Abstract
The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore1. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined2,3,4, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae5. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria6,7,8,9. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain–domain interfaces.
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Accession codes
Primary accessions
Protein Data Bank
Data deposits
The atomic coordinates and structure factor amplitudes of the following structures have been deposited in the Protein Data Bank: Ca2+ complex of BlrP1 at pH 4.5 (accession code 3GFX), metal-free complex of BlrP1 at pH 6.0 (3GFY), Mn2+ complex of BlrP1 at pH 6.0 (3GFZ), Mn2+ complex of BlrP1 at pH 9.0 (3GG0) and Ca2+ complex of BlrP1 at pH 8.0 (3GG1).
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Acknowledgements
We thank A. Jung and A. Schmidt for help during the initial stages of the project, C. Fang for c-di-GMP purification and Q. Wu and K. H. Gardner for discussions and for sharing unpublished data. We are grateful to I. Vetter for advice and support for the crystallographic software. Diffraction data were collected at the Swiss Light Source, beamline X10SA, Paul Scherrer Institute, Villigen, Switzerland. We thank the Dortmund–Heidelberg team for data collection and E. Pohl, A. Pauluhn and C. Schulze-Briese for their support in setting up the beamline. The K. pneumoniae genome was sequenced at the School of Medicine of the Washington University in St Louis. We acknowledge financial support from the Deutsche Forschungsgemeinschaft (Research Group FOR 526 ‘Sensory Blue Light Receptors’) to I.S., the US National Science Foundation (MCB 0645876) to M.G. and the Boehringer Ingelheim Fonds to J.J.G. I.S. and J.R. are members of the Cluster of Excellence CellNetworks, Heidelberg.
Author Contributions M.G. and I.S. designed the project; N.V.K. and M.G. identified and cloned blrP1; N.V.K. and D.A.R. purified BlrP1 and performed the initial biochemical analysis; J.J.G. constructed the expression clone and established the purification protocol of the protein used for crystallization and biochemical analysis; E.H. purified, crystallized and characterized wild-type and mutant BlrP1 by enzymatic and dynamic light scattering analysis; T.B. performed the spectroscopic analysis; T.R.M.B. determined the crystal structures; J.R. designed the kinetic experiments; R.L.S. performed the mass spectrometry and analytical ultracentrifugation; and I.S. did the practical crystallographic work. T.R.M.B., M.G. and I.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Barends, T., Hartmann, E., Griese, J. et al. Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase. Nature 459, 1015–1018 (2009). https://doi.org/10.1038/nature07966
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DOI: https://doi.org/10.1038/nature07966
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