IDENTIFICATION OF BINDING RESIDUES ON SURFACE CYTOCHROME POR

表面细胞色素 POR 上结合残基的鉴定

• 批准号: 7955531
• 负责人: VISHAL AGRAWAL
• 金额: $ 0.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955531
• 关键词: Active Sites; Affect; Amber; Americas; Binding; Binding Sites; Biological Assay; CYP17A1 gene; CYP3A4 gene; Charge; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Cytochrome P450; Docking; Drug usage; Electron Transport; Endocrinology; Enzymes; Erythromycin; Funding; Genets; Genomics; Grant; Hepatic; Human; Imagery; Informatics; Institution; Lyase; Mixed Function Oxygenases; Modeling; Oxidation-Reduction; Oxidoreductase; Pharmaceutical Preparations; Progesterone; Proteins; Research; Research Personnel; Resources; Science; Site-Directed Mutagenesis; Source; Structure; Surface; United States National Academy of Sciences; United States National Institutes of Health; Variant; Work; biocomputing; drug metabolism; in vitro testing; mutant; steroid hormone biosynthesis; ward

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. P450 oxidoreductase (POR) is the only know protein to transfer electrons to all the type II microsomal cytochrome P450�s, including those that metabolize more than 80% of clinically used drugs and are involved in the biosynthesis of steroid hormones (1). Previous work done in the lab with steroidogenic P450c17 has shown that selective mutants on the surface of P450c17 that reverse the surface charge in the POR-binding site of P450c17 can selectively ablate the 17,20 lyase activity while preserving the 17 α-hydroxylase activity (2). Our lab has previously built the model of P450c17 using the computational facility at CGL (3). P450 3A4 is responsible for around 40-50% of hepatic drug metabolism. Human P450 3A4 has been crystallized without substrate and bound to smaller substrates metyropone and progesterone (4); in presence of bigger substrate erythromycin (5,6). Depending on the size of bound substrate there were dramatic changes in the volume of the active site, but the description of these structures did not indicate whether these changes in the substrate-binding domains of 3A4 altered the geometry of the redox-partner binding site. A substrate induced conformational differences in 3A4 implies that different basic residues on P450 3A4 will interact with different acidic residues on POR, hence there will be fundamental differences in the mechanisms of electron transfer from POR to CYP3A4, depending upon the drug being metabolized. My previous work with POR variants has shown that POR variant affect the activity of different P450 differently (7-9), indicating that there is more than one way in which P450 and POR interact with each other The present work seeks to identify residues on the surface steroidogenic P450c17 and drug metabolizing P450 3A4 that interact with the reductase partner POR. This work will involve in silico docking POR and P450. The results from docking will be subsequently energy minimized using AMBER. The residues identified from in silico work will be tested in vitro through site-directed mutagenesis and enzyme assay for activity. Access to the computational facility at CGL will help me perform the necessary in silico work. 1. Miller, W. L. (2005) Endocrinology 146(6), 2544-2550 2. Geller, D. H., Auchus, R. J., Mendonca, B. B., and Miller, W. L. (1997) Nat Genet 17(2), 201-205 3. Auchus, R. J., and Miller, W. L. (1999) Mol Endocrinol 13(7), 1169-1182 4. Williams, P. A., Cosme, J., Vinkovic, D. M., Ward, A., Angove, H. C., Day, P. J., Vonrhein, C., Tickle, I. J., and Jhoti, H. (2004) Science 305(5684), 683-686 5. Ekroos, M., and Sjogren, T. (2006) Proc Natl Acad Sci U S A 103(37), 13682-13687 6. Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2004) J. Biol. Chem. 279(37), 38091-38094 7. Agrawal, V., Huang, N., and Miller, W. L. (2008) Pharmacogenet Genomics 18(7), 569-576 8. Huang, N., Agrawal, V., Giacomini, K. M., and Miller, W. L. (2008) Proceedings Of The National Academy Of Sciences Of The United States Of America 105(5), 1733-1738 9. Huang, N., Pandey, A. V., Agrawal, V., Reardon, W., Lapunzina, P. D., Mowat, D., Jabs, E. W., Van Vliet, G., Sack, J., Fluck, C. E., and Miller, W. L. (2005) Am J Hum Genet 76(5), 729-749

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代表。列出的机构是 对于中心，这不一定是调查员的机构。 P450氧化还原酶（POR）是将电子转移到所有II型微粒体细胞色素P450的唯一知道的蛋白质，包括代谢超过80％的临床使用药物并参与类固醇激素的生物合成的蛋白质（1）。使用类固醇生成P450C17在实验室完成的先前工作表明，在P450C17表面上的选择性突变体反转P450C17的POR结合位点的表面电荷可以选择性地烧光17,20裂解酶活性，同时保留17α-羟化酶活性（2）。我们的实验室以前已经使用CGL（3）的计算设施构建了P450C17的模型。 P450 3A4负责约40-50％的肝药物代谢。人P450 3A4已结晶而没有底物，并与较小的底物甲摩酮和孕酮结合（4）；存在较大的底物红霉素（5,6）。根据结合底物的大小，活动位点的体积发生了巨大变化，但是这些结构的描述并未指示3A4的底物结合域中的这些变化是否改变了氧化还原伴侣结合位点的几何形状。底物诱导的3A4中构象差异意味着P450 3A4上的不同基本残基将与POR上的不同酸性残基相互作用，因此，根据药物被代谢的药物，电子从POR到CYP3A4的机制将存在基本差异。我以前使用POR变体的工作表明，POR变体对不同的P450的活性有所不同（7-9），表明P450和POR相互作用有多种方式 目前的工作旨在确定与还原酶伴侣POR相互作用的表面类固醇生成P450C17和药物代谢P450 3A4的残基。这项工作将涉及硅离对接POR和P450。对接的结果将随后使用琥珀最小化。从计算机工作中鉴定出的残基将在体外通过位置定向的诱变和活性酶测定进行测试。访问CGL的计算设施将有助于我执行必要的计算机工作。 1。Miller，W。L.（2005）内分泌学146（6），2544-2550 2。Geller，D。H.，Auchus，R。J.，Mendonca，B。B.和Miller，W。L.（1997）Nat Genet 17（2），201-205 3。Auchus，R。J.和Miller，W。L.（1999）Mol Endocrinol 13（7），1169-1182 4。Williams，P.A.，Cosme，J.，Vinkovic，D.M.，Ward，A.，Angove，H.C.，Day，P.J.，Vonrhein，C.，Tickle，I.J。 5。Ekroos，M。和Sjogren，T。（2006）Proc Natl Acad Sci u S A 103（37），13682-13687 6。Yano，J。K.，Wester，M。R.，Schoch，G.A.，Griffin，K。J.，Stout，C。D.和Johnson，E。F.（2004）J。Biol。化学279（37），38091-38094 7。Agrawal，V.，Huang，N。和Miller，W。L.（2008）Pharmacogenet Genomics 18（7），569-576 8。Huang，N.，Agrawal，V.，Giacomini，K。M.和Miller，W。L.（2008）美国美国国家科学院的会议记录，美国美国国家科学院的会议记录。1733-1738，1733-1738 9。Huang，N.