Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation
Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation
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DOI：
10.1038/srep14557
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发表时间：
2015-09-28
影响因子：
4.6
通讯作者：
Kelekar, Ameeta
中科院分区：
文献类型：
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作者：
Karim, Christine B.;Espinoza-Fonseca, L. Michel;Kelekar, Ameeta
我们以前曾证明，在丝氨酸13(Ser13)处，一种54个残基的Bcl-2蛋白Noxa的磷酸化抑制了它通过与典型结合伙伴Mcl-1相互作用而促进细胞凋亡的能力。利用EPR光谱、分子动力学(MD)模拟和结合分析，我们提供了证据，表明磷酸化引起的结构变化部分掩盖了Noxa的BH3结构域，抑制了Noxa-Mcl-1的相互作用。未磷酸化的Noxa的EPR显示有序状态和动态无序状态之间的平衡，其中自旋标记的氨基酸TOAC包含在BH3结构域中。MCL-1进一步限制了非磷酸化Noxa的有序组分，但保持了pSer13 Noxa的轮廓不变。微秒MD模拟表明，未磷酸化的NoxA的BH3结构域位于一个柔性环中，连接两个反平行的β-折叠，两侧是无序的N-末端和C-末端，Ser13磷酸化创建了一个盐桥网络，促进了N-末端和BH3结构域之间的相互作用。EPR表明，插入在N-末端附近的自旋标记在未磷酸化的Noxa中被弱固定，与溶剂暴露的螺旋/环一致，但在pSer13 Noxa中受到强烈限制，表明如MD模拟所预测的那样，肽骨架更有序。总之，这些研究揭示了一种新的机制，通过这种机制，远端丝氨酸的磷酸化抑制了促凋亡的BH3结构域，并促进了细胞的存活。
We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics ( MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel beta-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N- terminus and the BH3 domain. EPR showed that a spin label inserted near the N- terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.