Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban
Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban
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DOI：
10.1021/bi047571e
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发表时间：
2005-03-22
期刊：
影响因子：
2.9
通讯作者：
Veglia, G
中科院分区：
文献类型：
--
作者：
Metcalfe, EE;Traaseth, NJ;Veglia, G
Phospholamban (PLB) 是心肌中肌（内）质钙腺苷三磷酸酶 (SERCA) 的 52 个氨基酸膜内源性调节剂。 PLB 在 S16 上的磷酸化和去磷酸化通过一种未确定的机制调节其对 SERCA 的调节作用。在本文中，我们利用多维H-1/N-15溶液NMR方法为PLB控制SERCA对S16磷酸化建立结构和动力学基础。在我们的研究中，我们使用 PLB 的单体、完全活性突变体，其中 C36、C41 和 C46 已分别突变为 A36、F41 和 A46。我们的数据表明，磷酸化破坏了单体 PLB 的“L 形”结构，导致细胞质螺旋（结构域 Ia）和连接该结构域与跨膜螺旋（结构域 Ib 和 II）的短环（残基 17-21）显着解旋。伴随着这种构象转变，我们还发现皮秒到纳秒和微秒到毫秒时间尺度的动力学发生显着变化。残基 1-25 的 H-1/N-15 异核 NOE 值显着低于未磷酸化 PLB，跨膜结构域的 NOE 值略低，反映出整个蛋白质的运动受限较少。这些数据得到了细胞质和环区域中存在的更快的自旋晶格弛豫率 (R-1) 以及跨膜域中观察到的增强的自旋-自旋横向弛豫率 (R-2) 的支持。这些结果表明，虽然 S16 磷酸化诱导局部结构转变，但 PLB 主链动力学的变化会在整个蛋白质主链中传播。我们提出 PLB 磷酸化的调节机制涉及有序到无序的转变，导致 PLB 对 SERCA 的抑制减弱。
Phospholamban (PLB) is a 52 amino acid membrane-endogenous regulator of the sarco(endo)-plasmic calcium adenosinetriphosphatase (SERCA) in cardiac muscle. PLB's phosphorylation and dephosphorylation at S16 modulate its regulatory effect on SERCA by an undetermined mechanism. In this paper, we use multidimensional H-1/N-15 solution NMR methods to establish the structural and dynamics basis for PLB's control of SERCA upon S16 phosphorylation. For our studies, we use a monomeric, fully active mutant of PLB, where C36, C41, and C46 have been mutated to A36, F41, and A46, respectively. Our data show that phosphorylation disrupts the "L-shaped" structure of monomeric PLB, causing significant unwinding of both the cytoplasmic helix (domain la) and the short loop (residues 17-21) connecting this domain to the transmembrane helix (domains Ib and II). Concomitant with this conformational transition, we also find pronounced changes in both the pico- to nanosecond and the micro- to millisecond time scale dynamics. The H-1/N-15 heteronuclear NOE values for residues 1-25 are significantly lower than those of unphosphorylated PLB, with slightly lower NOE values in the transmembrane domain, reflecting less restricted motion throughout the whole protein. These data are supported by the faster spin-lattice relaxation rates (R-1) present in both the cytoplasmic and loop regions and by the enhanced spin-spin transverse relaxation rates (R-2) observed in the transmembrane domain. These results demonstrate that while S16 phosphorylation induces a localized structural transition, changes in PLB's backbone dynamics are propagated throughout the protein backbone. We propose that the regulatory mechanism of PLB phosphorylation involves an order-to-disorder transition, resulting in a decrease in the PLB inhibition of SERCA.