Structure, identification and characterization of the RibD-enolase complex in Francisella

Francisella tularensis is a highly infectious bacterium, a Tier 1-select agent, and the causative agent of tularemia, a potentially fatal zoonotic disease. In this study originally aiming to identify anti-tularemia drug targets, we serendipitously determined the atomic structures and identified their components of the native RibD-enolase protein complex in Francisella novicida; and subsequently systematically characterized the catalytic functions of the RibD-enolase complex. Originally discovered as individually protein in Escherichia coli and yeast, respectively, RibD and Enolase are two essential Enzymes involved in distinct metabolic pathways, both of which could serve as potential therapeutic targets for tularemia treatment and prevention. Our biochemical validation using pull-down assays confirmed the formation of this complex in vivo, revealing that all eluted RibD is bound to Enolase, while the majority of Enolase remained uncomplexed. Structural analysis reveals unique features of the Francisella complex, including key RibD-enolase interactions that mediate complex assembly and β-strand swapping between RibD subunits. Furthermore, molecular dynamics simulations of the ligand-bound RibD-enolase complex highlight localized conformational changes within the substrate-binding sites and suggest a gating mechanism between RibD's substrate and cofactor-binding sites to ensure efficient uptake and turnover. Despite the physical association between RibD and Enolase, enzymatic assays indicated their catalytic activities are independent of each Other, thus the complex may have alternative functional roles that warrant further exploration. Our study provides the first structural and biochemical characterization of the RibD-enolase complex, establishing a foundation for further investigations into its functional significance in Francisella and potential Antibacterial development.

Francisella; RibD-enolase; cryoEM; enzyme complex.