Biochemical Insights Into the Coordination Plasticity of the Nitrate-binding Protein NreA From Staphylococcus Carnosus




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Nitrate is an oxyanion that is an integral part of the global nitrogen cycle. In bacteria it is an essential nutrient, as it can be incorporated into macromolecules or used as the terminal electron acceptor in anaerobic respiration. The reduction of nitrate to ammonia is regulated by its uptake at the membrane and detection in the cell. The NreABC pathway is an example of such regulation in Staphylococcus carnosus and related bacterial species. Key to this pathway is NreA, a soluble nitrate sensor. To understand how NreA can carry out its role, previous studies have crystallized NreA with both iodide and nitrate, the former as a surrogate. Interestingly, the protein bound both iodide and nitrate through the amide backbone of three residues with the side chain of a tryptophan residue. This discovery inspired in vivo testing, determining that anions activate the downstream pathway, increasing nitrate reduction. The molecular principles governing this binding plasticity were not studied, motivating us to further characterize NreA. We narrowed our focus to connect the rates of binding to the affinity of NreA for nitrate, iodide, and nitrite, as monitored by the intrinsic fluorescence of the binding site tryptophan. Tryptophan fluorescence was used to determine the affinity of binding to the three anions, providing the first quantitative measurements of the plasticity of this binding site. Nitrate was bound with the highest affinity, followed by iodide, and then nitrite. To complement these measurements, stopped-flow fluorimetry was used to determine the kinetic rates of binding, showing that the differences in affinity are predominately determined by the rate of dissociation of the anion from the protein. These experiments were conducted with chloride as a stabilizing ion in solution. Because chloride is an anion with similar properties to the tested anions, it is possible that chloride could interfere with the binding of nitrate and iodide, motivating additional testing. Because we could not remove chloride without affecting protein stability, we tested chloride’s effect by varying its concentration in the protein solution. These data showed that the binding of NreA to nitrate, iodide, and nitrite was impacted by the concentration of chloride, with higher levels of chloride resulting in a weaking of binding of each anion. However, this process was determined to be due to an equal contribution of both the rate of association and dissociation of binding for nitrate. These results have offered the first quantitative measurements of the binding of non-natural binding partners to NreA. The discovery that the relative affinities of different anions is correlated with their relative dissociation rates helps explain how this protein can selectively bind nitrate over similar anions. The interference of this binding by chloride also shows the importance of the protein’s environment, inspiring future testing to determine if chloride interacts specifically or through ionic strength effects. The discovered kinetic contributions can be complemented by thermodynamic measurements from isothermal titration calorimetry or by in silico techniques giving a better understanding of this anion-binding site, developing the general principles of selectively binding anions in proteins.



Chemistry, Biochemistry, Biology, Molecular