Journal
PROTEIN ENGINEERING DESIGN & SELECTION
Volume 29, Issue 11, Pages 541-549Publisher
OXFORD UNIV PRESS
DOI: 10.1093/protein/gzw046
Keywords
consensus design; loop grafting; protein engineering; stability-function trade-off; X-ray crystallography
Funding
- Australian Research Council [DP150101371]
- Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE)
- National Health and Medical Research Council [1022688, 1050146]
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The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-alpha-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-alpha-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-alpha-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-alpha-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-alpha-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-alpha-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-alpha-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.
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