Paul R. Rosevear

Binding of Levosimendan, a Calcium Sensitizer, to Cardiac Troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

NMR ANALYSIS OF CARDIAC TROPONIN C-TROPONIN I COMPLEXES: EFFECTS OF PHOSPHORYLATION

Phosphorylation of the cardiac specific amino‐terminus of troponin I has been demonstrated to reduce the Ca2+ affinity of the cardiac troponin C regulatory site. Recombinant N‐terminal cardiac troponin I proteins, cardiac troponin I(33–80), cardiac troponin I(1–80), cardiac troponin I(1–80)DD and cardiac troponin I(1–80)pp, phosphorylated by protein kinase A, were used to form stable binary complexes with recombinant cardiac troponin C. Cardiac troponin I(1–80)DD, having phosphorylated Ser residues mutated to Asp, provided a stable mimetic of the phosphorylated state. In all complexes, the N‐terminal domain of cardiac troponin I primarily makes contact with the C‐terminal domain of cardiac troponin C. The non‐phosphorylated cardiac specific amino‐terminus, cardiac troponin I(1–80), was found to make additional interactions with the N‐terminal domain of cardiac troponin C.

Effects of Troponin I Phosphorylation on Conformational Exchange in the Regulatory Domain of Cardiac Troponin C

Conformational exchange has been demonstrated within the regulatory domain of calcium-saturated cardiac troponin C when bound to the NH2-terminal domain of cardiac troponin I-(1–80), and cardiac troponin I-(1–80)DD, having serine residues 23 and 24 mutated to aspartate to mimic the phosphorylated form of the protein. Binding of cardiac troponin I-(1–80) decreases conformational exchange for residues 29, 32, and 34. Comparison of average transverse cross correlation rates show that both the NH2- and COOH-terminal domains of cardiac troponin C tumble with similar correlation times when bound to cardiac troponin I-(1–80). In contrast, the NH2- and COOH-terminal domains in free cardiac troponin C and cardiac troponin C bound cardiac troponin I-(1–80)DD tumble independently. These results suggest that the nonphosphorylated cardiac specific NH2 terminus of cardiac troponin I interacts with the NH2-terminal domain of cardiac troponin C.

Derivation of structural restraints using a thiol-reactive chelator

Recognition and identification of protein folds is a prerequisite for high‐throughput structural genomics. Here we demonstrate a simple protocol for covalent attachment of a short and more rigid metal‐chelating tag, thiol‐reactive EDTA, by chemical modification of the single cysteine residue in barnase(H102C). Conjugation of the metal‐chelating tag provides the advantage of allowing a greater range of paramagnetic metal substitutions. Substitution of Yb3+, Mn2+, and Co2+ permitted measurement of metal–amide proton distances, dipolar shifts, and residual dipolar couplings. Paramagnetic‐derived restraints are advantageous in the NMR structure elucidation of large protein complexes and are shown sufficient for validation of homology‐based fold predictions.

Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

Previously, we utilized 15N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca2+-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, M., and Rosevear, P.R. (1999) J. Biol. Chem.274, 16681–16684). Here we show a large decrease in cardiac troponin C linker flexibility, corresponding to residues 85–93, when bound to intact cardiac troponin I. The addition of 2 m urea to the intact cardiac troponin I-troponin C complex significantly increased linker flexibility. Conformational changes in the regulatory domain of cardiac troponin C were monitored in complexes with troponin I-(1–211), troponin I-(33–211), troponin I-(1–80) and bisphosphorylated troponin I-(1–80). The cardiac specific N terminus, residues 1–32, and the C-domain, residues 81–211, of troponin I are both capable of inducing conformational changes in the troponin C regulatory domain. Phosphorylation of the cardiac specific N terminus reversed its effects on the regulatory domain. These studies provide the first evidence that the cardiac specific N terminus can modulate the function of troponin C by altering the conformational equilibrium of the regulatory domain.

Structural insight into unique cardiac myosin-binding protein-C motif: a partially folded domain.

Background: Cardiac myosin-binding protein-C is a sarcomeric assembly protein necessary for the regulation of sarcomere structure and function. Results: The cMyBP-C motif is composed of two subdomains, a largely disordered N-terminal portion and a more ordered C-terminal subdomain. Conclusion: The C-terminal subdomain is capable of forming a three-helix bundle. Significance: The three-helix bundle may provide a platform for actin binding. The structural role of the unique myosin-binding motif (m-domain) of cardiac myosin-binding protein-C remains unclear. Functionally, the m-domain is thought to directly interact with myosin, whereas phosphorylation of the m-domain has been shown to modulate interactions between myosin and actin. Here we utilized NMR to analyze the structure and dynamics of the m-domain in solution. Our studies reveal that the m-domain is composed of two subdomains, a largely disordered N-terminal portion containing three known phosphorylation sites and a more ordered and folded C-terminal portion. Chemical shift analyses, dNN(i, i + 1) NOEs, and 15N{1H} heteronuclear NOE values show that the C-terminal subdomain (residues 315–351) is structured with three well defined helices spanning residues 317–322, 327–335, and 341–348. The tertiary structure was calculated with CS-Rosetta using complete 13Cα, 13Cβ, 13C′, 15N, 1Hα, and 1HN chemical shifts. An ensemble of 20 acceptable structures was selected to represent the C-terminal subdomain that exhibits a novel three-helix bundle fold. The solvent-exposed face of the third helix was found to contain the basic actin-binding motif LK(R/K)XK. In contrast, 15N{1H} heteronuclear NOE values for the N-terminal subdomain are consistent with a more conformationally flexible region. Secondary structure propensity scores indicate two transient helices spanning residues 265–268 and 293–295. The presence of both transient helices is supported by weak sequential dNN(i, i + 1) NOEs. Thus, the m-domain consists of an N-terminal subdomain that is flexible and largely disordered and a C-terminal subdomain having a three-helix bundle fold, potentially providing an actin-binding platform.

