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.

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

“…Single reversion mutants revealed the mutations that resulted in the greatest increases in affinity were most destabilizing. Similar results were found for an affinity-maturated fibronectin domain specific for lysozyme [22], and protein engineering approaches have been developed to overcome these trade-offs [23]. These and related findings [24, 25] demonstrate the generality of affinity/stability trade-offs during affinity maturation.…”

Consensus sequence design as a general strategy to create hyperstable, biologically active proteins

“…Importantly, all consensus proteins we assayed for function maintained some level of expected biological activities of both molecular recognition and enzymatic catalysis (Figures 4 and 5). This result, combined with previously reported studies showing consensus protein function (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), indicates that information necessary for protein function is retained in averaging over many sequences that each individually contain functionally important information.…”

“…There are a number of reports in the literature in which consensus design has successfully been used to produce proteins that adopt their archetypical fold. This approach has found recent success for linear repeat-protein targets (44)(45)(46)(47), but has also shown some success for globular protein targets (13,14,21,24,25). In most reports, globular consensus proteins have enhanced equilibrium stability, and sometimes retain biological activity.…”

“…The "wholesale" approach does just this, combining all substitutions toward consensus into a single consensus polypeptide composed of the most frequent amino acid at each position in sequence. The stabilities of several globular proteins and several repeat proteins have been increased using this approach (20)(21)(22)(23)(24)(25)(26)(27)(28)(29). An increase in thermodynamic stability is seen in most (but not all) cases, but effects on biological † These properties include rates of folding, unfolding, degradation, compartmentalization, oligomerization, and solubility.…”

Consensus sequence design as a general strategy to create hyperstable, biologically active proteins

“…have been increased using this approach (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). An increase in thermodynamic stability is seen in most (but not all) cases, but effects on biological activity are variable.…”