5j7k | pdb_00005j7k

Loop grafting onto a highly stable FN3 scaffold

spinqualitylabelsall models 5j7k, resolution 2.46Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5j7k is a 8 chain structure with sequence from Synthetic construct. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.46Å Ligands: ZN Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed 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. 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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