Alanine scanning of the yeast killer toxin K2 reveals key residues for activity, gain-of-function variants, and supports prediction of precursor processing and 3D structure

• Mutational scanning indicates more processing sites in K2 than initially recognized. • Insights into precursor processing help to predict the mature toxin structure. • Single mutations can enhance killer toxin function and change target specificity. • Yeast killer toxins are part of structural families despite low sequence identity. Yeast killer toxins (YKTs) are antimicrobial proteins secreted by yeast with potential applications ranging from food preservation to therapeutic agents in human health. However, the practical use of many YKTs is limited by specific pH requirements, low temperature stability, low production yields, and narrow target specificity. While protein engineering could potentially overcome these challenges, progress is hindered by a lack of detailed knowledge about sequence-function relationships and structural data for these often multi-step processed proteins. In this study, we focused on the YKT K2, encoded by the M2 satellite dsRNA in Saccharomyces cerevisiae . Using alanine scanning mutagenesis of the full open reading frame and structure predictions combined with molecular dynamics simulations, we generated a comprehensive sequence-structure–function map, refined the model for the proteolytic processing of the K2 precursor, and predicted the mature toxin structure. Our findings also demonstrate that K2 can be engineered toward enhanced toxicity and altered target specificity through single-site mutations. Furthermore, we identified structural homology between K2 and other killer toxins, including the SMK toxin from the yeast Millerozyma farinosa . Our cost-effective workflow provides a platform to broadly map YKT sequence-structure–function relationships.