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

Abstract

In contrast to globular proteins, much less is known about the mutational effects on the function of Intrinsically Disordered Proteins (IDPs). We employ Yeast Surface Display of a mutant library of the IDP CcdA, coupled to next generation sequencing to rapidly estimate apparent binding affinities of each library member to cognate target, CcdB. This yields insights into sequence-function relationships in disordered CcdA and also enables prediction of the interacting interface residues and the local structural signatures of CcdA in its bound form. We show that the non-interface residue, Gly63 with non-canonical backbone conformation, to be essential for optimal binding to the high affinity site on CcdB. Additionally, this data provides insights into the much-debated role of helicity and disorder in partner binding of IDPs. Relative to globular proteins, in the present case we observe much smaller effects on binding affinity for point mutations, presumably because of the extended binding interface. Based on this exhaustive mutational sensitivity data, a model was developed to predict mutational effects on binding affinity of IDPs forming alpha-helical structures upon binding.

The High Mutational Sensitivity of ccdA Antitoxin Is Linked to Codon Optimality

“…Such folding defects are unlikely in the case of CcdA, since its C-terminal domain, involved in CcdB binding, is intrinsically disordered and remains natively unfolded in unbound conditions. A more extensive DMS study of CcdA mutants in the YSD system indicates that mutations that affect the CcdB-binding affinity of CcdA primarily occur at residues directly involved in contact with CcdB ( Chandra et al . 2022 ).…”