title: Structural and functional consequences of the cardiac troponin C L48Q Ca(2+)-sensitizing mutation.
abstract: Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca(2+) sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca(2+) binding properties and studied the effects on the Ca(2+) sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341-35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca(2+) binding affinity and Ca(2+) sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca(2+) sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca(2+) and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca(2+) sensitivity can do so by modulating Ca(2+) and cTnI binding.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Ian M
authors.initials: IM
authors.lastname: Robertson
authors.forename: Monica X
authors.initials: MX
authors.lastname: Li
authors.forename: Michelle E
authors.initials: ME
authors.lastname: McCully
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Crane
authors.forename: Zhaoxiong
authors.initials: Z
authors.lastname: Luo
authors.forename: An-Yue
authors.initials: AY
authors.lastname: Tu
authors.forename: Valerie
authors.initials: V
authors.lastname: Daggett
authors.forename: Brian D
authors.initials: BD
authors.lastname: Sykes
authors.forename: Michael
authors.initials: M
authors.lastname: Regnier
mesh_terms: D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D006801:Humans; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D019209:Troponin C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism
chemical_list: D000577:Amides; D019209:Troponin C; D019210:Troponin I; D002118:Calcium