The cardiac Ca-sensitive regulatory switch, a system in dynamic equilibrium

TL;DR

This study develops a dynamic model of the cardiac Ca-sensitive regulatory switch, revealing the mechanistic steps of activation and deactivation, and characterizing the conformational states involved in cardiac muscle regulation.

Contribution

It introduces a new quantitative dynamic model of TnC-TnI allostery based on time-resolved FRET data, elucidating the mechanistic roles of calcium and TnI in cardiac regulation.

Findings

Ca-induced priming is the rate-limiting step in activation

Closing is the rate-limiting step in deactivation

Incomplete activation occurs when regulatory Ca is not bound

Abstract

The Ca-sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca, and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium FRET measurements suggest that the switch activates in two steps: a TnI-independent Ca-priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation following rapid addition of a lacking component (Ca or TnI) and deactivation following rapid chelation of Ca. The time-resolved measurements, stopped-flow measurements, and Ca-titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal (i) the Ca-induced priming step, which precedes opening, is the rate limiting step in activation, (ii) closing is the rate limiting step in deactivation, (iii) TnI induces opening, (iv) an incompletely deactivated population when regulatory Ca is not bound, which generates an accessory pathway of activation, and (v) incomplete activation by Ca--when regulatory Ca is bound, a 3:2 mixture of dynamically inter-converting open (active) and primed-closed (partially active) conformers is observed (15 C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermo-kinetic parametrization of opening. <Abstract Truncated>