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Solution structure of NPr, a bacterial signal-transducing protein that controls the phosphorylation state of the potassium transporter-regulating protein IIANtr

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Abstract

A nitrogen-related signal transduction pathway, consisting of the three phosphotransfer proteins EINtr, NPr, and IIANtr, was discovered recently to regulate the uptake of K+ in Escherichia coli. In particular, dephosphorylated IIANtr inhibits the activity of the K+ transporter TrkA. Since the phosphorylation state of IIANtr is partially determined by its reversible phosphorylation by NPr, we have determined the three-dimensional structure of NPr by solution NMR spectroscopy. In total, we obtained 973 NOE-derived distance restraints, 112 chemical shift-derived backbone angle restraints, and 35 hydrogen-bond restraints derived from temperature coefficients (wave). We propose that temperature wave is useful for identifying exposed beta-strands and assists in establishing protein folds based on chemical shifts. The deduced structure of NPr contains three α-helices and four β-strands with the three helices all packed on the same face of the β-sheet. The active site residue His16 of NPr for phosphoryl transfer was found to be neutral and in the Nε2-H tautomeric state. There appears to be increased motion in the active site region of NPr compared to HPr, a homologous protein involved in the uptake and regulation of carbohydrate utilization.

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Abbreviations

NPr:

Nitrogen-related HPr

HPr:

Histidine-containing protein

EINtr :

Nitrogen-related enzyme I

HSQC:

Heteronuclear single quantum coherence spectroscopy

IIANtr :

Nitrogen-related enzyme IIA

GAF:

cGMP-specific and –stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA

IPTG:

Isopropyl-β-D-thiogalactopyranoside

NMR:

Nuclear magnetic resonance

NOE:

Nuclear Overhauser effect

PCR:

Polymerase chain reaction

PEP:

Phosphoenolpyruvate

PTS:

Phosphoenolpyruvate:sugar phosphotransferase system

RCK:

Regulating conductance of K+

rmsd:

Root mean square deviation

SDS-PAGE:

Sodium dodecylsulfate-polyacrylamide gel electrophoresis

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Acknowledgments

This research was supported by the startup fund from the Eppley Institute of the University of Nebraska Medical Center (UNMC) to G.W. and, in part, by the Intramural Research Program of the NIH, NHLBI to A.P. The support of UNMC also includes the accessibility to the NMR Core Facility (supported by both a CORE grant from the National Cancer Institute-NIH and the Nebraska Research Initiative NRI). We are grateful to Frank Delaglio and Dan Garrett (NIH) for NMR software. We thank Paul Keifer for maintaining the NMR hardware during this study.

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Authors and Affiliations

  1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Room ECI3018, Omaha, NE, 68198-6805, USA

    Xia Li & Guangshun Wang

  2. Laboratory of Cell Biology, Building 50, National Heart, Lung and Blood Institute, National Institute of Health, Bethesda, MD, 20892, USA

    Alan Peterkofsky

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  1. Xia Li
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  2. Alan Peterkofsky
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Correspondence to Guangshun Wang.

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Li, X., Peterkofsky, A. & Wang, G. Solution structure of NPr, a bacterial signal-transducing protein that controls the phosphorylation state of the potassium transporter-regulating protein IIANtr . Amino Acids 35, 531–539 (2008). https://doi.org/10.1007/s00726-008-0079-9

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