Ca²⁺-induced PRE-NMR changes in the Troponin complex reveal the possessive nature of the cardiac isoform for Its regulatory switch

The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca²⁺) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca²⁺ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca²⁺-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca²⁺-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca²⁺ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of ¹H-¹⁵N-TROSY spectra of Ca²⁺-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (~10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca²⁺. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca²⁺ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.11 page(s