Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed
Jin Huang1, Koki Makabe, Matthew Biancalana
1Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
Journal of Molecular Biology
|August 4, 2009
Summary
A new protein engineering strategy, directed domain-interface evolution, creates synthetic affinity clamps by linking protein domains. These novel protein binders show enhanced affinity and tunable specificity for peptide targets.
Area of Science:
- Protein engineering
- Structural biology
- Biochemistry
Background:
- Protein domains are fundamental building blocks of proteins.
- Optimizing interactions between protein domains can create novel functionalities.
- Existing methods for protein engineering have limitations in creating tailored binding sites.
Purpose of the Study:
- To introduce a novel protein engineering strategy called directed domain-interface evolution.
- To generate and characterize synthetic two-domain affinity clamps with enhanced binding properties.
- To investigate the structural basis for altered specificity in engineered proteins.
Main Methods:
- Directed domain-interface evolution strategy.
- Construction of synthetic affinity clamps using PDZ and fibronectin type III (FN3) domains.
- Peptide phage-display libraries and scanning mutagenesis for specificity profiling.
- X-ray crystallography for structural analysis.
Main Results:
- Engineered affinity clamps exhibited significantly higher peptide-binding affinity than parent PDZ domains.
- Two distinct specificity profiles were observed: conserved and dramatically increased.
- High-specificity clamps demonstrated an enlarged recognition site, confirmed by structural analysis.
- Structural comparisons revealed domain interface plasticity influencing relative domain positioning and function.
Conclusions:
- Directed domain-interface evolution is an effective strategy for generating functional protein modules.
- The plasticity of domain interfaces enables the creation of diverse active-site topographies.
- This approach facilitates the facile generation of synthetic proteins with tailored functions.
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