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: 8780816
• 负责人: JENNIFER A MAYNARD
• 金额: $ 0.54万
• 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/8780816
• 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.

疏水膜蛋白在细胞中执行多种功能，但它们的结构是 众所周知，难以解决。因此，获得结构的膜蛋白晶体的新策略 决心至关重要。该提案的目标是开发伴侣的工具箱并使用它们 要结晶并解决从头开始的，两个信号肽肽酶（SPP）的高分辨率结构，该结构 使用催化天冬氨酸在脂质膜内进行水解。与使用亲和力的工作相反 引起的膜蛋白特异性的试剂，我们的较强的变化性侵蚀用途 超晶的单链抗体片段（SCFV）。我们的伴侣经过设计用于紧密绑定 可以将可以插入任何膜蛋白的短效率。我们期望我们紧密绑定的SCFV 伴侣将固定一个SPP环并提供稳定的晶体晶格，从而导致更好的衍射晶体。 SPPS修剪信号肽（SP）将其从内质网膜中解放出来。 spp 底物包括源自新的组织相容性复合物1B（MHC-1B）分子的SPS残留物。作为 一部分先天免疫，这些加工的肽呈现在细胞表面上，以识别自然 杀伤细胞表明细胞健康。此外，SPP底物包括来自涉及的蛋白质的SP 免疫反应和肌肉收缩。 SPP也被丙型肝炎病毒（HCV）劫持以进行复制， 并且与Presenilin有关，后者使用类似的化学物质在阿尔茨海默氏症中产生淀粉样蛋白生成肽 疾病。 SPP和Presenilin构成了膜内蛋白酶的三个超家族之一。 膜内蛋白水解的细节，从细胞生物信号传导到活性位点 化学既具有基本的生化重要性，又具有潜在的治疗应用。如何 在脂质膜的疏水空间的范围内提出并水解底物， 但是，在很大程度上仍然是一个谜。已经测序至少5个SPP变体位于不同区域 ER和SPP是保守的吞吐量生物学，但尚无晶体结构。 我们将首先解决与我们的伴侣伴侣复杂的结构，作为证明 原理，然后扩展为真核属，其生物医学相关活性已知。迄今为止，我们 已经设计了我们的第一个伴侣，并通过凝胶过滤隔离了与SPP的亲和力综合体。独立， 我们已经种植了伴侣和属的晶体。但是，SPP的晶体衍射不够好 为了确定结构，因此共和力化技术至关重要。 预期的结果是结晶伴侣的工具箱以及第一分子图片 SPP的of，包括活跃部位的位置和底物船坞贴片。综上所述，这个项目将 不仅为免疫调节和膜内蛋白水解的生物学做出贡献，而且还扩大了我们的 膜蛋白的知识并使其他膜蛋白结构得以解决。

项目成果

Real-time full-spectral imaging and affinity measurements from 50 microfluidic channels using nanohole surface plasmon resonance.

• DOI: 10.1039/c2lc40455a
• 发表时间: 2012-10-21
• 期刊: Lab on a chip
• 影响因子: 6.1
• 作者: Lee SH;Lindquist NC;Wittenberg NJ;Jordan LR;Oh SH
• 通讯作者: Oh SH

Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

• DOI: 10.1002/prot.24542
• 发表时间: 2014-09
• 期刊: PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
• 影响因子: 2.9
• 作者: Kalyoncu, Sibel;Hyun, Jeongmin;Pai, Jennifer C.;Johnson, Jennifer L.;Entzminger, Kevin;Jain, Avni;Heaner, David P., Jr.;Morales, Ivan A.;Truskett, Thomas M.;Maynard, Jennifer A.;Lieberman, Raquel L.
• 通讯作者: Lieberman, Raquel L.

Immunotherapeutic Approaches To Prevent Cytomegalovirus-Mediated Disease.

预防巨细胞病毒介导疾病的免疫治疗方法。

• DOI: 10.1128/microbiolspec.aid-0009-13
• 发表时间: 2014
• 期刊: Microbiology spectrum
• 影响因子: 3.7
• 作者: Seedah,EdithA;Frye,ZacharyP;Maynard,JenniferA
• 通讯作者: Maynard,JenniferA

Back to the future: recombinant polyclonal antibody therapeutics.

• DOI: 10.1016/j.coche.2013.08.005
• 发表时间: 2013-11
• 期刊: Current opinion in chemical engineering
• 影响因子: 6.6
• 作者: Wang XZ;Coljee VW;Maynard JA
• 通讯作者: Maynard JA

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配去污剂

• DOI: 10.3791/57901
• 发表时间: 2018
• 期刊: Journal of Visualized Experiments
• 作者: Oliver, Ryan C.;Naing, Swe-Htet;Weiss, Kevin L.;Pingali, Sai Venkatesh;Lieberman, Raquel L.;Urban, Volker S.
• 通讯作者: Urban, Volker S.