Molecular and thermodynamic determinants of self-assembly and hetero-oligomerization in the enterobacterial thermo-osmo-regulatory protein H-NS.

Environmentally regulated gene expression is critical for bacterial survival under stress conditions, including extremes in temperature, osmolarity and nutrient availability. Here, we dissect the thermo- and osmo-responsory behavior of the transcriptional repressor H-NS, an archetypal nucleoid-condensing sensory protein, ubiquitous in enterobacteria that infect the mammalian gut. Through experiments and thermodynamic modeling, we show that H-NS exhibits osmolarity, temperature and concentration dependent self-association, with a highly polydisperse native ensemble dominated by monomers, dimers, tetramers and octamers. The relative population of these oligomeric states is determined by an interplay between dimerization and higher-order oligomerization, which in turn drives a competition between weak homo- versus hetero-oligomerization of protein-protein and protein-DNA complexes. A phosphomimetic mutation, Y61E, fully eliminates higher-order self-assembly and preserves only dimerization while weakening DNA binding, highlighting that oligomerization is a prerequisite for strong DNA binding. We further demonstrate the presence of long-distance thermodynamic connectivity between dimerization and oligomerization sites on H-NS which influences the binding of the co-repressor Cnu, and switches the DNA binding mode of the hetero-oligomeric H-NS:Cnu complex. Our work thus uncovers important organizational principles in H-NS including a multi-layered thermodynamic control, and provides a molecular framework broadly applicable to other thermo-osmo sensory proteins that employ similar mechanisms to regulate gene expression.

Diverse Native Ensembles Dictate the Differential Functional Responses of Nuclear Receptor Ligand-Binding Domains.

Native states of folded proteins are characterized by a large ensemble of conformations whose relative populations and interconversion dynamics determine the functional output. This is more apparent in transcription factors that have evolved to be inherently sensitive to small perturbations, thus fine-tuning gene expression. To explore the extent to which such functional features are imprinted on the folding landscape of transcription factor ligand-binding domains (LBDs), we characterize paralogous LBDs of the nuclear receptor (NR) family employing an energetically detailed and ensemble-based Ising-like statistical mechanical model. We find that the native ensembles of the LBDs from glucocorticoid receptor, PPAγ, and thyroid hormone receptor display a remarkable diversity in the width of the native wells, the number and nature of partially structured states, and hence the degree of conformational order. Monte Carlo simulations employing the full state representation of the ensemble highlight that many of the functional conformations coexist in equilibrium, whose relative populations are sensitive to both temperature and the strength of ligand binding. Allosteric modulation of the degree of structure at a coregulator binding site on ligand binding is shown to arise via a redistribution of populations in the native ensembles of glucocorticoid and PPAγ LBDs. Our results illustrate how functional requirements can drive the evolution of conformationally diverse native ensembles in paralogs.

Correction: G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer's disease pathology.

[This corrects the article DOI: 10.1371/journal.pone.0225487.].

G82S RAGE polymorphism influences amyloid-RAGE interactions relevant in Alzheimer's disease pathology.

Receptor for advanced glycation end products (RAGE) has been implicated in the pathophysiology of Alzheimers disease(AD) due to its ability to bind amyloid-beta (Aß42) and mediate inflammatory response. G82S RAGE polymorphism is associated with AD but the molecular mechanism for this association is not understood. Our previous in silico study indicated a higher binding affinity for mutated G82S RAGE, which could be caused due to changes in N linked glycosylation at residue N81. To confirm this hypothesis, in the present study molecular dynamics (MD) simulations were used to simulate the wild type (WT) and G82S glycosylated structures of RAGE to identify the global structural changes and to find the binding efficiency with Aß42 peptide. Binding pocket analysis of the MD trajectory showed that cavity/binding pocket in mutant G82S glycosylated RAGE variants is more exposed and accessible to external ligands compared to WT RAGE, which can enhance the affinity of RAGE for Aß. To validate the above concept, an in vitro binding study was carried using SHSY5Y cell line expressing recombinant WT and mutated RAGE variant individually to which HiLyte Fluor labeled Aß42 was incubated at different concentrations. Saturated binding kinetics method was adopted to determine the Kd values for Aß42 binding to RAGE. The Kd value for Aß42- WT and Aß42-mutant RAGE binding were 92±40 nM (95% CI-52 to 152nM; R2-0.92) and 45±20 nM (95% CI -29 to 64nM; R2-0.93), respectively. The Kd value of <100nM observed for both variants implicates RAGE as a high-affinity receptor for Aß42 and mutant RAGE has higher affinity compared to WT. The alteration in binding affinity is responsible for activation of the inflammatory pathway as implicated by enhanced expression of TNFα and IL6 in mutant RAGE expressing cell line which gives a mechanistic view for the G82S RAGE association with AD.

Electrostatic Frustration Shapes Folding Mechanistic Differences in Paralogous Bacterial Stress Response Proteins.

Paralogous proteins play a vital role in evolutionary adaptation of organisms and species divergence. One outstanding question is the molecular basis for how folding mechanisms differ in paralogs that not only exhibit similar topologies but also evolve under near-identical selection pressures. Here, we address this question by studying a paralogous protein pair from enterobacteria, Hha and Cnu, combining experiments, simulations and statistical modeling. We find that Hha is less stable and folds an order of magnitude slower than Cnu despite similar packing and topological features. Differences in surface charge-charge interactions, however, promote a N-terminal biased unfolding mechanism in Hha unlike Cnu that unfolds via the C terminus. Our work highlights how electrostatic frustration contributes to the population of heterogeneous native ensembles in paralogs and the avenues through which evolutionary topological constraints could be overcome by modulating charge-charge interactions.