P450 and NO Synthase Regulation by Multiprotein Complexes

摘要 我们确定了含有Hsp 90/Hsp 70分子伴侣的多蛋白分子伴侣复合物在Hsp 90/Hsp 70分子伴侣中起着重要的作用。 在维持神经元NO合酶（nNOS）和其他P450细胞色素的蛋白质质量控制中的作用。在 在上一个资助周期，我们将研究重点转向了用单粒子电镜和冷冻电镜来确定nNOS的结构 方法，以帮助阐明是什么触发分子伴侣识别nNOS。在这些研究中，我们 偶然发现，全长CYP 2B 4：细胞色素P450还原酶（CPR）复合物可以形成 在作为表面活性剂的两性分子中，其在不存在下自组装并稳定膜蛋白， 清洁剂。值得注意的是，这些复合物是完全功能性的，稳定的，并且通过单个聚合物可以看到四聚体。 颗粒EM方法，并发现含有等摩尔量的P450和CPR。因此，在目前 建议，我们的目标是确定与CPR复合的微粒体P450的第一个结构。我们事先 通过这些单颗粒成功阐明了nNOS和P450 BM 3的第一个全长二聚体结构 方法为这项工作提供了信心。目前，我们已经解决了加氧酶的结构 全长nNOS的结构域，以105 μ m分辨率通过冷冻-EM方法。值得注意的是， P450：CPR复合物的二聚体结构类似于nNOS和BM 3的二聚体结构，两者都具有P450和 CPR结构域。我们有一个EM专家的协作团队，他们的知识将协同 凭借我们在P450和多蛋白质复合物方面的专业知识，我们可以解决这个令人兴奋但具有挑战性的项目。也 在之前的研究中，我们发现Hsp 90是血红素插入血红素缺陷型apo-nNOS所必需的 最近Stuehr实验室已经表明，Hsp 90是将血红素插入鸟苷酸环化酶所必需的， 血红蛋白和诱导型NO合酶。因此，前提存在的伴侣复合物是一个'血红素 插入酶。最近，我们发现与apo-nNOS免疫沉淀的细胞蛋白质是稳定的， 结合并催化血红素插入apo-nNOS。Hsp 90和Hsp 70与apo-nNOS结合，在apo-nNOS的表达中起关键作用 在血红素插入中的作用，但单独的Hsp 90和Hsp 70分子伴侣是不够的。在目前的提案中，我们 试图鉴定构成血红素插入酶活性的其他蛋白质，部分是通过鉴定 通过LC-MS/MS的免疫共沉淀蛋白。此外，我们将定义如何伴侣为基础的 装配血红素插入酶复合物。这些目标的成功完成将为我们提供一个 研究微粒体P450的新平台，阐明P450：CPR复合物的第一个结构， 表征将血红素插入血红素蛋白的蛋白质机制。

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.