The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C Peter M. Hwanga,b,1, Fangze Caib, Sandra E. Pineda-Sanabriab, David C. Corsonb, and Brian D. Sykesb Author Affiliations aDivision of General Internal Medicine, Department of Medicine, and bDepartment of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7 PNAS 2014 vol. 111 no. 40 14412–14417, doi: 10.1073/pnas.1410775111 pnas.org/content/111/40/14412.full 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. 1H-1H NOE Analysis of the cTnC–cTnI[1–73] Complex. Homonuclear 1H-1H NOEs can be observed between any two 1H atoms within ∼6 Å, critical for any NMR-based structure determination. The vast majority of intermolecular 1H-1H NOEs occur between cTnI[39–60] and cCTnC, consistent with extensive hydrophobic binding. There are few intermolecular NOEs observed between cNTnC and cTnI[1–73], but the most critical NOEs are between cNTnC Ala7 and cTnI Ala42/Ser43. The importance of this contact is underscored by the fact that a double Ser41/43 mutation to aspartate (mimicking phosphorylation) markedly decreases troponin calcium affinity (33). Because cTnI Ala42/Ser43 are part of a rigid alpha helix bound to cCTnC and Ala7 is part of cNTnC helix N, the interaction fixes the position of cNTnC relative to cCTnC. This contact is actually present in the X-ray crystal structure (Fig. 3 A and B), suggesting that the true interdomain orientation in solution is in fact very similar. This single contact point is alone insufficient to stably fix the domains in solution, but it is stabilized by crystal packing contacts in the X-ray structure. In solution, the orientation is bolstered by additional electrostatic interactions from the disordered N-terminal tail of cTnI. cTnI Ser41 is the N-terminal helix cap residue, but it is also part of a type VIII turn based on Ser38–Lys39–Ile40–Ser41 backbone chemical shifts, according to the Motif Identification from Chemical Shifts (MICS) program (22). Lys39 shows NOE contacts with Asp131 and Glu135 of helix G in cCTnC, whereas Ser38 has intermolecular NOEs to a lysine residue consistent with cNTnC Lys6 in the crystal structure (Fig. 3C). This brings Lys35–Lys36–Lys37 into contact with a prominent negatively charged surface that includes EF–hand loop II, the all-important calcium-binding loop (Fig. 3C). cTnI Lys35–37 display NOEs to residues with Asp- and Glu-like chemical shifts, but these could not be unambiguously separated from each other. In the crystal structure, there are two troponin complexes per asymmetric unit. In one complex, Lys35–37 are invisible, but in the other, they are modeled hovering over EF–hand loop II, with Lys37 closest to Asp75 of cNTnC. Finally, cTnI Arg21 and Arg26 also make weak NOEs to cTnC residues with Glu-like chemical shifts (Fig. S2), but these could not be unambiguously assigned either. The considerable chemical shift overlap for charged Lys, Arg, Asp, and Glu side chains makes unambiguous intermolecular NOE assignment near impossible. Nevertheless, these NOEs demonstrate specific and stable electrostatic interactions involving cTnI Arg21, Arg26, and part of Lys35–37. This is in marked contrast to the lack of NOEs involving hydrophobic residues, which are usually critical for defining intermolecular contacts. cTnI Pro17, Ile18, Tyr25, Ala27, Tyr28, and Ala29 showed no intermolecular NOEs (see Fig. S2 for an example). Classically, protein–protein interactions are dominated by hydrophobic interactions, which require close packing, exclusion of water, and a rigid structuring of the backbone, as exemplified by the interaction between cTnI[39–60] and cCTnC. In contrast, the electrostatic interactions involving cTnI[19–37] occur in a solvated environment that does not require as much rigidification of the backbone. It does require many positive charges, with as many as eight potentially involved (if His33 is included). Notably, Lys35 has been strongly implicated in autosomal dominant dilated cardiomyopathy (K36Q by the alternate numbering scheme), decreasing the calcium sensitivity of reconstituted thin filaments by 0.3–0.6 pCa units (14, 34). 