David C. Corson

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of human cardiac troponin C

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090±86 Å2 for apo-cNTnC to 3108±71 Å2 for Ca2+-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca2+-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.

The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C.

Significance Protein–protein interactions typically involve some degree of induced fit, producing complementary surfaces that account for high affinity and specificity. However, there are increasingly more examples of intrinsically disordered regions (IDRs) that exert important biologic effects despite never attaining a rigid structure. Here we show how a particularly disordered region of cardiac troponin I impacts the overall global conformation and function of its binding partner, cardiac troponin C. This newly described role for an IDR is accomplished through electrostatic interactions, which are particularly suited to IDRs. The regulation of electrostatic interactions in IDRs through phosphorylation is an emerging concept in cellular signaling, and troponin I is now another important example, one known by cardiac physiologists for 40 y. The cardiac isoform of troponin I (cTnI) has a unique 31-residue N-terminal region that binds cardiac troponin C (cTnC) to increase the calcium sensitivity of the sarcomere. The interaction can be abolished by cTnI phosphorylation at Ser22 and Ser23, an important mechanism for regulating cardiac contractility. cTnC contains two EF–hand domains (the N and C domain of cTnC, cNTnC and cCTnC) connected by a flexible linker. Calcium binding to either domain favors an “open” conformation, exposing a large hydrophobic surface that is stabilized by target binding, cTnI[148–158] for cNTnC and cTnI[39–60] for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI[1–73] in complex with cTnC. cTnI[39–60] binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI[41–67] and a type VIII turn in cTnI[38–41]. In contrast, cTnI[1–37] remains disordered, although cTnI[19–37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC. However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al. (2003) Nature 424(6944):35–41]. Domain positioning impacts the effective concentration of cTnI[148–158] presented to cNTnC, and this is how cTnI[19–37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank–Starling law of the heart.

13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.