NMR and computational strategies to dissect hydration dynamics in a metal-binding transcriptional regulator

VILLARRUEL-DUJOVNE, M.; BRINGAS, MAURO; RAVERA, E.; DI LELLA, SANTIAGO; CAPDEVILA, DAIANA

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Staphylococcus aureus CzrA is a paradigmatic member of the ArsR family of transcriptional metalloregulators, which are critical for the bacterial response to stress. Zn binding to CzrA, which induces DNA derrepresion, is entropically driven, as shown by calorimetry. A detailed equilibrium dynamics study of different allosteric states of CzrA reveals that Zn induces an entropy redistribution that, when perturbed, results in nearallosteric uncoupling; however, this change in conformational entropy makes only a small net contribution to the total entropy. This difference between the change in conformational entropy vs. total entropy of Zn binding implicates a significant contribution of solvent molecule rearrangements to this equilibrium. However, the absence of a major structural changes suggests that solvent rearrangements occur mainlyon the protein surface and/or from Zn desolvation, concomitant with a dynamical redistribution of conformational entropy. Previous results also suggest that Zn binding not only leads to a redistribution of protein internal dynamics, but also can lead to the release of waters from the protein surface, which may make a significant contribution to theallosteric response that results in dissociation from the DNA. Quantifying the differential hydration of two conformational states that share very similar crystal structures and then correlating this with the protein’s solvent entropy change constitutes a very challenging problem. Here, we present different avenues to dissect hydration dynamics in a metal-binding transcriptional regulator that provide different insights into this complex problem. We explore primary solution NMR tools for probing protein–water interactions are the laboratory frame nuclear Overhauser effect (NOE) and its rotating frame counterpart (ROE) of through-space dipolar magnetization transfer. The NOE/ROE ratio is an excellent tool for the detection of hydration dynamics near the surface of a protein, minimizing contamination from bulk solvent in a site specific manner. Molecular dynamics simulations and methods designed to provide a spatially resolved picture of solvent thermodynamics were also employed to provide a more complete panorama of solvent redistribution.