Structural and Functional Consequences of the Cardiac Troponin C L48Q Ca²⁺-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 Ca2+-sensitivity of the thin filament have been linked to disease. Tikunova and Davis have engineered a series of cNTnC mutations that altered Ca2+ binding properties and studied the effects on the Ca2+ sensitivity of the thin filament and contraction [Tikunova and Davis (2004) J Biol Chem279, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in cNTnC Ca2+ binding affinity and Ca2+ sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca2+ sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca2+ and cTnI to cTnC. NMR 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 Ca2+ sensitivity can do so by modulating Ca2+ and cTnI binding.

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation

“…I-9) at Cys-84 (cTnC IANBD C35S ) in the dark overnight at 4°C to monitor the Ca 2ϩ -cTn (K Ca ) and cTnC-cTnI (K C-I ) binding affinities, as described previously (7, 34 -36). The labeling efficiency was determined by measuring the IANBD fluorophore to protein molar concentration ratio (7,36). The concentration of protein was determined using Bio-Rad protein assay (based on Bradford method), and the IANBD concentration in the labeled protein was determined by dividing the absorbance of the labeled protein at the maximal absorbance for the fluorophore by the extinction coefficient of IANBD (21000…”

“…Whole cTn complexes were formed using rat cTnC (WT or cTnC IANBD C35S ), rat cTnI (WT, WT-Ser(P)-23/Ser(P)-24, WT-S23D/S24D, R146G, R146G/Ser(P)-23/Ser(P)-24, R21C, or R21C/S23D/S24D), and rat cTnT (WT) at a 1:1:1 molar ratio and then dialyzed through a series of buffers with gradually decreased KCl concentration at 4°C (without stirring) as described previously (38,39 fluorescence experiments were measured using an LS50B luminescence spectrometer (PerkinElmer Life Sciences) at 15°C as described previously (7,36,40 (43). After this, both ventricles were cut open, and the whole heart was demembranated in skinning solution containing (in mM): 100 KCl, 9 MgCl 2 , 4 Na 2 ATP, 5 K 2 EGTA, 10 MOPS, 1% Triton X-100, pH 7.0, 50% v/v glycerol, and 1:100 dilution "protease inhibitor mixture" (Sigma, catalog no.…”

“…Steady-state Fluorescence Measurements of K C-I and K CaThe effects of R146G or R21C mutation Ϯ PKA phosphorylation (or bis-phosphomimic substitutions) on cTn K Ca and the binding affinity of cTnC for cTnI (K C-I ) were determined by steady-state fluorescence measurements using a fluoroprobe IANBD, as described previously (7,36). IANBD, a sulfhydrylreactive and environment-sensitive extrinsic fluorophore, has been widely used to study the intra-molecular interactions of proteins, and labeling at Cys-84 of cTnC C35S reflects conformational and environmental changes of NcTnC that arise from Ca 2ϩ binding and/or interaction with cTnI (7,35,36,40 In view of the "gatekeeper" role of cTnC-cTnI interaction in translating the Ca 2ϩ signal to myofilament proteins to initiate cardiac muscle contraction, we also tested how the cTnI R146G mutation Ϯ PKA phosphorylation affected K C-I .…”

“…During the sampling process, the coordinates were saved every 10 ps. The stability between site II Ca 2ϩ and its coordinating residues (Asp-65, Asp-67, Ser-69, Thr-71, Asp-73, and Glu-76) of cTnC was monitored by calculating the following distances for each 150-ns simulation as described previously (8,36). Simulations were run in triplicate.…”

“…Contacts between two residues were defined as described previously (36), with a carbon-carbon distance of Յ5.4 Å and a distance between any other non-carbon atoms of Յ4.6 Å being a contact. Contacts between NcTnC-switch peptide of cTnI, and cTnC-inhibitory peptide of cTnI were monitored.…”