MOLECULAR DYNAMICS OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN, STAR

类固醇急性调节蛋白 STAR 的分子动力学

• 批准号: 7723505
• 负责人: WALTER L. MILLER
• 金额: $ 0.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723505
• 关键词: Amber; Binding; C-terminal; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Condition; Data; Disulfides; Floor; Funding; Grant; Helix (Snails); Hydrogen Bonding; In Vitro; Institution; Link; Liposomes; Location; Membrane; Membrane Lipids; Mitochondria; Modeling; Movement; Outer Mitochondrial Membrane; Pregnenolone; Proteins; Proteolysis; Research; Research Personnel; Resources; Shapes; Simulate; Source; Steroid biosynthesis; Sterols; Testing; United States National Institutes of Health; design; ear helix; intein; molecular dynamics; mutant; protein folding; protonation; size; steroidogenic acute regulatory protein; vector

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 类固醇生成急性调节蛋白（星星）是一种37 kDa的蛋白质，通过增加胆固醇从线粒体外膜（OMM）到内膜的流动来模拟类固醇生成，胆固醇在内膜中被P450 SCC转化为胆固醇烯醇酮。星星仅作用于OMM，在pH 3.5 ~ 4.0时部分解折叠成熔融球;有人提出，但未证明，这种熔融球转变是活性所必需的。 C-末端螺旋与OMM相互作用，因为由OMM脂质组成的脂质体仅保护该螺旋免受蛋白水解。 建模表明，这种C-螺旋，形成的甾醇结合口袋（SBP）的地板，是稳定的氢键相邻的环。我们将我们的模型的星星分子动力学3纳秒在300 K下使用AMBER 7.0在两个条件下：默认设置和质子化的D，E和H（模拟pH 4.0）。轨迹分析显示，W1环和C-螺旋在pH 4时移动的更多，打开和关闭SBP。 我们通过设计两个连接C-螺旋和W1环的二硫键突变体来测试这种运动对星星活性的影响。模型显示，D106 C/A268 C（DA）和S100 C/S261 C（SS）这两个配对突变体都没有破坏星星折叠或SBP的大小和形状。 用内含肽载体表达DA和SS突变体并纯化。MS分析证实了二硫化物的位置。 DA失去了所有的胆固醇结合能力和类固醇生成活性与离体线粒体在体外，和SS失去了约50%。通过用DTT破坏二硫键，使每一个都恢复完全结合和活性。这些数据支持这样一种模型，即星星活动需要在有机金属膜上经过一个依赖于pH值的过渡过程，形成一个熔融小球。