P450 and NO Synthase Regulation by Multiprotein Complexes

摘要 我们证实了含有Hsp90/Hsp70伴侣的多蛋白伴侣复合体在 神经元一氧化氮合酶(NNOS)和其他P450细胞色素在维持蛋白质质量控制中的作用。在 在上一个资助周期中，我们将重点转向用单粒子EM和冷冻EM来确定nNOS的结构 帮助阐明是什么触发了nNOS的伴侣识别。在这些研究过程中，我们 偶然发现全长的细胞色素P450还原酶(CPR)复合体可以形成 在两栖动物中，这是一种表面活性剂，可以在没有水的情况下自组装并稳定膜蛋白。 洗涤剂。值得注意的是，这些络合物功能齐全，稳定，通过单一的四聚体可见 颗粒EM法，发现含有等摩尔量的P450和CPR。因此，在当前 建议，我们的目标是确定微粒体P450与CPR复合体的第一结构。我们的前辈 用这些单颗粒成功地阐明了nNOS和P450 BM3的第一个全长二聚体结构 方法论为这项事业提供了信心。目前，我们已经解析了加氧酶的结构 通过冷冻-EM方法将全长nNOS的结构域转换为5？分辨率。值得注意的是，一个四聚络合物 P450：CPR复合体类似于nNOS和BM3的二聚体结构，都具有P450和 各单体中的CPR结构域。我们有一个由新兴市场专家组成的协作团队，他们的知识将协同 凭借我们在P450和多蛋白质复合体方面的专业知识来解决这一令人兴奋但具有挑战性的项目。也在 在之前的资助周期中，我们表明Hsp90是细胞内血红素插入缺乏血红素的apo-nNOS所必需的 最近，斯图尔实验室发现，Hsp90是将血红素插入鸟苷环化酶所必需的， 血红蛋白和诱导型一氧化氮合酶。因此，伴侣复合体存在的前提是它是一种‘亚铁血红素 插入酶‘。最近，我们发现与apo-nnos免疫沉淀的细胞蛋白是稳定的。 结合并催化血红素插入apo-nNOS。HSP90和HSP70与apo-nNOS结合并发挥关键作用 Hsp90和Hsp70在血红素插入中的作用，但仅有Hsp90和Hsp70是不够的。在目前的提案中，我们 寻求确定构成血红素插入酶活性的其他蛋白质，部分是通过鉴定 LC-MS/MS免疫共沉淀蛋白质的研究 血红素插入酶复合体组装完成。目标的成功完成将提供一个开创性的 研究微粒体P450的新平台，阐明P450的第一结构：CPR复合体，以及 描述将亚铁血红素插入到亚铁血红素蛋白中的蛋白质机制。

ABSTRACT We established that a multiprotein chaperone complex containing Hsp90/Hsp70 chaperones plays an essential role in maintaining protein quality control of neuronal NO synthase (nNOS) and other P450 cytochromes. In the last grant cycle, we turned our focus to determine the structure of nNOS by single particle EM and cryo-EM methods to help elucidate what triggers chaperone recognition of nNOS. In the course of these studies, we serendipitously discovered that full-length CYP2B4:cytochrome P450 reductase (CPR) complexes could form in amphipols, which are surfactants that self-assemble and stabilize membrane proteins in the absence of detergent. Remarkably, these complexes were fully functional, stable, and visible as tetramers by single particle EM methods, and found to contain equimolar amounts of P450 and CPR. Thus, in the current proposal, we aim to determine the first structure of a microsomal P450 in complex with CPR. Our prior success in elucidating the first full-length dimeric structure of nNOS and P450 BM3 by these single particle methods provides confidence in this undertaking. Currently we have resolved the structure of the oxygenase domain of full-length nNOS to 5 Å resolution by cryo-EM methods. It is noteworthy that a tetrameric complex of P450:CPR complex is analogous to the dimeric architecture of nNOS and BM3, both having a P450 and CPR domain in each monomer. We have a collaborative team of EM experts whose knowledge will synergize with our expertise in P450 and multi-protein complexes to tackle this exciting but challenging project. Also in prior grant cycles, we showed that Hsp90 is needed for cellular heme insertion into heme-deficient apo-nNOS and more recently the Stuehr lab has shown that Hsp90 is needed to insert heme into guanylate cyclase, hemoglobin, and inducible NO synthase. Thus, premise exists for a chaperone complex that is a ‘heme insertase’. Recently, we discovered that the cellular proteins that immunoprecipitate with apo-nNOS are stably bound and catalyze heme insertion into apo-nNOS. Hsp90 and Hsp70 are bound to apo-nNOS and play a key role in heme insertion but Hsp90 and Hsp70 chaperones alone are not sufficient. In the current proposal, we seek to identify the other proteins that make up the heme insertase activity, in part by identification of the co-immunoprecipitated proteins by LC-MS/MS. Moreover, we will define how the chaperone-based heme insertase complex is assembled. The successful completion of the aims will provide a groundbreaking new platform for the study of microsomal P450s, elucidate the first structure of a P450:CPR complex, and characterize the protein machinery that inserts heme into hemeproteins.