Pierdominici-Sottile, G. | Search bioRxiv

bioRxiv2025

Towards the Rational Design of RsmE Small-RNA Binders: Insights from Molecular Dynamics Simulations

The RsmZ-RsmE interaction is a key element in post-transcriptional regulation in Pseudomonas species. Experimental studies have shown that alternative fragments of RsmZ, despite displaying only subtle sequence and structural differences, exhibit markedly distinct binding affinities for RsmE. To complicate matters further, the affinities measured for isolated fragments differ substantially from those observed when the same segments are embedded in the full-length sRNA molecule. To explore the origin of these discrepancies, we generated computational models of RsmE dimers bound to one or two sRNA stem loops, including several experimentally studied variants, a couple of truncated forms, and a synthetic construct created by linking two native stem loops with an also native single-stranded region. The unbinding of the RNA fragments from these complexes was studied using Umbrella Sampling simulations, which revealed that base pairs located in the stems, as well as the presence of a linker region, shape the interaction landscape between the protein and RNA, thereby modulating binding affinities. Thus, our findings offer a structural and mechanistic basis for interpreting the experimentally observed differences among RsmZ fragments and establish a conceptual foundation for the future computational design of synthetic sRNAs capable of fine-tuning the RsmZ/RsmE regulatory system in a predictable manner.

biophysics↗

bioRxiv2025

Bayesian inference of functional asymmetry in a ligand-gated ion channel

Ligand-gated ion channels enable rapid cellular signaling by coupling extracellular cues to conformational transitions and ionic fluxes[1-5]. ATP-activated P2X receptors, with their minimalist trimeric architecture, serve as models for studying allosteric activation[6-8]. Although high-resolution structures reveal closed and open states[8, 9], the physical basis of the "flip state" and the emergence of negative cooperativity remain unresolved. Here we combine a recursive Bayesian framework (MacroIR) applied to outside-out patch-clamp recordings, with molecular dynamics simulations, to show that P2x2 activation proceeds via a directional, asymmetric coupling mechanism. ATP binding selectively lowers the energetic barrier for rotation of one subunit at the binding interface, promoting partial activation and substantial conductance, while minimally affecting its neighbor. This rotation, in turn, increases the barrier for subsequent ATP binding, providing a mechanistic explanation for negative cooperativity[7]. In the absence of ligand, elevated barriers stabilize the closed state, quantitatively accounting for spontaneous current fluctuations. These findings overturn the prevailing assumption of symmetric, concerted activation, and demonstrate that the classical flip state arises as a necessary physical intermediate. By showing that ligand-induced modulation of activation barriers can drive symmetry breaking in homomeric channels, our results establish a general principle for dynamic protein assemblies, and provide a conceptual basis for designing conformation-selective modulators in pain and inflammation.

biophysics↗