Prediction of Residue-specific Contributions to Binding and Thermal Stability Using Yeast Surface Display

Abstract

Accurate prediction of residue burial as well as quantitative prediction of residue-specific contributions to protein stability and activity is challenging, especially in the absence of experimental structural information. This is important for prediction and understanding of disease causing mutations, and for protein stabilization and design. Using yeast surface display of a saturation mutagenesis library of the bacterial toxin CcdB, we probe the relationship between ligand binding and expression level of displayed protein, with in vivo solubility in E. coli and in vitro thermal stability. We find that both the stability and solubility correlate well with the total amount of active protein on the yeast cell surface but not with total amount of expressed protein. We coupled FACS and deep sequencing to reconstruct the binding and expression mean fluorescent intensity of each mutant. The reconstructed mean fluorescence intensity (MFIseq) was used to differentiate between buried site, exposed non active-site and exposed active-site positions with high accuracy. The MFIseq was also used as a criterion to identify destabilized as well as stabilized mutants in the library, and to predict the melting temperatures of destabilized mutants. These predictions were experimentally validated and were more accurate than those of various computational predictors. The approach was extended to successfully identify buried and active-site residues in the receptor binding domain of the spike protein of SARS-CoV-2, suggesting it has general applicability.

Stabilizing proteins through saturation suppressor mutagenesis

“…Different populations of SSSM libraries were sorted based on the expression and binding histograms of the libraries (Supporting Figure S2). Populations were subjected to deep sequencing and the mean fluorescence intensities of binding (MFI seq (bind)) and expression (MFI seq (expr)) were estimated as described (22). Values of normalized MFI seq (bind) and normalized MFI seq (expr) were used to predict stabilized mutants.…”

“…Interestingly none of the suppressors were found at the residues directly involved in GyrA binding. In any case, we have recently shown that such active-site residues can be identified based on the pattern of MFI seq (bind) and MFI seq (expr) in deep mutational scanning libraries and removed from the set of putative global suppressors (22).…”

“…Instead, we observed that MFI seq (bind) is a better predictor of protein stability than MFI seq (expr). Previously, we found that for stable proteins it is difficult to isolate stabilizing mutants using yeast surface display, as the surface expression and binding of all the mutants above a threshold stability are often similar (22). To overcome this problem, we introduced a PIM in the DMS library of CcdB which reduced binding of all the mutants present in the library and then selected for suppressors which showed improved binding compared to the PIM.…”

“…Yeast surface display and flowcytometric analysis were performed as explained earlier (22). Briefly, Saccharomyces cerevisiae EBY100 cells containing WT ccdB or mutant plasmids were grown in SDCAA (glucose 20g/L, yeast nitrogen base 6.7g/L, casamino acid 5g/L, citrate 4.3g/L, sodium citrate dihydrate 14.3g/L) media for 16 hours and induced in SGCAA (galactose 20g/L, yeast nitrogen base 6.7g/L, casamino acid 5g/L, citrate 4.3g/L, sodium citrate dihydrate 14.3g/L) media for an additional 16 hours at 30 º C. Ten million cells were taken for FACS sample preparation.…”

“…It is also unclear whether it will always be possible to isolate suppressors for every PIM. In the present study, we have modified this approach by introducing PIMs in the background of a deep mutational scanning library of bacterial toxin CcdB, sorted different populations, subjected each population to deep sequencing of the ccdb gene and reconstructed the mean fluorescent intensity (MFI) of each mutant as described (22) (see also Experimental Procedures section). We use both the reconstructed binding MFI (MFI seq (bind)) and expression MFI (MFI seq (expr)) as criteria to differentiate between stabilized mutants, WT-like and destabilized mutants.…”