Switching Functional DNA-Binding Modes by Tuning Protein Order-Disorder Equilibria

Abstract

Physical remodeling of chromatin by non-histone architectural proteins of the High Mobility Group B (HMGB) family is central to eukaryotic transcriptional regulation. Nhp6A, the prototypical single-HMG-box protein from yeast, harbors both ordered and disordered regions enabling it to bind and bend DNA without sequence specificity. Here, we integrate ensemble experiments, single-molecule FRET, statistical mechanical modeling and atomistic simulations to dissect the structural and functional consequences of context-dependent phosphorylation in the ordered domain and its interplay with the intrinsically disordered region in Nhp6A. We find that Nhp6A occupies a narrow thermodynamic window, with a melting temperature close to the growth temperature of its host organism and high unfolding cooperativity, a feature conserved across the HMG-box family. Phosphorylation extents - mimicked by multisite phosphomimetic substitutions at residue positions conserved across fungal taxa - smoothly tuning the conformational equilibria between at least two different substates in the native ensemble, apart from the unfolded state. This intrinsic plasticity enables close packing of Nhp6A on DNA through two degenerate binding modes, accompanied by two distinct DNA bending geometries. DNA rescues a strongly destabilized mutant, T63D, through favorable intermolecular interactions, thus effectively acting as a chaperone driving folding. Our findings thus reveal a conserved sequence-ensemble-dynamics code in Nhp6A wherein not just stability, but also phosphorylation-induced conformational switching, disordered tail dynamics, and DNA binding-bending closely coordinate chromatin accessibility. The combination of marginal stability, large cooperativity and electrostatic frustration emerges as a design principle to encode charge sensitivity into proteins, and may represent a general strategy for multisite post-translational regulation.