Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes. Nucleic Acids Res

Deep mutational scanning has emerged as a promis-ing tool for mapping sequence–activity relationships in proteins, ribonucleic acid and deoxyribonucleic acid. In this approach, diverse variants of a se-quence of interest are first ranked according to their activities in a relevant assay, and this ranking is then used to infer the shape of the fitness land-scape around the wild-type sequence. Little is cur-rently known, however, about the degree to which such fitness landscapes are dependent on the spe-cific assay conditions from which they are inferred. To explore this issue, we performed comprehensive single-substitution mutational scanning of APH(3′)II, a Tn5 transposon-derived kinase that confers resis-tance to aminoglycoside antibiotics, in Escherichia coli under selection with each of six structurally di-verse antibiotics at a range of inhibitory concentra-tions. We found that the resulting local fitness land-scapes showed significant dependence on both an-tibiotic structure and concentration, and that this de-pendence can be exploited to guide protein engineer-ing. Specifically, we found that differential analysis of fitness landscapes allowed us to generate synthetic APH(3′)II variants with orthogonal substrate speci-ficities