15N Relaxation of cTnI[1–73] Free and in Complex with cTnC. 15N relaxation rates provide a window into nanosecond to picosecond (10−9 to 10−12 s) timescale conformational fluctuations in a protein. Structured proteins tumble with a global rotational correlation time, τC, on the order of several nanoseconds for a small domain, with τC scaling roughly proportionally to molecular weight. In rigidly structured regions, the overall motion is approximated by the global correlation time. As structural flexibility increases, for example, toward the N or C terminus, faster internal motions begin to dominate NMR relaxation behavior. Binding causes a decrease in internal motions that can be detected via changes in relaxation. 15N backbone relaxation studies were obtained for cTnI[1–73] both free and bound to C35S,C84S–cTnC (Fig. 4). The transverse relaxation rate, R2, is roughly proportional to the weighted average correlation time (including global tumbling and internal motions) at each backbone amide site. R1 relaxation is most effectively induced by motions with a timescale near 2–3 ns (15N nucleus on a 600-MHz spectrometer). A negative 1H-15N heteronuclear NOE is most effectively induced by motions with a timescale near 0.2–0.3 ns. Looking at the plots of R1, R2, and heteronuclear NOEs in free cTnI[1–73], it becomes evident that residues 45–67 are the most rigid in all of cTnI[1–73], with the highest R1, R2, and least negative heteronuclear NOE values, consistent with nascent helix formation. When this region binds to cTnC, it appears more massive and tumbles with a correlation time of about 15 ns (calculated using a model-free analysis: R2, ∼29 s−1; R1, ∼0.9 s−1; NOE, ∼0.8). The 15-ns correlation time is consistent with a 28-kDa protein. This suggests that not only is cTnI[1–73] tightly bound to cCTnC, but cNTnC is immobilized as well, with the whole complex tumbling as a single 28-kDa unit. R2 values for cTnI[1–73] are consistent with those measured by Rosevear and coworkers for cTnC bound to cTnI[1–80] (35). In the absence of cTnI[1–80], the two domains of cTnC tumbled independently (like two smaller proteins) (mean R2, 13 s−1). Upon addition of cTnI[1–80], the two domains became rigidly fixed and tumbled as a single unit (mean R2, 31 s−1). Pseudophosphorylation of cTnI Ser22/Ser23 by mutation to aspartate caused the two domains of cTnC to tumble as two separate proteins (mean R2, 15 s−1). This work, along with the present study, shows that interaction with the cardiac-specific N-terminal extension of cTnI fixes the position of the cNTnC regulatory domain relative to the rest of the troponin complex, whereas phosphorylation of cTnI Ser22/Ser23 abolishes this. The relaxation data for cTnI[19–37] are quite informative. Upon addition of cTnC, the most striking change is the plateau of increased R2 values in this region (Fig. 4). This indicates that although cTnI[19–37] is intrinsically disordered with no secondary structure preference, there is a substantial restriction of mobility. This would be expected if Arg19–21, Arg26, and Lys35–37 are electrostatically tethered, but there is likely transient structuring occurring in the intervening segments as well. Faint intermediate-range (i,i+3 and i,i+4) intramolecular 1H-1H NOEs are scattered throughout residues 18–32, indicating a transient helix- or turn-like structure, although chemical shift analysis indicates no net helical preference. Interestingly, the MICS Protein Structural Motif Prediction program indicates some potential turn structure at residues 22–25 and 31–34. The increased R2 values in cTnI[19–37] could result from fast conformational exchange (microsecond to millisecond timescale) and/or conformational restriction on the nanosecond timescale. Support for the latter comes from accompanying changes in R1 and heteronuclear NOE values. Note that the R1 values for cTnI[19–37] are the highest in all of cTnI, and these could not arise from an equilibrium between a strongly bound structured state (which would have a correlation time of 15 ns and R1 < 1 s−1) and a free state (with R1 < 1.5 s−1 from Fig. 4). If this were the case, the R1 values would be an average of these two states and significantly lower (so long as the exchange time constants were significantly longer than the nanosecond timescale correlation times). Instead, the high R1 values (up to 2 s−1) suggest a single partially structured state (or ensemble of very rapidly