH2螺旋的因素。在MgADP和MgATPγS存在下，扇贝S1的单功能标记速率增加，但受到MgADP·V(i)和肌动蛋白的抑制。S1的ATP酶活性的变化是SH2而非SH1修饰的症状，这通过质谱分析得到证实。对于各种长度的双功能试剂，在没有核苷酸的情况下，短时间内不会发生交联。在MgADP存在下，所有试剂的交联都大大增强。这些反应以及SH1和SH2之间二硫键的形成在扇贝S1·ADP中比在兔骨骼肌S1·ADP中快得多，并且受试剂与扇贝S1的初始结合限速。通过质谱将交联位点定位到SH1和SH2。这些结果揭示了SH1 - SH2螺旋在构象和动力学上的同工型特异性差异，为该螺旋在某些扇贝S1中不稳定而在其他S1同工型结构中稳定提供了可能的解释。
