5j7c
A picomolar affinity FN3 domain in complex with hen egg-white lysozyme
Structural highlights
FunctionLYSC_CHICK Lysozymes have primarily a bacteriolytic function; those in tissues and body fluids are associated with the monocyte-macrophage system and enhance the activity of immunoagents. Has bacteriolytic activity against M.luteus.[1] Publication Abstract from PubMedThe 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. Circumventing the stability-function trade-off in an engineered FN3 domain.,Porebski BT, Conroy PJ, Drinkwater N, Schofield P, Vazquez-Lombardi R, Hunter MR, Hoke DE, Christ D, McGowan S, Buckle AM Protein Eng Des Sel. 2016 Aug 29. PMID:27578887[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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