Investigating the Structural Basis of Antibody Steroselectivity (AREA)

描述（由申请人提供）：生理过程总是依赖于生物结合配偶体之间的特异性相互作用。由于受体、转运蛋白、酶和抗体能够区分手性化合物的立体异构体，因此一种立体异构体通常在例如，代谢途径和细胞通讯。因此，生物体在几乎所有的相互作用水平上区分内源性和外源性化合物（如药物）的对映体，并对它们作出不同的反应。虽然药物的一种对映异构体可能表现出所需的活性，但另一种可能引起严重的药理学和毒理学副作用，或作为拮抗剂。尽管世纪前人们就已经认识到蛋白质-配体相互作用的立体选择性，但这种手性区分的分子基础还不清楚。我们的长期目标是通过研究抗体和低分子量手性化合物之间的立体选择性相互作用作为模型系统，阐明在分子水平上控制手性歧视的结构要素。将立体选择性识别氨基酸或羟基酸的D-或L-对映异构体的几种抗体的抗原结合片段结晶，用于通过X射线晶体学进行结构分析。虽然比较的结构，例如，分别与D-和L-氨基酸复合的抗-D-氨基酸抗体和抗-L-氨基酸抗体将揭示抗体结合位点中的哪些残基介导与配体的立体选择性相互作用，对具有和不具有配体的单个抗-氨基酸抗体的评估将允许我们评估立体选择性结合的构象变化的重要性。用抗羟基酸抗体进行的类似研究将提供关于取代立体中心的官能团的影响的额外信息。利用所研究的抗体的类别特异性，与不同配体复合的所选抗体的结构将揭示特定残基对立体选择性和总体结合活性的贡献。单链可变片段的产生及其通过定点诱变的结构指导操作将用于测试我们的发现的有效性，并产生具有定制结合特性的抗体。该项目的成功将为生物识别的分子基础以及对基础科学和应用科学以及人类健康的影响提供前所未有的新见解。例如，更好地理解蛋白质立体选择性的分子基础对于合理设计更有效的药物和开发用于分析、分离和合成立体异构化合物的定制手性主体分子将是有价值的。

DESCRIPTION (provided by applicant): Physiological processes invariably depend on specific interactions between biological binding partners. Since receptors, transport proteins, enzymes, and antibodies are able to differentiate between the stereoisomers of chiral compounds, one stereoisomer is generally preferred in, e.g., metabolic pathways and cellular communication. Thus, living organisms discriminate between the enantiomers of endogenous and exogenous compounds, such as drugs, at virtually all levels of interaction, and respond differently to them. While one enantiomer of a drug may exhibit a desired activity, the other may cause severe pharmacologic and toxicologic side effects, or act as an antagonist. Despite the fact that the stereoselectivity of protein-ligand interactions was first recognized more than a century ago, the molecular basis of such chiral discrimination is not yet understood. Our long-term objective is to elucidate the structural elements that govern chiral discrimination at the molecular level by studying the stereoselective interaction between antibodies and low-molecular weight chiral compounds as a model system. The antigen binding fragments of several antibodies that stereoselectively recognize the D- or L-enantiomers of amino acids or hydroxy acids will be crystallized for structural analysis by X-ray crystallography. While the comparison of the structures of, e.g., anti-D-amino acid antibodies and anti-L-amino acid antibodies in complex with D- and L-amino acids, respectively, will reveal, which residues in the antibody binding site mediate stereoselective interaction with the ligand, the assessment of individual anti-amino acid antibodies with and without ligand will allow us to evaluate the importance of conformational changes for stereoselective binding. Analogous studies with the anti-hydroxy acid antibodies will provide additional information about the effect of substituting functional groups about the stereogenic center. Utilizing the class-specificity of the antibodies under study, structures of selected antibodies in complex with different ligands will disclose the contribution of particular residues to stereoselectivity and overall binding activity. The production of single chain variable fragments and their structure-guided manipulation by site-directed mutagenesis will be used to test the validity of our findings and to create antibodies with tailor-made binding properties. Success in this project will provide new, unprecedented insights into the molecular basis of biorecognition and impact upon both, basic and applied science, and thus human health. A better comprehension of the molecular basis of protein stereoselectivity will, for example, be valuable for the rational design of more efficient drugs and the development of tailor-made chiral host molecules for the analysis, separation, and synthesis of stereoisomeric compounds.