Structural and functional effects of myosin-binding protein-C phosphorylation in heart muscle are not mimicked by serine-to-aspartate substitutions

Abstract

Myosin-binding protein-C (cMyBP-C) is a key regulator of contractility in heart muscle, and its regulatory function is controlled in turn by phosphorylation of multiple serines in its m-domain. The structural and functional effects of m-domain phosphorylation have often been inferred from those of the corresponding serine-to-aspartate (Ser–Asp) substitutions, in both in vivo and in vitro studies. Here, using a combination of in vitro binding assays and in situ structural and functional assays in ventricular trabeculae of rat heart and the expressed C1mC2 region of cMyBP-C, containing the m-domain flanked by domains C1 and C2, we tested whether these substitutions do in fact mimic the effects of phosphorylation. In situ changes in thin and thick filament structure were determined from changes in polarized fluorescence from bifunctional probes attached to troponin C or myosin regulatory light chain, respectively. We show that both the action of exogenous C1mC2 to activate contraction in the absence of calcium and the accompanying change in thin filament structure are abolished by tris-phosphorylation of the m-domain, but unaffected by the corresponding Ser–Asp substitutions. The latter produced an intermediate change in thick filament structure. Both tris-phosphorylation and Ser–Asp substitutions abolished the interaction between C1mC2 and myosin sub-fragment 2 (myosin S2) in vitro, but yielded different effects on thin filament binding. These results suggest that some previous inferences from the effects of Ser–Asp substitutions in cMyBP-C should be reconsidered and that the distinct effects of tris-phosphorylation and Ser–Asp substitutions on cMyBP-C may provide a useful basis for future studies.

Cardiac myosin binding protein-C phosphorylation regulates the super-relaxed state of myosin

“…It was apparent that the amino acid substitutions used to mimic phosphorylation of cMyBP-C might be completely representative of the structural changes accompanying endogenous serine phosphorylation. Indeed, a recent report suggests that this may be the case (20). Thus, to test whether destabilization of the SRX, as observed in phosphomimetic hearts, was representative of endogenously phosphorylated cMyBP-C, WT preparations were pretreated with PKA to phosphorylate myofilament targets before performing single-nucleotide turnover assays.…”

Role of intrinsic disorder in muscle sarcomeres

“…However, one must be careful with conclusions made on Ser-to-Asp phosphomimetic molecules as it has been argued that not all effects of Ser phosphorylation are reproduced by Ser substitution with Asp. 51 In another study, the structural effects of the M-domain phosphorylation were tested in modified mouse cMyBP-C fragment C0C2 with fluorescent and spin labels placed on Cys residues in C1 (Cys248) and C2 (Ser440Cys) domains. 52 The interprobe distance distribution was quantitated with time-resolved fluorescence resonance energy transfer (TR-FRET) and dipolar electron paramagnetic resonance (DEER).…”

“…Quantitative microscale thermophoresis measurements gave K d for complexes between cMyBP-C and S2Δ. 51 The K d for an isolated M-domain was ~40 μM, for the C1C2 ~20 μM, and for C1 linked to C2 by a flexible unrelated linker ~480 μM. This study established that although Ig domains C1 and C2 slightly improve the binding of the M-domain by a factor of ~2, the most important region providing interacting contacts with S2Δ is the M-domain itself.…”

Skeletal myosin binding protein-C: An increasingly important regulator of striated muscle physiology

“…The cMyBP-C protein has also been described as a modulator of length- dependent activation of myocardium in a manner independent of cTnI phosphorylation [70, 71]. Using N-terminal fragments of cMyBP-C, it was reported that the serine-to- aspartate substitution widely used to mimic phosphorylation in transgenic mice is unable to recapitulate all aspects of PKA-mediated cMyBP-C phosphorylation [72]. This is a very important consideration in interpreting future results; however, to date, no other method has been able to isolate the functional roles of phosphorylation sites of endogenous proteins, particularly in vivo .…”