Molecular basis of a million-fold affinity maturation process in a protein-protein interaction |
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Authors: | Bonsor Daniel A Postel Sandra Pierce Brian G Wang Ningyan Zhu Penny Buonpane Rebecca A Weng Zhiping Kranz David M Sundberg Eric J |
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Affiliation: | 1 Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA2 Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA3 Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA |
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Abstract: | Protein engineering is becoming increasingly important for pharmaceutical applications where controlling the specificity and affinity of engineered proteins is required to create targeted protein therapeutics. Affinity increases of several thousand-fold are now routine for a variety of protein engineering approaches, and the structural and energetic bases of affinity maturation have been investigated in a number of such cases. Previously, a 3-million-fold affinity maturation process was achieved in a protein-protein interaction composed of a variant T-cell receptor fragment and a bacterial superantigen. Here, we present the molecular basis of this affinity increase. Using X-ray crystallography, shotgun reversion/replacement scanning mutagenesis, and computational analysis, we describe, in molecular detail, a process by which extrainterfacial regions of a protein complex can be rationally manipulated to significantly improve protein engineering outcomes. |
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Keywords: | CDR, complementarity-determining region TCR, T-cell receptor SAG, superantigen mVβ8.2, mouse TCR Vβ8.2 SEB, staphylococcal enterotoxin B hVβ2.1, human TCR Vβ2.1 SpeC, streptococcal pyrogenic exotoxin C |
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