Structure of the NPr:EINNtr Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems

Madeleine Strickland; Ann Marie Stanley; Guangshun Wang; Istvan Botos; Charles D Schwieters; Susan K Buchanan; Alan Peterkofsky; Nico Tjandra

doi:10.1016/j.str.2016.10.007

Structure of the NPr:EIN^Ntr Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems

Structure. 2016 Dec 6;24(12):2127-2137. doi: 10.1016/j.str.2016.10.007. Epub 2016 Nov 10.

Authors

Madeleine Strickland¹, Ann Marie Stanley², Guangshun Wang³, Istvan Botos², Charles D Schwieters⁴, Susan K Buchanan², Alan Peterkofsky⁵, Nico Tjandra⁶

Affiliations

¹ Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
² Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
⁴ Office of Intramural Research, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA.
⁵ Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: peterkofsky@nih.gov.
⁶ Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: tjandran@nhlbi.nih.gov.

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:EI^sugar and NPr:EI^Ntr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EI^sugar interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EI^Ntr complex 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 EI^Ntr (EIN^Ntr), 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.

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.

Published by Elsevier Ltd.

MeSH terms

Carrier Proteins / chemistry*
Carrier Proteins / metabolism*
Catalytic Domain
Crystallography, X-Ray
Escherichia coli Proteins / chemistry*
Escherichia coli Proteins / metabolism*
Models, Molecular
Molecular Docking Simulation
Monosaccharides / metabolism
Nitrogen / metabolism
Nuclear Magnetic Resonance, Biomolecular
Peptide Fragments / chemistry*
Peptide Fragments / metabolism*
Phosphate-Binding Proteins
Phosphoenolpyruvate Sugar Phosphotransferase System / chemistry*
Phosphoenolpyruvate Sugar Phosphotransferase System / metabolism*
Phosphorylation
Protein Binding
Protein Domains
Structural Homology, Protein

Substances

Carrier Proteins
Escherichia coli Proteins
Monosaccharides
NPr protein, E coli
Peptide Fragments
Phosphate-Binding Proteins
Phosphoenolpyruvate Sugar Phosphotransferase System
EIN protein, E coli
Nitrogen

Grants and funding

Z01 HL001048-11/ImNIH/Intramural NIH HHS/United States