Vadim Gaponenko

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.

The hypervariable region of K-Ras4B is responsible for its specific interactions with Calmodulin

K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival, and motility. The p21 Ras proteins, such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry, we demonstrate that the hypervariable region of K-Ras4B contributes in a major way to the interaction with calmodulin, while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca(2+)-loaded calmodulin with micromolar affinity, while the GTP-gamma-S-loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin.

Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

Background: Conserved Asp-137 destabilizes the hydrophobic core of the coiled-coil tropomyosin. Results: Leu substitution of Asp-137 decreases flexibility of tropomyosin and causes long range structural rearrangements; mouse hearts expressing this variant show altered function. Conclusion: Residue Asp-137 is important for regulatory function of tropomyosin in the heart. Significance: Our data support the hypothesis that tropomyosin flexibility regulates cardiac function in vivo. α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca2+ sensitivity and cardiomyocyte contractility without changes in intracellular Ca2+ transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.

Cardiac troponin I inhibitory peptide: location of interaction sites on troponin C

Cardiac troponin I(129–149) binds to the calcium saturated cardiac troponin C/troponin I(1–80) complex at two distinct sites. Binding of the first equivalent of troponin I(129–149) was found to primarily affect amide proton chemical shifts in the regulatory domain, while the second equivalent perturbed amide proton chemical shifts within the D/E linker region. Nitrogen‐15 transverse relaxation rates showed that binding the first equivalent of inhibitory peptide to the regulatory domain decreased conformational exchange in defunct calcium binding site I and that addition of the second equivalent of inhibitory peptide decreased flexibility in the D/E linker region. No interactions between the inhibitory peptide and the C‐domain of cardiac troponin C were detected by these methods demonstrating that the inhibitory peptide cannot displace cTnI(1–80) from the C‐domain.

Application of Reductive 13C-Methylation of Lysines to Enhance the Sensitivity of Conventional NMR Methods

NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high concentrations, proteins or peptides tend to aggregate. In order to overcome this problem, we make use of reductive 13C-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one 13CH3 signal arising from two carbons and six protons, as compared to one carbon and three protons in aliphatic amino acids. The improved sensitivity allows us to study protein-protein or protein-peptide interactions at very low micromolar concentrations. We demonstrate the utility of this method by studying the interaction between the post-translationally lipidated hypervariable region of a human proto-oncogenic GTPase K-Ras and a calcium sensor protein calmodulin. Calmodulin specifically binds K-Ras and modulates its downstream signaling. This binding specificity is attributed to the unique lipidated hypervariable region of K-Ras. At low micromolar concentrations, the post-translationally modified hypervariable region of K-Ras aggregates and binds calmodulin in a non-specific manner, hence conventional NMR techniques cannot be used for studying this interaction, however, upon reductively methylating the lysines of calmodulin, we detected signals of the lipidated hypervariable region of K-Ras at physiologically relevant nanomolar concentrations. Thus, we utilize 13C-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.

The Use of Reductive Methylation of Lysine Residues to Study Protein-Protein Interactions in High Molecular Weight Complexes by Solution NMR

While solution state NMR is very well suited for analysis of protein-protein interactions occurring with a wide range of affinities, it suffers from one significant weakness, known as the molecular weight limitation. This limitation stems from the efficient nuclear relaxation processes in macromolecules larger than 30 kDa (Wider & Wuthrich, 1999). These relaxation processes cause rapid decay of NMR signals. Although the use of transverse relaxation optimized spectroscopy (TROSY) approaches has made solution state NMR of large proteins and protein-protein complexes more feasible, it is still limited by the ability to produce isotope enriched proteins (Pervushin et al., 1997). However, there is a significant number of proteins for which no convenient system for stable isotope incorporation exists. We recently utilized reductive methylation methodology to demonstrate that it is possible to introduce 13C-enriched methyl groups into lysine residues in otherwise unlabeled proteins with the purpose of studying protein-ligand and protein-protein interactions by NMR (Abraham et al., 2008).