Two-component regulatory systems are one of the most common mechanisms for signal transduction in bacteria and have recently been found in eukaryotes. In these systems, a histidine kinase autophosphorylates in response to an envoironmental stimulus, providing the phoshate for the response regulator which, subsequently, transduces the signal to a downstream target. Nothing is known about the structural basis of the activation of response regulators upon phosphorylation because the lifetime of the phosphorylated protein is prohibitively short for structural analysis. We are using the response regulator, NTRC, which controls nitrogen metabolism, as a model system. The structure of the unphosphorylated form has been determined in our lab (Volkman, et al., Biochemistry 34, 1413-1424). We have recently obtained conditions which maintain the phosphorylated state long enough for structure determination by NMR. This is done by creating a steady state equilibrium using a small molecule as a phosphodonor. However, due to aggregation and fast turnover, the protein concentration is limited to 0.5 mM. Therefore, structure determination would be greatly facilitated by the higher sensitivity of a 750 MHz magnet and an 8 mm probe. HSQC's taken in our laboratory indicate that the conformational change upon phosphorylation involves only a portion of the molecule so that the known NMR data on the unphosphorylated form will be helpful in analyzing the activated, phosphorylated form. This work should provide the first example of structural understanding of activation via phosphorylation in the response regulator superfamily. Given the high sequence and structural homology among two-component systems, the structure/function relationships in NTRC should be general for other response regulators. Furthermore, the structural changes of a protein triggered by phosphorylation is of interest because this is one of the most common covalent modifications used for modulation of protein function.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
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University of Wisconsin Madison
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Travers, Timothy; López, Cesar A; Van, Que N et al. (2018) Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain. Sci Rep 8:8461
Thomas, Nathan E; Wu, Chao; Morrison, Emma A et al. (2018) The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release. J Biol Chem 293:19137-19147
Assadi-Porter, Fariba M; Radek, James; Rao, Hongyu et al. (2018) Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties. Molecules 23:
Wijayatunga, Nadeeja N; Sams, Valerie G; Dawson, John A et al. (2018) Roux-en-Y gastric bypass surgery alters serum metabolites and fatty acids in patients with morbid obesity. Diabetes Metab Res Rev 34:e3045
Assadi-Porter, Fariba M; Reiland, Hannah; Sabatini, Martina et al. (2018) Metabolic Reprogramming by 3-Iodothyronamine (T1AM): A New Perspective to Reverse Obesity through Co-Regulation of Sirtuin 4 and 6 Expression. Int J Mol Sci 19:
Dominguez, Eddie; Zarnowski, Robert; Sanchez, Hiram et al. (2018) Conservation and Divergence in the Candida Species Biofilm Matrix Mannan-Glucan Complex Structure, Function, and Genetic Control. MBio 9:
Franco, Aldo; Dovell, Sanaz; Möller, Carolina et al. (2018) Structural plasticity of mini-M conotoxins - expression of all mini-M subtypes by Conus regius. FEBS J 285:887-902
Wales, Jessica A; Chen, Cheng-Yu; Breci, Linda et al. (2018) Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase. J Biol Chem 293:1850-1864
Selen Alpergin, Ebru S; Bolandnazar, Zeinab; Sabatini, Martina et al. (2017) Metabolic profiling reveals reprogramming of lipid metabolic pathways in treatment of polycystic ovary syndrome with 3-iodothyronamine. Physiol Rep 5:
Mong, Surin K; Cochran, Frank V; Yu, Hongtao et al. (2017) Heterochiral Knottin Protein: Folding and Solution Structure. Biochemistry 56:5720-5725

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