Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02903, USA.
J Am Chem Soc. 2011 Jan 12;133(1):73-80. doi: 10.1021/ja107810r. Epub 2010 Dec 10.
Muscle relaxation is triggered by the dephosphorylation of Ser19 in the myosin regulatory light chain. This reaction is catalyzed by the holoenzyme myosin phosphatase (MP), which includes the catalytic subunit protein phosphatase 1 (PP1) and the regulatory targeting subunit (MYPT). MYPT1 (myosin phosphatase targeting subunit 1) is responsible for both targeting the holoenzyme to subcellular compartments in the muscle and directing PP1 specificity toward myosin. To understand the molecular events leading to the MYPT1-PP1 holoenzyme formation, we used NMR spectroscopy to determine the structural and dynamic characteristics of unbound MYPT1. This allowed the conformations of MYPT1 in the free, unbound state to be directly compared to the PP1-bound state. Our results show that MYPT1(1-98) behaves like a two-domain protein in solution. The first 40 residues of MYPT1(1-98), the disordered region, are intrinsically disordered and highly dynamic, whereas residues 41-98, the folded ankyrin-repeat region, are well-structured and rigid. Furthermore, the integrated use of NMR and biophysical data enabled us to calculate an ensemble model for MYPT1(1-98). The most prominent structural feature of the MYPT1(1-98) ensemble is a 25% populated transient α-helix in the disordered region of MYPT1(1-98). This α-helix becomes fully populated when bound to PP1 and, as we show, likely plays a central role in the formation of the MYPT1-PP1 holoenzyme complex. Finally, this combined analysis shows that the structural and dynamic behaviors exhibited by MYPT1 for PP1 are distinct from those of any other previously analyzed PP1 regulatory protein. Collectively, these data enable us to present a new model of the molecular events that drive MYPT1-PP1 holoenzyme formation and demonstrate that there are structural differences in unbound PP1 regulators that have not been previously observed. Thus, this work adds significant insights to the currently limited data for molecular structures and dynamics of PP1 regulators.
肌松是由肌球蛋白调节轻链 Ser19 的去磷酸化触发的。该反应由全酶肌球蛋白磷酸酶 (MP) 催化，其中包括催化亚基蛋白磷酸酶 1 (PP1) 和调节靶向亚基 (MYPT)。MYPT1(肌球蛋白磷酸酶靶向亚基 1) 负责将全酶靶向肌肉中的亚细胞区室，并指导 PP1 特异性针对肌球蛋白。为了了解导致 MYPT1-PP1 全酶形成的分子事件，我们使用 NMR 光谱学来确定未结合的 MYPT1 的结构和动态特征。这使得可以直接比较 MYPT1 在游离、未结合状态下的构象与与 PP1 结合的状态。我们的结果表明，MYPT1(1-98)在溶液中表现为一种两域蛋白。MYPT1(1-98)的前 40 个残基，即无序区，是无规卷曲的，高度动态的，而残基 41-98，即折叠的锚蛋白重复区，是结构良好的刚性结构。此外，NMR 和生物物理数据的综合使用使我们能够为 MYPT1(1-98)计算一个整体模型。MYPT1(1-98)整体模型的最突出的结构特征是 MYPT1(1-98)无序区中 25%的瞬态 α-螺旋。当与 PP1 结合时，该 α-螺旋完全被占据，并且正如我们所示，它可能在 MYPT1-PP1 全酶复合物的形成中起核心作用。最后，这种综合分析表明，MYPT1 对 PP1 表现出的结构和动态行为与任何其他以前分析的 PP1 调节蛋白都不同。总的来说，这些数据使我们能够提出一个新的模型，用于驱动 MYPT1-PP1 全酶形成的分子事件，并证明在未结合的 PP1 调节蛋白中存在以前未观察到的结构差异。因此，这项工作为目前有限的 PP1 调节蛋白的分子结构和动力学数据提供了重要的见解。
