Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch

•Structural analysis of a glutamine riboswitch in free and L-glutamine-bound states•L-glutamine complex formation induces a tuning fork to L-shaped conformational transition•A long-range linchpin Watson-Crick G-C pair-capping interaction drives complex formation•The conformational change underlies low-affinity, high-selectivity L-glutamine binding

SummaryNaturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg2+-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range “linchpin” Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg2+ solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

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