title: 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-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-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.
authors: Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.|Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, CambridgeCB2 0QH, UK.
authors: Benjamin T
authors: BT
authors: Porebski
authors: Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
authors: Paul J
authors: PJ
authors: Conroy
authors: Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
authors: Nyssa
authors: N
authors: Drinkwater
authors: Department of Immunology, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St Vincent's Clinical School, The University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors: Peter
authors: P
authors: Schofield
authors: Department of Immunology, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St Vincent's Clinical School, The University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors: Rodrigo
authors: R
authors: Vazquez-Lombardi
authors: Department of Pathology, University of Cambridge, CambridgeCB2 1QP, UK.
authors: Morag R
authors: MR
authors: Hunter
authors: Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
authors: David E
authors: DE
authors: Hoke
authors: Department of Immunology, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.|Faculty of Medicine, St Vincent's Clinical School, The University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia.
authors: Daniel
authors: D
authors: Christ
authors: Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
authors: Sheena
authors: S
authors: McGowan
authors: Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
authors: Ashley M
authors: AM
authors: Buckle
keywords: consensus design, loop grafting, protein engineering, stability-function
                      trade-off, X-ray crystallography