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.

Keywords: consensus design, loop grafting, protein engineering, stability-function trade-off, X-ray crystallography.

Fig. 1

Biophysical characterization of FNfn10-α-lys and FN3con-α-lys. CD thermal melts of (A) FNfn10-α-lys (T_m of 43 ± 2°C) and (B) FN3con-α-lys (T_m of 101 ± 3°C). SEC, revealing complex formation for (C) FNfn10-α-lys and (D) FN3con-α-lys with lysozyme. Representative BLI sensograms of (E) FNfn10-α-lys and (F) FN3con-α-lys with titrations of the respective FN3 protein against an immobilized HEL surface (fit for each concentration as a thin black line).

Fig. 2

The FNfn10-α-lys-lysozyme complex reveals a tight binding interface that makes use of framework residues. (A) The loop residues (red) of FNfn10-α-lys (gray) as previously evolved for lysozyme (tan) binding (Hackel et al., 2008). (B) The actual binding interface residues (red) of FNfn10-α-lys (gray) with lysozyme (tan) as determined by crystal structure and the PDBePISA web server (Krissinel and Henrick, 2007). (C) A sequence alignment of FN3con-α-lys with FNfn10-α-lys, highlighting the B/C, D/E and F/G loops (blue, purple and orange) that were previously evolved for lysozyme binding (Hackel et al., 2008), the actual residues involved in the binding interface (red) and positions of the FNfn10-α-lys framework mutations previously introduced (Hackel et al., 2008).

Fig. 3

Structural comparison between FNfn10-α-lys and FN3con-α-lys reveals framework incompatibilities that likely prevent tight complex formation. A conformational change is observed between FNfn10-α-lys (A) and FNfn10 (B) resulting in a 180° flip and +1 register shift of strand D that is also lacking in the unbound FN3con-α-lys crystal structure (C). Differences in framework residues of the lysozyme-binding interface (tan region) between FNfn10-α-lys (D) and FN3con-α-lys (E) highlight the potential for cavity formation due to the lack of Y31 (G30 in FN3con-α-lys) and steric clashes as a result of R32 and R71 in FN3con-α-lys. These characteristics may impact the formation of a tight binding interface.

Fig. 4

Framework residues in the lysozyme-binding interface of FN3con-α-lys were redesigned by alignment of FNfn10-α-lys and the FN3con-α-lys crystal structures. Redesign restored binding at the cost of thermodynamic stability. (A) The crystal structure of FNfn10-α-lys showing the paratope surface residues (tan) and surrounds; (B) the composite crystal structure of FN3con-α-lys showing the paratope surface residues (tan) and surrounds; (C) a homology model of the redesigned FN3con-α-lys.v2 based on FNfn10-α-lys showing the redesigned binding interface residues (tan); (D) SEC complex formation shift of FN3con-α-lys.v2; (E) representative BLI sensograms of FN3con-α-lys.v2 titrations against an immobilized HEL surface (fit for each concentration as a thin black line); (F) variable temperature CD melt of FN3con-α-lys.v2 showing a T_m of 87 ± 2°C and incomplete reversible folding.