title: Structure of the NPr:EINNtrComplex: Mechanism for Specificity in Paralogous Phosphotransferase Systems.
abstract: Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EIsugarand NPr:EINtr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EIsugarinteraction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EINtrcomplex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domain of EINtr(EINNtr), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Strickland
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Stanley
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Guangshun
authors.initials: G
authors.lastname: Wang
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Istvan
authors.initials: I
authors.lastname: Botos
authors.affiliation: Office of Intramural Research, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Charles D
authors.initials: CD
authors.lastname: Schwieters
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Buchanan
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: peterkofsky@nih.gov.
authors.forename: Alan
authors.initials: A
authors.lastname: Peterkofsky
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: tjandran@nhlbi.nih.gov.
authors.forename: Nico
authors.initials: N
authors.lastname: Tjandra
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009005:Monosaccharides / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D028044:Phosphate-Binding Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D040681:Structural Homology, Protein
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D009005:Monosaccharides; C502371:NPr protein, E coli; D010446:Peptide Fragments; D028044:Phosphate-Binding Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C439615:EIN protein, E coli; D009584:Nitrogen
keywords: Enzyme I; NPr; X-ray crystallography; nuclear magnetic resonance; phosphotransferase system; pseudocontact shifts; residual dipolar couplings; small-angle X-ray scattering; specificity; surface potential