Cross-linking between the regulatory regions of troponin-I and troponin-C abolishes the inhibitory function of troponin.
We reported previously that both residues 48 and 82 on opposite sides of troponin-C's (TnC's) N-terminal regulatory hydrophobic cleft photo-cross-linked to Met121 of troponin-I (TnI) [Luo, Y., Leszyk, J., Qian, Y., Gergely, J., and Tao, T. (1999) Biochemistry 38, 6678-6688]. Here we report that the Ca2+-absent inhibitory activity of troponin (Tn) was progressively lost as the extent of photo-cross-linking increased. To extend these studies, we constructed a mutant TnI with a single cysteine at residue 121 (TnI121). In Tn complexes containing TnI121 and mutant TnCs with a single cysteine at positions 12, 48, 82, 98, or 125 (TnC12, TnC48 etc.), TnI121 formed disulfide cross-links primarily with TnC48 and TnC82 when Ca2+ was present, and with only TnC48 when Ca2+ was absent. These results indicate that TnI Met121 is situated within the N-domain hydrophobic cleft of TnC in the presence of Ca2+, and that it moves out of the cleft upon Ca2+ removal but remains within the vicinity of TnC. Activity assays revealed that the Met121 to Cys mutation in TnI121 reduced the Ca2+-present activation of Tn, indicating that Met121 is important in hydrophobic interactions between this TnI region and TnC's N-domain cleft. The formation of a disulfide cross-link between TnI121 and TnC48 or TnC82 abolished the Ca2+-absent inhibitory activity of Tn, indicating that the movement of the Met121 region of TnI out of TnC's N-domain cleft is essential for the occurrence of further events in the inhibitory process of skeletal muscle contraction. On the basis of these and other results, a simple mechanism for Ca2+ regulation of skeletal muscle contraction is presented and discussed.
Luo
Yin
Y
Muscle and Motility Group, Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Li
Bing
B
Yang
Guang
G
Gergely
John
J
Tao
Terence
T
D000818:Animals; D001388:Aziridines / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D003545:Cysteine / Q000235:genetics; D003619:Dansyl Compounds / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008715:Methionine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011817:Rabbits; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014466:Ultraviolet Rays
D001388:Aziridines; D003619:Dansyl Compounds; D004220:Disulfides; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D019209:Troponin C; D019210:Troponin I; C017173:dansylaziridine; D008715:Methionine; D003545:Cysteine; D002118:Calcium