Jack W. Howarth

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.

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)

Phosphorylation modulates the mechanical stability of the cardiac myosin-binding protein C motif.

Cardiac myosin-binding protein C (cMyBP-C) is a thick-filament-associated protein that modulates cardiac contractility through interactions of its N-terminal immunoglobulin (Ig)-like C0-C2 domains with actin and/or myosin. These interactions are modified by the phosphorylation of at least four serines located within the motif linker between domains C1 and C2. We investigated whether motif phosphorylation alters its mechanical properties by characterizing force-extension relations using atomic force spectroscopy of expressed mouse N-terminal cMyBP-C fragments (i.e., C0-C3). Protein kinase A phosphorylation or serine replacement with aspartic acids did not affect persistence length (0.43 ± 0.04 nm), individual Ig-like domain unfolding forces (118 ± 3 pN), or Ig extension due to unfolding (30 ± 0.38 nm). However, phosphorylation did significantly decrease the C0-C3 mean contour length by 24 ± 2 nm. These results suggest that upon phosphorylation, the motif, which is freely extensible in the nonphosphorylated state, adopts a more stable and/or different structure. Circular dichroism and dynamic light scattering data for shorter expressed C1-C2 fragments with all four serines replaced by aspartic acids confirmed that the motif did adopt a more stable structure that was not apparent in the nonphosphorylated motif. These biophysical data provide both a mechanical and structural basis for cMyBP-C regulation by motif phosphorylation.