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

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein-protein interactions are crucial for biological processes.
• Interfaces mediating these interactions can exhibit compositional plasticity.
• Understanding this plasticity can reveal alternative binding strategies.

Purpose of the Study:

• To investigate the plasticity of the TEM1 β-lactamase and BLIP inhibitor interface.
• To identify mutations that maintain high-affinity binding despite interface composition changes.
• To develop novel methods for selecting faster protein-ligand association rates.

Main Methods:

• Yeast surface display for high-throughput screening.
• Low-stringency selection of a random TEM1 β-lactamase mutant library.
• Preequilibrium selection by drastically reducing incubation time.

Main Results:

• Most interfacial residues of TEM1 β-lactamase can be mutated without compromising binding affinity, stability, or enzymatic activity.
• Many identified mutations enhanced the association rate of the protein complex.
• Preequilibrium selection effectively isolated variants with faster association kinetics.

Conclusions:

• The TEM1-BLIP interface demonstrates significant plasticity, supporting high-affinity binding through diverse compositions.
• Faster association rates can be achieved through specific interfacial mutations.
• Preequilibrium selection is a novel and effective method for isolating rapidly associating protein complexes.