title: Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function.
abstract: During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state.
authors.affiliation: Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, VT 05405;
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Previs
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655;
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Mun
authors.affiliation: Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, VT 05405;
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Michalek
authors.affiliation: Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, VT 05405;
authors.forename: Samantha Beck
authors.initials: SB
authors.lastname: Previs
authors.affiliation: Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors.forename: James
authors.initials: J
authors.lastname: Gulick
authors.affiliation: Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Robbins
authors.affiliation: Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, VT 05405; david.warshaw@uvm.edu roger.craig@umassmed.edu.
authors.forename: David M
authors.initials: DM
authors.lastname: Warshaw
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655; david.warshaw@uvm.edu roger.craig@umassmed.edu.
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D000099163:Myosin Binding Protein C
chemical_list: D002118:Calcium; D002352:Carrier Proteins; D000099163:Myosin Binding Protein C
keywords: cMyBP-C; muscle activation; muscle regulation; structure-function