Structure of a transiently phosphorylated switch in bacterial signal transduction

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Abstract

Receiver domains are the dominant molecular switches in bacterial signalling1,2. Although several structures of non-phosphorylated receiver domains have been reported3,4,5,6,7,8, a detailed structural understanding of the activation arising from phosphorylation has been impeded by the very short half-lives of the aspartyl-phosphate linkages. Here we present the first structure of a receiver domain in its active state, the phosphorylated receiver domain of the bacterial enhancer-binding protein NtrC (nitrogen regulatory protein C). Nuclear magnetic resonance spectra were taken during steady-state autophosphorylation/dephosphorylation, and three-dimensional spectra from multiple samples were combined. Phosphorylation induces a large conformational change involving a displacement of β-strands 4 and 5 and α-helices 3 and 4 away from the active site, a register shift and an axial rotation in helix 4. This creates an exposed hydrophobic surface that is likely to transmit the signal to the transcriptional activation domain.

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Figure 1: Gain in signal-to-noise ratio by adding multiple three-dimensional NMR data taken on different samples.
Figure 2: The structure of the phosphorylated receiver domain of NtrC.
Figure 3: Molecular switch upon phosphorylation of D54 in NtrCr.
Figure 4: Formation of an exposed hydrophobic surface on helix 4 upon phosphorylation.

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Acknowledgements

We thank D. King for analysis by mass spectrometry. This work was supported by the Director, Office of Biological & Environmental Research, Office of Energy Research of the US Department of Energy, and through instrumentation grants from the NSF D.E.W. would also like to thank the Miller Institute for support during part of this work. NMR studies were carried out at the National Magnetic Resonance Facility at Madison with support from the NIH Biomedical Technology Program and additional equipment funding from the University of Wisconsin, NSF Academic Infrastructure Program, NIH Shared Instrumentation Program, NSF Biological Instrumentation Program, and US Department of Agriculture.

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Correspondence to Dorothee Kern.

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Kern, D., Volkman, B., Luginbühl, P. et al. Structure of a transiently phosphorylated switch in bacterial signal transduction. Nature 402, 894–898 (1999) doi:10.1038/47273

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