The molecular recognition of proteins by antibodies: A model for rational design

抗体对蛋白质的分子识别：合理设计的模型

• 批准号: 7592805
• 负责人: Sandra Smith-Gill
• 金额: $ 90.47万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592805
• 关键词: Address; Affect; Affinity; Antibodies; Antibody Affinity; Antigen-Antibody Complex; Antigens; Binding; Biological Models; Characteristics; Complex; Crystallization; Data; Databases; Drug Design; Electrostatics; Engineering; Evolution; Fluorine; Goals; Hydrophobic Interactions; Immune response; Immunoglobulin Constant Region; Immunology; Immunotherapy; Kinetics; Label; Lead; Ligands; Light; Link; Methodology; Modeling; Molecular Evolution; Molecular Target; Monitor; Monoclonal Antibodies; NMR Spectroscopy; Play; Pliability; Process; Property; Proteins; Protocols documentation; Rate; Role; Sodium Chloride; Somatic Mutation; Specificity; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; System; Techniques; Therapeutic antibodies; Thermodynamics; Time; Tryptophan; Vi antigen; Water; Water Movements; antibody engineering; antigen antibody binding; antigen binding; base; cancer cell; comparative; cross reactivity; design; drug discovery; inhibitor/antagonist; insight; intermolecular interaction; molecular modeling; molecular recognition; novel; protein protein interaction; receptor; research study; response; simulation; structural biology

The Structural Immunology Section investigates molecular recognition in antibody complexes with proteins as model systems to elucidate the general principles of protein target recognition by antibodies. The results to date have provided new paradigms on the mechanism of antibody-antigen binding, and in addition have provided new methodology for examining molecular interaction networks. We have developed novel protocols for surface plasmon resonance (SPR) analysis which reveal that (i) Antibody-antigen bimolecular association is a time-dependent 2-step binding process; (ii) Kinetics, thermodynamics, and water movements accompanying the 2 steps are distinctly different, and define which of the steps are rate limiting, information which informs the effective design of competitive inhibitors; (iii) Antibody affinity maturation, a protype of molecular evolution, is driven by thermodynamics, thus thermodynamics inform the rational design of antibodies; (iv) Affinity and specificity of protein-protein interactions are determined by inherent protein flexibility, thus receptor and ligand flexibility must be considered in structure-based drug discovery; (v) Intramolecular salt link networks provide strong electrostatic interactions which can be significantly stabilizing and can modulate the dynamics of antibody recognition and binding to antigen; (vi) Molecular modeling and dynamics simulations can identify many significant intermolecular interactions which can be confirmed experimentally. The thermodynamic studies have revealed properties which we believe to be predictive of binding characteristics including long term complex stability and likely cross-reactivity with related antigens, and we are developing a protocol for assessing these properties which would be valuable in selection of lead therapeutic antibodies. The insight(s) gained by analyses of complex kinetics and thermodynamics provide a framework and rationale antibody engineering, informing the design of antibodies of predefined specificity for immunotherapy, and will also lead to better strategies for structure-based drug design and selection of lead compounds in molecular targeting efforts. In addition, the methodology and insight from this project inform design of experiments to study in molecular interaction networks in normal and cancer cells. We have initiated two new structural biology studies to inform our interpretation of structure-activity relationships. We have incorporated fluorinated tryptohan at six tryptophan residues in a single-chain Fv antibody and are using F-19 NMR to study flexibility and conformational changes upon antigen binding. While it often assumed that fluorine labeling proteins does little to perturb the structure, 3 lines of evidence from our data to date indicate that the structure of the labeled protein differs significantly from that of the unlabeled: (1) the CD spectra are significantly different; (2) the binding kinetics and affinties of the the labeled and unlabeled proteins are significantly different (3) shifts in the F-19 spectra strongly suggest that the F-19 alters with association of the heavy and light chains of the Fv. We have begun crystallization trials, in order to compare the structures of both the wild type and the fluorinated ScFv complexes. This would be only the second F-19 incorporated protein in the Protein Data Base, and would yield important information about the conformational effects of F-19 incorporation into proteins. This information is of broad significance because this technique is commonly used for NMR spectroscopy.

结构免疫学部分研究了与蛋白质作为模型系统的抗体复合物中的分子识别，以阐明抗体蛋白靶标识别的一般原理。迄今为止的结果为抗体 - 抗原结合机理提供了新的范式，此外，还为检查分子相互作用网络提供了新的方法。我们已经开发了用于表面等离子体共振（SPR）分析的新型方案，该方案表明（i）抗体 - 抗原双分子缔合是一个时间依赖的2步结合过程； （ii）伴随两个步骤的动力学，热力学和水运动明显不同，并定义了哪个步骤是限制速率的，这些信息可为有效设计竞争性抑制剂设计； （iii）抗体亲和力成熟是一种分子进化的原型，由热力学驱动，因此热力学为抗体的合理设计提供了启用。 （iv）蛋白质蛋白相互作用的亲和力和特异性取决于固有的蛋白质柔韧性，因此在基于结构的药物发现中必须考虑受体和配体柔韧性； （v）分子内盐连接网络提供强大的静电相互作用，可以显着稳定，并可以调节抗体识别的动力学并与抗原结合； （vi）分子建模和动力学模拟可以识别许多可以通过实验确认的明显分子间相互作用。热力学研究揭示了我们认为的特性，这些特性可以预测结合特征，包括长期复杂稳定性和可能与相关抗原的交叉反应性，并且我们正在开发一种评估这些特性的方案，这些属性对于选择铅疗法抗体的有价值。通过对复杂动力学和热力学的分析获得的洞察力提供了一个框架和基本原理抗体工程，为免疫疗法预定义特异性的抗体设计介绍了设计，还将为基于结构的药物设计和分子靶向工作中铅化合物的选择提供更好的策略。此外，该项目的方法和洞察力为实验设计提供了正常和癌细胞中分子相互作用网络中的研究。我们已经启动了两项新的结构生物学研究，以告知我们对结构活性关系的解释。我们已经在单链FV抗体中掺入了六个色氨酸残基的氟化色氨酸，并使用F-19 NMR研究抗原结合时的柔韧性和构象变化。尽管它通常假设氟标记蛋白对结构的扰动几乎没有作用，但迄今为止，来自我们数据的3条证据表明，标记蛋白的结构与未标记的蛋白质明显不同：（1）CD光谱显着不同； （2）标记和未标记的蛋白质的结合动力学和字体显着差异（3）F-19光谱的变化强烈表明，F-19与FV的重链和轻度链的关联变化。我们已经开始进行结晶试验，以比较野生型和氟化SCFV复合物的结构。这将只是蛋白质数据库中的第二个F-19掺入蛋白，并且会产生有关F-19掺入蛋白质的构象作用的重要信息。此信息具有广泛的意义，因为该技术通常用于NMR光谱。