Poster number 82 submitted by Ingrid Kilde
Exploring structural determinants of ligand selectivity in cobalamin riboswitches
Ingrid Kilde (Chemistry, University of Michigan Ann Arbor), Elizabeth Tidwell (Biophysics, University of Michigan Ann Arbor), Suada Leskaj (Biochemistry, University of Michigan Ann Arbor), Markos Koutmos (Chemistry and Biophysics, University of Michigan Ann Arbor)
Cobamides are a family of complex organometallic coenzymes involved in numerous metabolic pathways across all domains of life. Within microbial communities, some bacterial strains are capable of cobamide synthesis (cobamide producers) whereas others (cobamide scavengers) must rely on environmental sources of cobamides including cross-feeding, resulting in a complex and dynamic network of nutrient sharing that is incompletely understood. Both producers and scavengers employ riboswitches (RSs) as a strategy to attenuate gene expression based on environmental cobamide concentrations. The first cobamide RS identified was found to bind adenosylcobalamin and thus RSs identified with similar aptamer domains were classified as cobalamin (Cbl) RSs. However, further study is required to assess the ability of RSs in this class to respond to or differentiate between cobamides besides Cbl. In order to further our understanding of ligand specificity and molecular recognition by Cbl RSs, we aim to characterize Cbl RSs likely to differentiate between cobamides. We seek to validate putative RSs and determine their ligand specificity via binding assays. Following this, we aim to identify the structural foundation of this selectivity using high resolution methods including x-ray crystallography and Cryo-EM. Additionally, we are beginning to use differential scanning fluorimetry as a label-free, highly parallel method to monitor the impact of different cobamides on RS stability. This work could help gain insight into the mechanisms which regulate cobamide metabolism in prokaryotes, further subclassify Cbl RSs, and answer fundamental questions about molecular recognition by RNA.
Keywords: Riboswitch, Cobalamin, Structure