title: Characterization of elements involved in allosteric light regulation of phosphodiesterase activity by comparison of different functional BlrP1 states.
abstract: Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions. One interesting example in this context is the blue-light-regulated phosphodiesterase 1 (BlrP1) of Klebsiella pneumoniae. This covalently linked system of a sensor of blue light using FAD (BLUF) and an EAL phosphodiesterase domain orchestrates the light-dependent down-regulation of c-di-GMP levels. To reveal details of light-induced structural changes involved in EAL activity regulation, we extended previous crystallographic studies with hydrogen-deuterium exchange experiments and small-angle X-ray scattering analysis of different functional BlrP1 states. The combination of hydrogen-deuterium exchange and small-angle X-ray scattering allows the integration of local and global structural changes and provides an improved understanding of light signaling via an allosteric communication pathway between the BLUF and EAL domains. This model is supported by results from a mutational analysis of the EAL dimerization region and the analysis of metal-coordination effects of the EAL active site on the dark-state recovery kinetics of the BLUF domain. In combination with structural information from other EAL domains, the observed bidirectional communication points to a general mechanism of EAL activity regulation and suggests that a similar allosteric coupling is maintained in catalytically inactive EAL domains that retain a regulatory function.
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Electronic address: Andreas.Winkler@mpimf-heidelberg.mpg.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Winkler
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Electronic address: Aniko.Udvarhelyi@mpimf-heidelberg.mpg.de.
authors.forename: Anikó
authors.initials: A
authors.lastname: Udvarhelyi
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Electronic address: Elisabeth.Hartmann@mpimf-heidelberg.mpg.de.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Hartmann
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Electronic address: Jochen.Reinstein@mpimf-heidelberg.mpg.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Reinstein
authors.affiliation: Paul Scherrer Institut, 5232 Villigen PSI, Switzerland. Electronic address: Andreas.Menzel@psi.ch.
authors.forename: Andreas
authors.initials: A
authors.lastname: Menzel
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Electronic address: Robert.Shoeman@mpimf-heidelberg.mpg.de.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Shoeman
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Electronic address: Ilme.Schlichting@mpimf-heidelberg.mpg.de.
authors.forename: Ilme
authors.initials: I
authors.lastname: Schlichting
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D015398:Signal Transduction; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP; D002118:Calcium
keywords: BLUF; BLUF-photoreceptor; BlrP1; EAL; EDTA; HDX; LED; MS; NMA; PDE; SAXS; Sensor of blue light using FAD; allostery; blue-light-regulated phosphodiesterase 1; c-di-GMP; cyclic dimeric GMP; ethylenediaminetetraacetic acid; hydrogen–deuterium exchange; light-emitting diode; mass spectrometry; normal mode analysis; phosphodiesterase; small-angle X-ray scattering