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Phosphorylation and Calcium Antagonistically Tune Myosin‐binding Protein C's Molecular Structure and Function
Author(s) -
Previs Michael J,
Mun Ji Young,
Michalek Arthur J,
Previs Samantha Beck,
Gulick James,
Robbins Jeffrey,
Warshaw David M,
Craig Roger
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1012.2
Subject(s) - phosphorylation , biophysics , myosin , actin , myosin light chain kinase , c terminus , chemistry , calcium , protein filament , microbiology and biotechnology , biochemistry , biology , amino acid , organic chemistry
During each heartbeat, calcium activates the sliding of actin‐thin filaments towards the centers of myosin‐thick filaments to shorten the overall length of cardiac muscle cells. Cardiac myosin binding protein C (cMyBP‐C) tunes these interactions throughout the contractile cycle. cMyBP‐C's C terminus is strongly bound to the thick filament backbone and its N‐terminal domains extend away and transiently interact with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocities. Both mechanisms are tunable by phosphorylation of 4 serines within an extensible, intrinsically disordered region of cMyBP‐C's N‐terminus, the M‐domain. Does phosphorylation impact the structure and molecular mechanics of cMyBP‐C's N terminus to tune it's function? Atomic force spectroscopy studies of N‐terminal fragments (domains C1–C2), showed that phosphorylation reduced the M‐domain's contour length and extensibility. Rotary shadowing electron microscopy showed that M‐domain phosphorylation caused N‐terminal fragments (C0–C3) to shift from an extended, rod‐like structure to a compact conformation. Taken together, M‐domain phosphorylation and its impact on cMyBP‐C's N‐terminal structure suggest a mechanism for tuning cMyBP‐C's function in motility assays. Interestingly, we found that free calcium (0.1 mM), necessary to fully activate the thin‐filament, mitigated the structural effects of phosphorylation by increasing the free‐extensibility of the phosphorylated M‐domain and shifting the phosphorylated N‐terminal fragments back to the extended, rod‐like state, as if unphosphorylated. Functionally, even though phosphorylation reduced cMyBP‐C's ability to inhibit actin filament sliding velocity in the motility assay (24% vs. 40% inhibition), addition of calcium ablated the impact of phosphorylation, fully restoring cMyBP‐C's inhibitory capacity. We conclude that phosphorylation of MyBP‐C's M‐domain may have its greatest effect on cardiac contractility by tuning cMyBP‐C's ability to sensitize actin‐thin filaments to calcium at the low levels present during the onset of contraction. Importantly, calcium levels at the peak contraction would allow cMyBP‐C to remain a potent contractile modulator, regardless of the cMyBP‐C's phosphorylation state. Support or Funding Information NIH grants HL124041 and HL059408 supported these studies.