Towards the rational design of RsmE small-RNA binders: insights from molecular dynamics simulations

Abstract

RsmZ is a small RNA (sRNA) that regulates gene expression in Pseudomonas through binding to the dimeric protein RsmE. Remarkably, closely related RsmZ fragments with only minor sequence and structural differences display widely different binding affinities. Moreover, affinities measured for isolated fragments differ substantially from those observed when the same segments are embedded in the full-length sRNA. To uncover the physicochemical basis of these discrepancies, we constructed computational models of RsmE dimers bound to one or two sRNA stem loops, including experimentally characterised variants, truncated forms, and a synthetic construct linking two native stem loops via a single-stranded region. Umbrella sampling simulations of RNA unbinding reveal that stem base pairing and the presence of a linker region reshape the interaction landscape, thereby modulating binding affinities. These results provide a structural and mechanistic framework for rationalising the diverse binding behaviour of RsmZ fragments and establish a basis for the computational design of synthetic sRNAs capable of predictably tuning the RsmZ/RsmE regulatory system.