interconverting partially structured states). Residues 2–18 of cTnI appear to be very mobile with considerable subnanosecond timescale motions and increasing mobility toward the N terminus. Upon addition of cTnC, there is no change in the rapid motions (followed by R1 and NOEs) in this region, suggesting that it is not tethered in the same way as cTnI[19–37]. Calcium Titration of cTnI[1–73]–cTnC. The calcium binding affinity of the 2-(4′- (iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS)–Cys84 conjugate of C35S–cTnC was studied using fluorescence, in complex with either cTnI[1–73] or cTnI[34–71] (Fig. S3). cTnI[1–73]–cTnC had a calcium pCa50 of 6.04 ± 0.03, and cTnI[34–71]–cTnC, 6.04 ± 0.04. Thus, there was no measurable difference between the calcium affinities of the complexes. Thus, the electrostatic interaction between cTnI[19–37] and cNTnC does not exert a direct effect on the calcium binding affinity of cNTnC. NMR Spectroscopy and Data Analysis. The 3D backbone assignment spectra were recorded on a Varian Inova 600 spectrometer. All experiments were from Agilent BioPack (VnmrJ 3.2D) unless otherwise specified. The experiments used for backbone assignment were HNCA, HN(CO)CA, HN(CA)CO, and HNCO. The molecular weight of the cTnC–cTnI[1–73] complex was 28 kDa, and sensitivity was further limited by exchange broadening. Additional experiments were used to assign the flexible regions of cTnI[1–73]: HNCACB, H(CCO)NH-TOCSY, and (H)C(CO)NH-TOCSY. To obtain the complete assignments of cTnC, HN(CA)HA and HA(CACO)NH (49) experiments were recorded, in-house modified from the BioPack HNCACB and CBCA(CO)NH experiments, respectively. 15N-edited nuclear Overhauser enhancement spectroscopy (NOESY)–HSQC spectra were also used for backbone assignment. Enhanced sensitivity and gradient selection was used in the backbone assignment triple resonance experiments, but TROSY was not used for the 15N and 1H dimensions. The 3D NOESY spectra were acquired on a Varian Inova 800 spectrometer equipped with cryoprobe. 13C-edited HMQC–NOESY spectra were run on 13C,15N-labeled samples in H2O, although the majority of intramolecular NOE data were obtained from 13C-edited NOESY–HSQC spectra acquired in D2O, and intermolecular NOEs were obtained from 12C-filtered, 13C-edited NOESY–HSQC spectra in D2O. NMR data were processed using NMRPipe (50) software and visualized and analyzed with NMRViewJ (51). 15N T1, T2, and 1H-15N NOE experiments were conducted using 2H,15N-labeled cTnI[1,73] with unlabeled aCys–cTnC. TROSY was used in the 1H and 15N dimensions. A 6-s saturation time or recycle delay was used in the NOE experiments. For T1 and T2, curves were fit using a mono exponential decay function using the simplex minimization algorithm in MATLAB. The variance for each time point was estimated from the sum of the squares of the residuals divided by (N – 2), where N is the number of data points for each curve and 2 is the number of fitting parameters. A Monte Carlo method was then applied to obtain error estimates for R1 and R2. For 1H-15N NOE, the error estimate was based on the ratio of spectral noise to signal intensity in the reference spectrum, multiplied by a factor of √2 to reflect the fact that the 1H-15N NOE is a ratio. 15N R1, R2, and 1H-15N NOE data were further analyzed using a model-free analysis of internal motions (52). Ribbon diagrams of the troponin complex drawn by PyMol and derived from ref. 15. cNTnC is shown in magenta, and cCTnC is shown in cyan. Calcium ions are shown as green spheres. Troponin T, residues 226–271, is shown in yellow. cTnI, residues 34–135 and 147–161, is shown in orange. (A and B) The key contact that fixes the position of cNTnC relative to cCTnC is between A7 of cNTnC and A42/S43 of cTnI, shown as space-filling spheres. The side chains of cTnI[35–39] are also shown in stick figures, with K39 of cTnI contacting D131 and E135 of cCTnC (shown in sticks) and K35-S38 hovering over cNTnC. (C) Electrostatic surface representation of cNTnC showing the negatively charged surface that interacts with cTnI[19–37]. 15N backbone relaxation data for cTnI[1–73]; “x” denotes free, and “o” denotes bound to C35S,C84S–cTnC.
Posted on: Tue, 11 Nov 2014 17:21:10 +0000
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