Prediction of residue-specific contributions to binding and thermal stability using yeast surface display

Abstract

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.

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern

“…Yeast surface display and flow cytometric analysis were performed as described earlier ( 34 ). Briefly, Saccharomyces cerevisiae EBY100 cells containing WT RBD or mutant plasmids were grown in SDCAA media for 16 h and induced in SGCAA media for 16 h at 30°C.…”

“…The principle of SSSM is as follows. We have previously shown in the context of unstable proteins displayed on the yeast surface that the relative amount of properly folded mutant proteins displayed on the yeast surface correlates with the thermal stability of the corresponding purified mutant measured in vitro ( 34 ). However, once the stability crosses a certain threshold, further stability increases are not accompanied by increased binding on the yeast surface; hence, it is challenging to isolate mutants with higher stability than the wild type from single-site saturation mutagenesis (SSM) libraries using this approach.…”

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“…The binding of intrinsically disordered MazE6 antitoxin with MazF6 toxin was measured using YSD. The full-length MazE6 antitoxin was expressed on the yeast cell surface and the level of expression was measured using anti-HA antibody and goat anti-chicken conjugated to Alexa fluor 488 incubated with the yeast cell surface displayed protein as explained earlier (Najar et al, 2017;Ahmed et al, 2021). Binding was measured by incubating the yeast cell expressing full-length MazE6 with 1 nM of its cognate partner MazF6 toxin, having 3X FLAG tag.…”

“…An extended analysis of scanning mutagenesis data from multiple protein systems, revealed the difficulty of distinguishing between buried and active-site residues in globular proteins, purely from phenotypic data on protein function, as both classes of residues are affected by mutation (Bhasin and Varadarajan, 2021). In a recent study, we show that by combining mutational effects on expression and binding/function, it is possible to distinguish between these two classes of residues (Ahmed et al, 2021). The problem, is in principle simpler for IDPs, where most substitutions are not expected to affect expression.…”

“…Binding is maximally affected at those sites which become buried on partner binding. Prior mutational data with globular proteins showed aspartate followed by arginine to be the best mutagenic probe of residue burial (Bajaj et al, 2005;Tripathi et al, 2016;Ahmed et al, 2021). We therefore used this approach to identify binding interface residues in the case of the MazE6 antitoxin.…”