Previs Michael J, Mun Ji Young, Michalek Arthur J, Previs Samantha Beck, Gulick James, Robbins Jeffrey, Warshaw David M, Craig Roger
Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, University of Vermont, Burlington, VT 05405;
Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655;
Proc Natl Acad Sci U S A. 2016 Mar 22;113(12):3239-44. doi: 10.1073/pnas.1522236113. Epub 2016 Feb 23.
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.
在每次心跳过程中，心肌收缩力源于肌动蛋白细肌丝在钙激活下朝着肌球蛋白粗肌丝中心滑动，从而使细胞长度缩短。心肌肌球蛋白结合蛋白C（cMyBP-C）是粗肌丝的一个组成部分，它似乎通过其N端结构域与肌动蛋白和/或肌球蛋白S2结构域短暂相互作用来调节这些机械化学相互作用，使细肌丝对钙敏感并控制最大滑动速度。这两种功能机制都可能通过cMyBP-C N端一个内在无序、可延伸区域（M结构域）的磷酸化进一步调节。利用原子力光谱、电子显微镜和突变蛋白表达，我们证明磷酸化降低了M结构域的可延伸性，并使N端结构域的构象从伸展结构转变为紧凑构型。结合运动分析数据，M结构域磷酸化的这些结构效应提示了一种降低单个cMyBP-C分子功能效力的机制。有趣的是，我们发现最大程度激活细肌丝所需的钙水平通过增加M结构域的可延伸性并将磷酸化的N端片段转变回伸展状态（就好像未磷酸化一样）减轻了磷酸化的结构效应。在功能上，向运动分析中添加钙消除了磷酸化对最大滑动速度的影响，完全恢复了cMyBP-C的抑制能力。我们得出结论，M结构域磷酸化可能在收缩之间的低钙水平下对调节cMyBP-C对细肌丝的钙敏化作用影响最大。重要的是，收缩峰值时的钙水平将使cMyBP-C无论其磷酸化状态如何都能保持为一种有效的收缩调节因子。
