Ca2+-Induced PRE-NMR Changes in the Troponin Complex Reveal the Possessive Nature of the Cardiac Isoform for Its Regulatory Switch

Abstract

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.

Order–Disorder Transitions in the Cardiac Troponin Complex

“…25; 29 In the closed state, the switch peptide adopts a helical conformation and binds the hydrophobic cleft of the TnC N , 23 moving approximately 10 Å from its blocked state position. 29 TnI N is also bound to TnC N , 25 and TnI C is bound to tropomyosin but likely in a different conformation. 30 The IR is likely not bound to the thin filament in the closed state.…”

Molecular Effects of cTnC DCM Mutations on Calcium Sensitivity and Myofilament Activation—An Integrated Multiscale Modeling Study

“…This increase in inter-calcium distance was caused by a straightening of the central cTnC helix, leading to an increased distance between NcTnC and CcTnC (see Figure 3B). Experimentally, it has been demonstrated that isolated cTnC takes on a more extended form than cTnC anchored in the cTn complex 38 . Similar trends are observed for the homologous calmodulin protein, for which the Ca 2+ -bound state assumes an elongated conformation that maximally-separates the N- and C-terminal globular domains in absence of a target protein, but subsequently collapses when a target protein is presented 39 .…”

Full Atom Simulations of Spin Label Conformations

“…Since the early 1970s label motion has been modeled in terms of “wobble within a cone” model (Berliner, 1976),(Borbat, McHaourab, & Freed, 2002) or as an excluded volume (Cordina et al, 2014). These non-atomistic models overestimated the label motion, erring on the conservative side.…”

“…The extent of that relaxation enhancement is distance dependent, thus if the label tilts towards one side of the backbone, we expect those residues to be affected more than the residues the spin label tilts away from. We have one case of a spin labeled protein, TnC, for which preNMR was performed using the MTSSL label attached to position 136 (Cordina et al, 2014). The C-terminal domain of TnC is fairly rigid, and we can assume that the backbone structure in the NMR experiments is the same as the structure observed in the crystals.…”

“…Comparison of the distances observed by paramagnetic relaxation enhancement NMR ( squares ) on spin labeled troponin C (site 136) from (Cordina et al, 2014) and the distances calculated for the most prevalent conformation of the label from SS simulations ( circles ). On the left , comparison of the same NMR distances with the distances for canonical rotamers mmmtt, pmptt, tmmtt .…”