Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch

Abstract

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch conformational ensembles and that Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the translation-inhibiting state of the switching sequence followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from μs to ms. These dynamics are at least an order of magnitude faster than folding and unfolding kinetics associated with translation-state switching in the switching sequence. Moreover, we propose that two pathways exist for ligand recognition. Together, our results suggest a dynamic ensemble switching of the aptamer domain that may lead to the translation-inhibiting state of the riboswitch. Additionally, our results suggest that multiple configurations could enable inhibitory tuning manifested through ligand-dependent changes via ensemble switching and kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

Integrative structural dynamics probing of the conformational heterogeneity in synaptosomal-associated protein 25

“…Similar workflows resolved the structural dynamics in enzymes, 36 the dynamic heterogeneity in multi-domain proteins, 18 and other intrinsically disordered proteins, 37 , 38 and has been used for determining the ensemble switching mechanism of eukaryotic thiamin riboswitch. 39 Furthermore, the integrative structural modeling using DMD and label-based experiments can solve the remaining challenges in highly dynamic systems such as IDPs, IDRs, and complex supertertiary structural dynamics.…”

Riboswitch Folds to Holo-Form Like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration

“…In this study, we showed that TPP AD populates intermediates when its folding is induced by Mg 2+ in agreement with the experiments. 12 We found that at physiological [Mg 2+ ] (≈ 4 mM), AD populated a closed-arm Y-shaped and a holo-form like folded conformations. The holo-form like conformation is not the global minimum in the FES at around physiological [Mg 2+ ] but becomes the global minimum in higher [Mg 2+ ] (≥ 5 mM) even in the absence of the TPP ligand.…”

“…44,45 Experiments studying TPP AD folding also suggest that multiple structural ensembles are populated depending on [Mg 2+ ] and TPP-ligand. 12 Further, the timescales associated with the transitions among these ensembles varied from µs to s, indicating significant variations in the barrier heights separating the basins.…”

“…Single-molecule FRET 12,18 and SAXS [41][42][43] studies have shown that physiological [Mg 2+ ] is indispensable for the cognate ligand to bind to the TPP AD. Single-molecule FRET 18 study proposed that a Y-shaped intermediate mediated by Mg 2+ gets populated during the initial stages of folding even in the absence of the cognate TPP ligand.…”