标题：Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1997
卷号：94
期号：22
页码：11899-11904
DOI：10.1073/pnas.94.22.11899
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an in vitro motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaD{Delta}24-53 lacking the residues 24-53 to myosin compared with the level of binding of CaD{Delta}54-85 missing the adjacent residues 54-85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24-53 nearly abolished the ability of CaD to inhibit actin filament velocity in the in vitro motility experiments, whereas CaD{Delta}54-85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1-597, which lacks the major actin-binding site(s), did not inhibit actin-filament velocity despite the presence of the major myosin-binding site. These data provide direct evidence for the inhibition of actin filament velocity in the in vitro motility assay caused by the tethering of myosin to actin through binding of both the CaD N-terminal region to myosin and the C-terminal region to actin.
