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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 类固醇生成的急性调节蛋白（StAR）是一种37 kDa蛋白，通过增加胆固醇的流量从线粒体外膜（OMM）增加到内膜，从而模拟类固醇生成，在该膜通过P450SCC转化为妊娠烯醇酮。恒星仅作用于pH 3.5〜4.0处的OMM，部分展开至熔融球。有人建议但尚未证明这种熔融的大球过渡是活动所必需的。 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不受蛋白水解的影响。 建模表明，形成固醇结合口袋（SBP）地板的C-螺旋稳定在相邻环上。在两个条件下，我们使用Amber 7.0在300K下将恒星模型进行了300K的分子动力学：D，E和H的默认设置和质子化（模拟pH 4.0）。轨迹分析表明，W1环路和C-螺旋在pH 4时移动得多，打开和关闭SBP。 我们通过设计两个将C螺旋与W1环连接的二硫化物突变体设计来测试该运动对恒星活动的影响。建模表明，这些配对的突变体D106C/A268C（DA）和S100C/S261C（SS）都没有，恒星折叠或SBP的大小和形状。 DA和SS突变体用内部载体表达并纯化。 MS分析证实了二硫化物的位置。 DA在体外丧失了所有胆固醇结合能力和类固醇活性，并且在体外孤立的线粒体失去了胆固醇的能力，而SS则损失了约50％的两者。通过用DTT破坏二硫化物来恢复完全结合和活性。这些数据支持恒星活性需要pH依赖性的过渡到OMM上的熔融球。