Troponin regulatory function and dynamics revealed by H/D exchange-mass spectrometry

Abstract

Muscle contraction is tightly regulated by Ca(2+) binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca(2+) binding site in the NH(2) domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D(2)O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D(2)O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca(2+) binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca(2+)-saturated TnC NH(2) domain. More notably, Ca(2+) also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca(2+) sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.

FIGURE 1.

Proteolytic troponin fragments used to characterize H/D exchange in native troponin. Fragments indicated by solid lines were characterized kinetically in the presence of both high and low Ca²⁺ concentrations. Short and long dashed lines indicate, respectively, peptides measured only in the presence of high or low Ca²⁺.

FIGURE 2.

Kinetics of H/D exchange distant from the regulatory Ca²⁺ binding site. A, TnT 244–250, 251–263, and 243–263; B and C, TnI 97–109 and 97–116; D, TnI 117–124; E, TnI 54–66 and 54–78; F, TnI 62–85 and 78–88; G, TnC 154–161. Dashed lines indicate the increase in mass corresponding to full exchange for the indicated peptides. Solid lines are non-linear least square fit curves. The words high and low refer to alternate Ca²⁺ concentration conditions. In panels A and D–G both high Ca²⁺ data (filled symbols), and low Ca²⁺ data (open symbols) are shown. In A, exchange was modest regardless of condition, and the high and low Ca²⁺ data are merged. Panels B and C contrast high (B) and low (C) Ca²⁺ data for the same two peptides.

FIGURE 3.

Kinetics of H/D exchange within regions more proximal to the regulatory Ca²⁺ binding site. A, TnC 1–12; B, TnC 13–24; C (high Ca²⁺); and D (low Ca²⁺) - TnI 156–162 and 162–169; data; E, (high Ca²⁺) and F (low Ca²⁺) - TnI 125–134 and 125–152. High Ca²⁺, filled symbols. Low Ca²⁺, open symbols. Dashed and solid lines, and empty and filled symbols are as in Fig. 2.

FIGURE 4.

Primary structure mapping of native state troponin H/D exchange. Results indicate the measured degree of exchange rate slowing relative to that estimated (20) for the unfolded state, and are color coded on a logarithmic scale. Both high Ca²⁺ and low Ca²⁺ findings are shown. Violet regions are the least dynamic, most protected from exchange (generally not exchanging after 6 h). Reddish-brown regions are the most dynamic, evidencing no protection (exchange completed by 5 s). This indicates either weak or absent folding within native troponin. In the TnC NH₂ domain, the relative location of exchange transitions could not be determined within residues 14–24 and also within residues 26–57; the colors may be scrambled.

FIGURE 5.

Three-dimensional structure mapping of H/D exchange protection. A, high Ca²⁺ results from Fig. 4 are mapped onto the Ca²⁺-saturated structure of the cardiac troponin core domain. Gray indicates sections where no H/D exchange data were obtained. B, map of troponin regions with exchange rates altered by Ca²⁺ saturation of TnC site II. In high Ca²⁺ conditions relative to low Ca²⁺ conditions, blue indicates slower exchange, red indicates faster exchange, silver (light blue) indicates statistically insignificant alteration, and green indicates regions without data for comparison between high and low Ca²⁺. C, low Ca²⁺ results from Fig. 4 are mapped onto the proposed model of skeletal muscle troponin core domain under low Ca²⁺ conditions (Mg²⁺/EGTA) (5). The orientation in panel C is different from panels A and B, and only partial superposition is possible in any orientation. The indicated TnI segment is the inhibitory region, which is disordered so not shown in panels A and B.

FIGURE 6.

Illustration of the proposed effects of Ca²⁺ on troponin. Top, selected, indicated regions from the high resolution Ca²⁺-saturated skeletal muscle troponin structure. Bottom, same regions are shown within the proposed structure of Mg²⁺/EGTA skeletal muscle troponin, which was derived from intermediate resolution data in the same study (5). Cardiac TnI regions with Ca²⁺-sensitive H/D exchange rates are shown in hot pink, including the inhibitory region (stick representation), the switch helix, and much of the coiled-coil. A glycine at the coiled-coil terminus is shown in spherical representation, to indicate where the Ca²⁺-sensitive changes in the TnI inhibitory region commences in the proposed model. In high Ca²⁺, the cardiac TnI inhibitory region exhibits H/D exchange protection, as would be predicted from this skeletal muscle troponin structure, but not from the cardiac troponin x-ray structure (Fig. 5). Several changes occur when Ca²⁺ dissociates from the TnC NH₂ domain. The TnI switch helix dissociates and becomes disordered. The inhibitory region turns to tightly interact with the end of the coiled coil and changes its position greatly. Interactions with TnC helix H are altered. The stabilizing effects of the inhibitory region on the D/E helix are lost.