Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser

Riboswitches are structured 160;cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the 160;add 160;adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the 160;add 160;adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall 160;molecule exhibits a motion at 160;kex?~?319 160;s-1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with 160;kex~?83 160;s-1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 160;s-1. The 160; 181;s-ms 160;motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation,?~?50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

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