Molecular Basis of a Million-Fold Affinity Maturation Process in a Protein–Protein Interaction

• Published in: Journal of molecular biology 2011-08, Vol.411 (2), p.321-328
• Main Authors: Bonsor, Daniel A., Postel, Sandra, Pierce, Brian G., Wang, Ningyan, Zhu, Penny, Buonpane, Rebecca A., Weng, Zhiping, Kranz, David M., Sundberg, Eric J.
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; AFFINITY; Bacterial Proteins; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; BIOLOGY; chemistry; computational biology; CRYSTALLOGRAPHY; Crystallography, X-Ray; DRUGS; metabolism; Models, Molecular; MUTAGENESIS; Mutant Proteins; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Protein Binding; PROTEIN ENGINEERING; Protein Interaction Mapping; Protein Structure, Quaternary; protein-protein interactions; PROTEINS; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell - chemistry; Receptors, Antigen, T-Cell - metabolism; SPECIFICITY; therapeutics; X-ray crystallography; X-ray diffraction; yeast display; YEASTS
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2011.06.009

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. [Display omitted] ► Protein engineering approaches regularly achieve thousand-fold affinity increases. ► Rational manipulation of extrainterfacial residues can increase affinity gains. ► We previously achieved a 3-million-fold affinity increase by such an approach. ► The structural and energetic bases of this affinity increase are presented here. ► We detail a mechanism to significantly improve protein engineering outcomes.