Structural and functional significance of aspartic acid 89 of the troponin C central helix in Ca2+ signaling

Sundaram Ramakrishnan, Sarah E. Hitchcock-DeGregori

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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 α-helix (αh89A) consisting of 87AEAALKAAMEA97; and (3) A89 of αh89A was replaced with D (αh89D). D89A and αh89A 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 αh89D had higher activity (113 ± 3%). Both αh89A and D89A had apparently normal interactions with TnI and TnT whereas αh89D 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 αh89A 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 Ca2+-dependent binding to TnI or TnT. The results suggest that D89 is required for Ca2+-dependent signal transduction, an event that can be dissociated from Ca2+-dependent binding to TnC targets on the thin filament.

Original languageEnglish (US)
Pages (from-to)15515-15521
Number of pages7
JournalBiochemistry
Volume35
Issue number48
DOIs
StatePublished - Dec 17 1996
Externally publishedYes

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Troponin C
Myosins
Aspartic Acid
Signal transduction
Divalent Cations
Amino Acid Sequence
Signal Transduction
Chemical activation
Amino Acids
Mutation
polyproline

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural and functional significance of aspartic acid 89 of the troponin C central helix in Ca2+ signaling. / Ramakrishnan, Sundaram; Hitchcock-DeGregori, Sarah E.

In: Biochemistry, Vol. 35, No. 48, 17.12.1996, p. 15515-15521.

Research output: Contribution to journalArticle

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abstract = "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 α-helix (αh89A) consisting of 87AEAALKAAMEA97; and (3) A89 of αh89A was replaced with D (αh89D). D89A and αh89A 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 αh89D had higher activity (113 ± 3{\%}). Both αh89A and D89A had apparently normal interactions with TnI and TnT whereas αh89D 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 αh89A 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 Ca2+-dependent binding to TnI or TnT. The results suggest that D89 is required for Ca2+-dependent signal transduction, an event that can be dissociated from Ca2+-dependent binding to TnC targets on the thin filament.",
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AB - 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 α-helix (αh89A) consisting of 87AEAALKAAMEA97; and (3) A89 of αh89A was replaced with D (αh89D). D89A and αh89A 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 αh89D had higher activity (113 ± 3%). Both αh89A and D89A had apparently normal interactions with TnI and TnT whereas αh89D 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 αh89A 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 Ca2+-dependent binding to TnI or TnT. The results suggest that D89 is required for Ca2+-dependent signal transduction, an event that can be dissociated from Ca2+-dependent binding to TnC targets on the thin filament.

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