Structural and functional significance of aspartic acid 89 of the troponin C central helix in Ca2+ signaling.
The central helix of troponin C is highly conserved in length and amino acid sequence. In this region, D89 is conserved and specific to TnC. To investigate its significance, three mutations were made in avian fast troponin C: (1) D89 was replaced with A (D89A); (2) the central helix was replaced with a designed alpha-helix (alpha h89A) consisting of 87AEAALKAAMEA97; and (3) A89 of alpha h89A was replaced with D (alpha h89D). D89A and alpha h89A activated the regulated actomyosin ATPase poorly in the presence of Ca2+ (24 +/- 1.0% and 14 +/- 2.0%, respectively, of the wild type maximal activity) whereas alpha h89D had higher activity (113 +/- 3%). Both alpha h89A and D89A had apparently normal interactions with TnI and TnT whereas alpha h89D formed a complex with TnT even in the absence of Ca2+. The central helix was also replaced with a flexible random coil and rigid polyproline linkers in which D89 was Arg or Pro, respectively. Like alpha h89A and D89A, both mutants were defective in activation of the actomyosin ATPase in the presence of Ca2+. Changes in regulatory function of the mutants did not correlate with altered Ca2+ affinity, altered conformational changes upon binding divalent cations, or Ca(2+)-dependent binding to TnI or TnT. The results suggest that D89 is required for Ca(2+)-dependent signal transduction, an event that can be dissociated from Ca(2+)-dependent binding to TnC targets on the thin filament.
Ramakrishnan
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Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854, USA.
Hitchcock-DeGregori
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D000818:Animals; D001224:Aspartic Acid; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000378:metabolism; D011487:Protein Conformation; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014336:Troponin / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T
D015415:Biomarkers; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D001224:Aspartic Acid; D009218:Myosins; D002118:Calcium