Engineered antibody fragments for cocrystallization with signal peptide peptidase

用于与信号肽肽酶共结晶的工程化抗体片段

• 批准号: 8520337
• 负责人: JENNIFER A MAYNARD
• 金额: $ 28.81万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8520337
• 关键词: Active Sites; Address; Affinity; Alzheimer' s Disease; Antibodies; Aspartate; Binding; Biochemical; Bioinformatics; Biological; Biology; Cell surface; Cells; Chemistry; Cocrystallography; Complex; Crystallization; Development; Disease; Distant; Docking; Endoplasmic Reticulum; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Escherichia coli; Gel Chromatography; Hepatitis C Therapy; Hepatitis C virus; His-His-His-His-His-His; Homologous Gene; Hydrolysis; Immune response; Immunity; Immunoglobulin Fragments; Integral Membrane Protein; Knowledge; Location; Major Histocompatibility Complex; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular; Molecular Chaperones; Muscle Contraction; Mutagenesis; Natural Immunity; Natural Killer Cells; Outcome; Parents; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Phage Display; Process; Proteins; Proteolysis; Randomized; Reagent; Resolution; Role; Route; Signal Transduction; Site-Directed Mutagenesis; Structure; Technology; Therapeutic; Variant; Virus Replication; Work; directed evolution; experience; immunoregulation; inhibitor/antagonist; interest; new technology; novel strategies; presenilin; protein structure; signal peptide peptidase; virus pathogenesis

Hydrophobic membrane proteins perform a variety of functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. The objectives of this proposal are to develop a toolbox of chaperones and use them to crystallize and solve the de novo, high resolution structure of two signal peptide peptidases (SPPs), which use catalytic aspartates to conduct hydrolysis within the lipid membrane. In contrast to work employing affinity reagents specific to the membrane protein of interest, our potentialy transformative aproach uses hypercrystallizable single chain antibody fragments (scFvs). Our chaperones are engineered for tight binding to a short epitope that can be inserted into any membrane protein. We expect that our tightly bound scFv chaperone will immobilize an SPP loop and provide a stable crystal lattice, leading to better diffracting crystals. SPPs trim signal peptides (SPs) to liberate them from the endoplasmic reticulum membrane. SPP substrates include SPs remnants derived from new histocompatibility complex 1b (MHC-1b) molecules. As a part of innate immunity, these processed peptides are presented on cell surfaces for recognition by Natural Killer cells to indicate that the cell is healthy. In addition, SPP substrates include SPs from proteins involved in immune response and muscle contraction. SPP is also hijacked by the Hepatitis C virus (HCV) for replication, and is related to presenilin, which uses similar chemistry to generate amyloidogenic peptides in Alzheimer Disease. SPP and presenilin comprise one of just three superfamilies of intramembrane proteases. 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, however, remain largely a mystery. At least 5 SPP variants have been sequenced, located in different regions of ER, and SPPs are conserved throughput biology, but there is no crystal structure yet. We will start by solving the structure an archeal homolog in complex with our chaperones as proof-of- principle, and then expand to a eukaryotic SPP, whose biomedical relevant activity is known. To date, we have engineered our first chaperone and isolated an affinity complex with SPP by gel filtration. Independently, we have grown crystals of the chaperone and SPP. However, the crystals of SPP do not diffract well enough for structure determination, and thus the cocrystalllization technology is critical. The expected outcomes are a toolbox of crystallization chaperones as well as the first molecular picture of SPP, including the location of the active site and substrate-docking patches. Taken together, this project will contribute not only to the biology of immunoregulation and intramembrane proteolysis, but also broaden our knowledge of membrane proteins and enable other membrane protein structures to be solved.

疏水性膜蛋白在细胞中执行各种功能，但它们的结构是 出了名的难以解决。因此，获得用于结构的膜蛋白晶体的新策略 决心是至关重要的。这项提议的目标是开发一个监护人工具箱并使用它们 为了结晶和解决两个信号肽酶(SPPs)的从头开始的高分辨结构， 使用催化天冬氨酸在脂膜内进行水解。与使用亲和力的工作相反 特定于感兴趣的膜蛋白的试剂，我们潜在的转化方法使用 超结晶性单链抗体片段(ScFv)。我们的监护人专为紧密捆绑而设计 到一个可以插入任何膜蛋白的短表位。我们希望我们紧密绑定的scFv 伴侣分子将固定SPP环并提供稳定的晶格，从而产生更好的衍射晶体。 SPPS修剪信号肽(SPS)，将它们从内质网膜中释放出来。SPP 底物包括来自新的组织相容性复合体1b(MHC-1b)分子的SPS残基。作为一名 作为先天免疫的一部分，这些经过处理的多肽被呈现在细胞表面，供Natural识别 杀伤细胞来表明细胞是健康的。此外，SPP底物包括来自参与 免疫反应和肌肉收缩。SPP还被丙型肝炎病毒(丙型肝炎病毒)劫持进行复制， 与早老素有关，早老素使用类似的化学物质在阿尔茨海默病中产生淀粉样多肽 疾病。SPP和早老素是膜内蛋白水解酶的三个超家族之一。 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，既具有基本的生物化学重要性，又具有潜在的治疗应用。多么 底物被呈现并在脂膜的疏水空间的范围内被水解， 然而，这在很大程度上仍然是一个谜。至少有5个SPP变异体被测序，分布在不同的地区 内质网和SPP是保守的透过性生物学，但目前还没有晶体结构。 我们将从解决结构和我们的伴侣作为证据的复杂的元古同源开始- 原理，然后扩展到真核SPP，其生物医学相关活性已知。迄今为止，我们 设计了我们的第一个伴侣，并通过凝胶过滤分离了与SPP的亲和复合物。独立地， 我们已经生长了伴侣和SPP的晶体。然而，SPP的晶体并不能很好地衍射。 对于结构测定，共结晶技术是至关重要的。 预期的结果是一个结晶伴侣的工具箱以及第一张分子图片 SPP的位置，包括活性部位和底物对接贴片的位置。总而言之，这个项目将 不仅有助于免疫调节和膜内蛋白分解的生物学，而且还拓宽了我们的 膜蛋白的知识，并使其他膜蛋白结构得以解决。