Peripheral positions encode transport specificity in the small multidrug resistance exporters

• 成果类型: Article
• 署名作者: Burata, Olive E.; O'Donnell, Ever; Hyun, Jeonghoon; Lucero, Rachael M.; Thomas, Junius E.; Gibbs, Ethan M.; Reacher, Isabella; Carney, Nolan A.; Stockbridge, Randy B.
• 署名单位: University of Michigan System; University of Michigan; University of Michigan System; University of Michigan
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10006
• DOI: 10.1073/pnas.2403273121
• 发表日期: 2024-06-18
• 关键词: escherichia-coli; emre provides; EVOLUTION; proteins; symmetry; FAMILY; ligand

摘要

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this physiology, binding assays, and a proteoliposome- based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.