Crystal structure of signal peptide peptidase with engineered antibody fragment

描述(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呈递自然杀伤细胞识别的特定多肽来报告其蛋白质折叠机制的保真度。这些多肽是来自新合成的组织相容性复合体1b(MHC-1b)分子的信号肽(SP)的残留物，向NK细胞发出蛋白质合成正常的信号。负责将信号肽从内质网中释放出来的酶是一种膜包埋的天冬氨酸蛋白酶，信号肽酶(SPP)。除了MHC Class 1b外，人类SPP底物还包括来自参与免疫反应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多肽。SPP和早老素形成了仅有的三个膜内蛋白水解酶超家族之一。这项提议的目的是解决SPP的第一个高分辨率X射线晶体结构，提供第一个膜内天冬氨酸蛋白酶(IAP)的快照。调控膜内蛋白分解的细节，从细胞生物信号到活性部位化学，都具有基本的生化重要性和潜在的治疗应用。底物如何在脂膜疏水空间的范围内被呈现和水解在很大程度上仍然是一个谜。SPP的结构不仅有助于蛋白平衡和膜内蛋白分解的生物学，而且有助于我们了解膜蛋白，其中很少有结构是高分辨率的。我们将SPP的结构解析为具有单链抗体片段(ScFv)伴侣的复合体。ScFv将被设计成与多肽表位紧密结合，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。我们预计，我们紧密结合的结晶伴侣将固定SPP环，并提供稳定的晶格，导致更好的衍射晶体。与以前使用针对相关膜蛋白的亲和试剂的结晶伴侣不同，这项技术具有潜在的变革性，因为表位可以整合到任何膜蛋白结晶靶标中。预期的结果是IAP的分子结构，包括活性部位的位置和任何底物对接补丁。SPP的结构将提供对生化和功能的洞察，以便与研究得很好的可溶性天冬氨酸蛋白酶进行比较，并为定向生化研究评估调节催化的关键残基奠定基础。从长远来看，这一知识可能有助于开发免疫调节药物或抗丙型肝炎病毒的药物。 与公众健康相关：信号肽酶是一种参与天然免疫和获得性免疫的重要脂沉酶，与多种疾病有关，包括阿尔茨海默氏症和丙型肝炎病毒感染。这项工作旨在确定SPP的第一分子结构，以了解其在健康和疾病中的作用，并帮助开发潜在的阿尔茨海默氏症和丙型肝炎治疗药物。

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.