title: Quantitative Description of a Protein Fitness Landscape Based on Molecular Features.
abstract: Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo-β-lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo-β-lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo-β-lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters.
authors.affiliation: Laboratory of Metalloproteins, Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR) and Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: María-Rocío
authors.initials: MR
authors.lastname: Meini
authors.affiliation: Laboratory of Metalloproteins, Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR) and Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Pablo E
authors.initials: PE
authors.lastname: Tomatis
authors.affiliation: Department of Ecology and Evolutionary Biology, and Center for Computational Molecular Biology, Brown University.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Weinreich
authors.affiliation: Laboratory of Metalloproteins, Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR) and Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina vila@ibr-conicet.gov.ar.
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Vila
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D002506:Cephalexin / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004843:Epistasis, Genetic; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D019143:Evolution, Molecular; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D019897:Periplasm / Q000378:metabolism; D013696:Temperature; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001618:beta-Lactamases; D015032:Zinc; D002506:Cephalexin
keywords: antibiotic resistance; epistasis; metallo-ß-lactamase; protein evolution