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首页> 外文期刊>Biochemistry >Combinations of Affinity-Enhancing Mutations in a T Cell Receptor Reveal Highly Nonadditive Effects within and between Complementarity Determining Regions and Chains
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Combinations of Affinity-Enhancing Mutations in a T Cell Receptor Reveal Highly Nonadditive Effects within and between Complementarity Determining Regions and Chains

机译：T细胞受体中亲和力增强突变的组合显示互补决定区域和链内和之间的高度非累加效应。

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Understanding the energetic and structural response to multiple mutations in a protein-proteinninterface is a key aspect of rational protein design. Here we investigate the cooperativity of combinationsnof point mutations of a T cell receptor (TCR) that binds in vivo to HLA-A2 MHC and a viral peptide.nThe mutations were obtained from two sources: a structure-based design study on the TCR R chainn(nine mutations) and an in vitro selection study on the TCR β chain (four mutations). In addition to combiningnthe highest-affinity variants from each chain, we tested other combinations of mutations within and among thenchains, for a total of 23 TCR mutants that we measured for binding kinetics to the peptide and majornhistocompatibility complex. A wide range of binding affinities was observed, from 2- to 1000-fold bindingnimprovement versus that of thewild type,with significant nonadditive effects observedwithin and betweenTCRnchains. This included an amino acid-dependent cooperative interaction betweenCDR1 and CDR3 residues thatnare separated by more than 9 A in the wild-type complex. When analyzing the kinetics of the mutations, wenfound that the association rates were primarily responsible for the cooperativity, while the dissociation ratesnwere responsible for the anticooperativity (less-than-additive energetics). On the basis of structural modeling ofnanticooperative mutants, we determined that side chain clash between proximal mutants likely led to non-nadditive binding energies. These results highlight the complex nature of TCRassociation and binding andwill beninformative in future design efforts that combine multiple mutant residues.
机译：了解蛋白质-蛋白质界面中多个突变的能量和结构响应是合理蛋白质设计的关键方面。在这里，我们研究了结合HLA-A2 MHC和病毒肽的T细胞受体（TCR）点突变的组合的协同作用.n突变是从两个来源获得的：基于结构的TCR R链的设计研究（9个突变）和TCRβ链的体外选择研究（4个突变）。除了结合来自每条链的最高亲和力变体外，我们还测试了随后链内和链间突变的其他组合，共测量了23种TCR突变体，我们对其与肽和主要组织相容性复合物的结合动力学进行了测量。观察到广泛的结合亲和力，与野生型相比，结合亲和力提高了2至1000倍，并且在TCRn链内和之间观察到显着的非累加作用。这包括在野生型复合物中，CDR1和CDR3残基之间的氨基酸依赖性协作相互作用，这些残基之间相隔9 A以上。在分析突变的动力学时，温特发现缔合速率主要负责协同作用，而解离速率则负责反协同作用（小于加成能量）。根据nanticooperative突变体的结构模型，我们确定近端突变体之间的侧链冲突可能导致非nadditive结合能。这些结果突出了TCR缔合和结合的复杂性质，并且在结合多个突变残基的未来设计工作中将是有益的。

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    《Biochemistry》 |2010年第33期|p.7050-7059|共10页
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    Brian G. Pierce Jaafar N. Haidar Yong Yu and Zhiping Weng;

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    ‡Bioinformatics Program, Boston University, Boston, Massachusetts 02215,§Program in Bioinformatics and Integrative Biology,University of Massachusetts Medical School, Worcester, Massachusetts 01605, and ) Department of Biomedical Engineering,Boston University, Boston, Massachusetts 02215.^Present address: Imclone Systems, New York, NY 10014.@Present address: Novartis Institutes for Biomedical Research, Cambridge, MA 02139-4229.;

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