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Circumventing the stability-function trade-off in an engineered FN3 domain.

ABSTRACT: 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-?-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-?-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-?-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-?-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-?-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-?-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-?-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.

SUBMITTER: Porebski BT

PROVIDER: S-EPMC5081044 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Circumventing the stability-function trade-off in an engineered FN3 domain.

Porebski Benjamin T BT Conroy Paul J PJ Drinkwater Nyssa N Schofield Peter P Vazquez-Lombardi Rodrigo R Hunter Morag R MR Hoke David E DE Christ Daniel D McGowan Sheena S Buckle Ashley M AM

Protein engineering, design & selection : PEDS 20161101 11

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-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromi ...[more]

PMID: 27578887

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Circumventing the stability-function trade-off in an engineered FN3 domain.

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Circumventing the stability-function trade-off in an engineered FN3 domain.

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