Crystal structure of signal peptide peptidase with engineered antibody fragment

描述（由申请人提供）：作为先天免疫的一部分，真核细胞通过在其细胞表面上呈递被自然杀伤细胞识别的特异性肽来报告其蛋白质折叠机制的保真度。这些肽是来自新合成的组织相容性复合物1b（MHC-1b）分子的信号肽（SP）的残余物，并向NK细胞发出蛋白质合成正常的信号。负责修剪信号肽以将其从ER膜释放的酶是膜包埋的乙酰基蛋白酶，信号肽肽酶（SPP）。除了MHC 1b类，人SPP底物还包括来自参与免疫应答和肌肉收缩的蛋白质的SP。SPP也被丙型肝炎病毒劫持进行复制，并且与早老素有关，早老素在阿尔茨海默病中使用类似的化学物质产生淀粉样蛋白生成肽。SPP和早老素形成仅有的三个膜内蛋白酶超家族之一。 该提案的目的是解决SPP的第一个高分辨率X射线晶体结构，提供膜内乙酰基蛋白酶（IAP）的第一个快照。从细胞生物学信号到活性位点化学，受调控的膜内蛋白水解的细节具有基本的生物化学重要性和潜在的治疗应用。底物是如何在脂膜的疏水空间内呈现和水解的，这在很大程度上仍然是个谜。SPP的结构不仅有助于蛋白质稳定和膜内蛋白水解的生物学，而且有助于我们对膜蛋白的了解，其中很少有结构是已知的高分辨率。 我们将解决SPP的结构作为与单链抗体片段（scFv）伴侣的复合物。scFv将被工程化以紧密结合肽表位，同时保留已知使用远离表位结合位点的晶体接触的scFv的支架。我们期望紧密结合的结晶分子伴侣将抑制SPP环并提供稳定的晶格，从而产生更好的衍射晶体。与先前使用结晶分子伴侣的工作（其采用对感兴趣的膜蛋白特异性的亲和试剂）相比，该技术具有潜在的变革性，因为表位可以并入任何膜蛋白结晶靶标中。 预期的结果是IAP的分子结构，包括活性位点和任何底物对接补丁的位置。SPP的结构将提供深入了解生物化学和功能的比较，充分研究的可溶性乙酰基蛋白酶和定向生化研究的基础，以评估关键残基，调节催化。从长远来看，这些知识可能有助于开发用于免疫调节或抗丙型肝炎病毒的药物。 公共卫生相关性：信号肽肽酶是参与先天性和适应性免疫的必需的脂质浸没酶，并且与多种疾病（包括阿尔茨海默氏症和丙型肝炎病毒感染）有关。本文提出的工作旨在确定SPP的第一个分子结构，以了解其在健康和疾病中的作用，并帮助开发特异性抑制剂作为潜在的阿尔茨海默氏症和丙型肝炎疗法。

DESCRIPTION (provided by applicant): As a part of innate immunity, the eukaryotic cell reports on the fidelity of its protein folding machinery by presenting specific peptides on its cell surface that are recognized by Natural Killer cells. These peptides are remnants of signal peptides (SPs) derived from newly synthesized histocompatibility complex 1b (MHC-1b) molecules, and signal to NK cells that protein synthesis is normal. The enzyme responsible for trimming signal peptides to liberate them from the ER membrane is a membrane-embedded aspartyl protease, signal peptide peptidase (SPP). In addition MHC Class 1b, human SPP substrates include SPs from proteins involved in immune response and muscle contraction. SPP is also hijacked by the Hepatitis c virus for replication, and is related to presenilin, which uses similar chemistry to generate amyloidogenic peptides in Alzheimer Disease. SPP and presenilin form one of just three superfamilies of intramembrane proteases. The objective of this proposal is to solve the first high resolution X-ray crystal structure of SPP, providing the first snapshot of an intramembrane aspartyl protease (IAP). The details of regulated intramembrane proteolysis, from cell biological signaling to active site chemistry, are of both fundamental biochemical importance and potential therapeutic application. How substrates are presented and hydrolyzed within the confines of the hydrophobic space of the lipid membrane remain largely a mystery. The structure of SPP will contribute not only to the biology of proteostasis and intramembrane proteolysis, but also to our knowledge of membrane proteins, few structures of which are known to high resolution. We will solve the structure of SPP as a complex with a single chain antibody fragment (scFv) chaperone. The scFv will be engineered for tight binding to a peptide epitope while retaining the scaffold of an scFv known to use crystal contacts remote from the epitope binding site. We expect that our tightly bound crystallization chaperone will immobilize an SPP loop and provide a stable crystal lattice, leading to better diffracting crystals. In contrast to previous work with crystallization chaperones, which employed affinity reagents specific to the membrane protein of interest, this technology is potentially transformative as the epitope can be incorporated into any membrane protein crystallization target. The expected outcome is the molecular architecture of IAPs, including the location of the active site and any substrate-docking patches. The SPP structure will provide insight into biochemistry and function for comparison with well-studied soluble aspartyl proteases and a basis for directed biochemical studies to evaluate key residues that modulate catalysis. In the long-term this knowledge is likely to facilitate drug development for immune modulation or agents against the Hepatitis C virus. PUBLIC HEALTH RELEVANCE: Signal peptide peptidase is an essential lipid-submerged enzyme involved in innate and adaptive immunity, and is implicated in a variety of diseases, including Alzheimer's and Hepatitis C viral infection. The work proposed here aims to determine the first molecular structure of SPP in order to understand its role in health and disease, and aid in development of specific inhibitors as potential Alzheimer's and Hepatitis C therapies.