MOLECULAR DYNAMICS OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN, STAR

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。类固醇合成急性调节蛋白(STAR)是一种37 kDa的蛋白质，它通过增加胆固醇从线粒体膜外膜到内膜的流量来模拟类固醇的形成，在内膜上被P450scc转化为孕烯醇酮。恒星只作用于OMM，并在pH 3.5~4.0的范围内部分展开为熔球；有人提出，但尚未得到证实，这种熔球转变是活动所必需的。C-末端螺旋与OMM相互作用，因为由OMM脂类组成的脂质体仅保护该螺旋免受蛋白质降解。建模表明，形成固醇结合口袋(SBP)底部的这个C-螺旋是通过与相邻环的氢键稳定的。我们使用Amber 7.0在两种条件下对我们的STAR模型进行了3nSec的分子动力学实验：默认设置以及D、E和H的质子化(模拟pH 4.0)。轨迹分析表明，在pH为4时，W1环和C-螺旋的流动性更强，从而打开和关闭SBP。我们通过设计两个将C-螺旋与W1环连接起来的二硫键突变体来测试这一运动对恒星活动的影响。模型显示，D106C/A268C(DA)和S100C/S261C(SS)这两对突变体都没有破坏星状折叠或SBP的大小和形状。用内含素载体表达并纯化了DA和SS突变体。MS分析确认了二硫化物的位置。DA在体外失去了与线粒体的所有胆固醇结合能力和类固醇合成活性，SS失去了约50%的胆固醇结合能力和类固醇生成活性。通过用DTT破坏二硫键来恢复每个分子的完全结合和活性。这些数据支持这样的模型，即恒星活动需要与pH有关的转变为OMM上的熔化球体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Steroidogenic acute regulatory protein (StAR) is a 37 kDa protein that simulates steroidogenesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane where it is converted to pregnenolone by P450scc. StAR acts exclusively on the OMM and partially unfolds to a molten globule at pH 3.5 ~ 4.0; it has been suggested, but not proven, that this molten globule transition is required for activity. The C-terminal helix interacts with the OMM, as liposomes composed of OMM lipids protect only this helix from proteolysis. Modeling shows that this C-helix, which forms the floor of the sterol-binding pocket (SBP), is stabilized by hydrogen bonding to adjacent loops. We subjected our model of StAR to molecular dynamics for 3 nsec at 300K using AMBER 7.0 under two conditions: default settings and protonation of D, E and H (to mimic pH 4.0). Trajectory analysis shows the W1 loop and C-helix are much more mobile at pH 4, opening and closing the SBP. We tested the effect of this movement on StAR activity by designing two disulfide mutants linking the C- helix to the W1 loop. Modeling showed that neither of these paired mutants, D106C/A268C (DA) and S100C/S261C (SS), disrupted StAR folding or the size and shape of the SBP. The DA and SS mutants were expressed with an intein vector and purified. MS analysis confirmed the locations of the disulfides. DA lost all cholesterol-binding capacity and steroidogenic activity with isolated mitochondria in vitro, and SS lost ~50% of both. Full binding and activity was restored to each by disrupting the disulfides with DTT. These data support the model that StAR activity requires a pH-dependant transition to a molten globule on the OMM.