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.
Highlights
The results presented above show that serine-to-aspartate substitution of the three phosphorylation sites in the m-motif of cMyBP-C fully mimic the effects of phosphorylation of those sites with respect to binding to myosin S2⌬ but fail to mimic its effects on the interaction between cMyBP-C and the thin filament
The ionic strength dependence of the binding between cMyBP-C and myosin S2⌬ suggests that it is mainly driven by ionic interactions between the m-motif and myosin S2⌬, consistent with the fact that it can be abolished by the introduction of additional negative charges, by either phosphorylation or Ser–Asp substitutions
Regulatory interactions of cMyBP-C with Native thin filaments (NTF) have been localized to both domain C1 and the m-motif [17]
Summary
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. 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. We combined these structural measurements with measurements of active isometric force and with biochemical characterization of the interaction between C1mC2 and myosin S2, and C1mC2 and native thin filaments (NTF) in vitro, to determine whether Ser–Asp substitutions reproduce the structural and functional effects of phosphorylation
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