title: Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes.
abstract: Deep mutational scanning has emerged as a promising tool for mapping sequence-activity relationships in proteins, ribonucleic acid and deoxyribonucleic acid. In this approach, diverse variants of a sequence 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 landscape around the wild-type sequence. Little is currently known, however, about the degree to which such fitness landscapes are dependent on the specific 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 resistance to aminoglycoside antibiotics, in Escherichia coli under selection with each of six structurally diverse antibiotics at a range of inhibitory concentrations. We found that the resulting local fitness landscapes showed significant dependence on both antibiotic structure and concentration, and that this dependence can be exploited to guide protein engineering. Specifically, we found that differential analysis of fitness landscapes allowed us to generate synthetic APH(3')II variants with orthogonal substrate specificities.
authors.affiliation: Broad Technology Labs, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Melnikov
authors.affiliation: Broad Technology Labs, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Rogov
authors.affiliation: Broad Technology Labs, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Broad Technology Labs, Broad Institute, Cambridge, MA 02142, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gnirke
authors.affiliation: Broad Technology Labs, Broad Institute, Cambridge, MA 02142, USA tarjei@broadinstitute.org.
authors.forename: Tarjei S
authors.initials: TS
authors.lastname: Mikkelsen
mesh_terms: D019943:Amino Acid Substitution; D000617:Aminoglycosides / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects; D019868:Kanamycin Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D019868:Kanamycin Kinase