Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

Proc Natl Acad Sci U S A. 2016 Dec 27;113(52):14982-14987. doi: 10.1073/pnas.1613122113. Epub 2016 Dec 12.

Abstract

Protein-protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 β-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the library-ligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes.

Keywords: association rate; in vitro evolution; interface plasticity; protein–protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Gene Library
  • Mutation*
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Mapping
  • Sequence Analysis, DNA
  • Two-Hybrid System Techniques
  • beta-Lactamase Inhibitors / chemistry*
  • beta-Lactamases / chemistry*

Substances

  • Bacterial Proteins
  • beta-Lactamase Inhibitors
  • beta-Lactamases