Role of the Acidic N′-Region of Cardiac Troponin I In Regulating Myocardial Function*

Cardiac troponin I (cTnI) phosphorylation modulates myocardial contractility and relaxation during β‐adrenergic stimulation. cTnI differs from the skeletal isoform in that it has a cardiac specific N′ extension of 32 residues (N′ extension). The role of the acidic N′ region in modulating cardiac contractility has not been fully defined. To test the hypothesis that the acidic N′ region of cTnI helps regulate myocardial function, we generated cardiac‐specific transgenic mice in which residues 2–11 (cTnIΔ2–11) were deleted. The hearts displayed significantly decreased contraction and relaxation under basal and β‐adrenergic stress compared to nontransgenic hearts, with a reduction in maximal Ca2+‐dependent force and maximal Ca2+‐activated Mg2+‐ATPase activity. However, Ca2+ sensitivity of force development and cTnI‐Ser23/24 phosphorylation were not affected. Chemical shift mapping shows that both cTnI and cTnIΔ2–11 interact with the N lobe of cardiac troponin C (cTnC) and that phosphor‐ylation at Ser23/24 weakens these interactions. These observations suggest that residues 2–11 of cTnI, comprising the acidic N′ region, do not play a direct role in the calcium‐induced transition in the cardiac regulatory or N lobe of cTnC. We hypothesized that phosphorylation at Ser23/24 induces a large conformational change positioning the conserved acidic N region to compete with actin for the inhibitory region of cTnI. Consistent with this hypothesis, deletion of the conserved acidic N′ region results in a decrease in myocardial contractility in the cTnIΔ2–11 mice demonstrating the importance of acidic N′ region in regulating myocardial contractility and mediating the response of the heart to β‐AR stimulation. Sadayappan, S., Finley, N., Howarth, J. W., Osinska, H., Klevitsky, R., Lorenz, J. N., Rosevear, P. R., Robbins, J. Role of the acidic N′ region of cardiac troponin I in regulating myocardial function. FASEB J. 22, 1246–1257 (2008)

Ligand conformations and ligand-enzyme interactions as studied by the nuclear Overhauser effect

Publisher Summary The nuclear Overhauser effect (NOE) has proved to be a powerful tool for the elucidation of molecular structure. Limitations of the NOE method for determining the conformation of a bound ligand result from the assumptions of fixed interproton distances and a single correlation time for all internuclear vectors. In many cases, the validity of these assumptions can be tested by experiment. However, if several bound conformations coexist with different interproton distances, the NOE method will yield a nonlinear average conformation skewed toward those conformations having the shortest interproton distances. Multiple conformations for the bound ligand can usually be detected by observing two or more interproton NOEs that are not mutually consistent with a single conformation. However, a major advance in biological nuclear magnetic resonance (NMR) will be the application of one- and two-dimensional isotope-filtered NOEs along with the selective labeling of the protein and ligand. This methodology will greatly increase the selectivity, identification, and resolution of the NOE experiment and thus our ability to determine the conformations and arrangements of bound ligands and the amino acid environment provided by the protein.

[3] 13C NMR Analysis of Complex Carbohydrates

Publisher Summary This chapter describes the applications of 13 C nuclear magnetic resonance (NMR) using higher field NMR spectrometers to the study of oligosaccharides containing a number of different monosaccharide units with special attention given to the examination of solution conformations. Typically, 13 C NMR spectra are obtained using broadband decoupling of protons. Spectra are relatively simple, with a single sharp resonance for each carbon in the compound. At natural abundance levels, 13 C– 13 C coupling is not observable. Proton-coupled 13 C spectra are much more complex and show the number of hydrogens covalently bonded to each carbon because each 13 C resonance is split by covalently bonded hydrogens into n + 1 lines. Additional splittings or line-broadening because of 2- and 3-bond and long-range coupling also add to the complexity of 1 H-coupled 13 C spectra. Several experimental options are available on most high-field FT spectrometers that facilitate assignment of 13 C resonances and of 13 C– 1 H coupling constants. These include: gated 1 H decoupling, off-resonance 1 H decoupling, selective 13 C saturation combined with gated 1 H decoupling and FT difference spectroscopy, and two-dimensional J spectroscopy. The latter may be particularly valuable for complex molecules because it permits 13 C chemical shifts and 13 C– 1 H coupling of each 13 C nucleus to be displayed separately.

Computational studies of the effect of the S23D/S24D troponin i mutation on cardiac troponin structural dynamics

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1-40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1-161, cTnI 1-172, and cTnT 236-285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca(2+) binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca(2+) binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.