Mutational scan inferred binding energetics and structure in intrinsically disordered protein CcdA

Abstract

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well‐studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence‐function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation. In contrast to previous titration‐based approaches which use a number of ligand concentrations, we show that use of a single rationally chosen ligand concentration can provide quantitative estimates of relative binding constants for large numbers of protein variants. This is because the extended interface of IDP ensures that energetic effects of point mutations are spread over a much smaller range than for globular proteins. Our data also provides insights into the much‐debated role of helicity and disorder in partner binding of IDPs. Based on this exhaustive mutational sensitivity dataset, a rudimentary model was developed in an attempt to predict mutational effects on binding affinity of IDPs that form alpha‐helical structures upon binding.

Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants

“…Most of the serum samples neutralized Delta and Kappa variants better than WA.1 (Figure 1C, heatmap) due to the high similarity between the spikes of both variants 35 . Interestingly, certain sera (BCRTH 29,30,33,35,45,46) showed potent neutralization against WA.1 but failed to neutralize other tested VOCs, especially at higher dilutions (Figure 1C). This suggests that vaccination and subsequent infections have primed a strong immune response towards the parental strain but failed to generate antibodies that are broadly cross reactive against the VOCs, as also reported previously 36 .…”

“…Additionally, a highly conserved class 5 epitope has been recently identified and characterized as a target for broadly neutralizing antibodies 27,28 . 29,30 , a library of individual charged substitutions at all the solvent-exposed positions of the wild-type RBD was generated and displayed on the yeast surfaces to map the epitopes targeted by the polyclonal sera. A high fraction of antibodies targeting either class 1 epitopes or the rare cryptic class 5 epitope, also denoted as RBD8 43 , were observed in all three sera tested.…”