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Structure-function analysis of phototropin blue-light receptors
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S225–S233
P2.7 Structure-function analysis of phototropin blue-light receptors J. Christie, M. Jones, S. Sullivan, C. Thomson, (University of Glasgow, United Kingdom) Phototropins (phot1 and phot2) are blue light-activated serine/ threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis, and is necessary to activate phot1 kinase activity through lightinduced structural changes within a conserved Éø-helix situated C-terminal to LOV2. We have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln575) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN-binding. These findings, in combination with double mutant analyses, indicate that Gln575 plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain. Additional structure-function analysis of Arabidopsis phot1 will also be presented. doi:10.1016/j.cbpa.2007.01.499
P2.8 A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain J. Kennis, M. Alexandre, J. Arents, R. van Grondelle, (Vrije Universiteit (Vrije University), The Netherlands); K. Hellingwerf, (University of Amsterdam, The Netherlands) Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the Cterminal part of Avena sativa phototropin 1. The N-terminal part contains two Light, Oxygen or Voltage (LOV) sensing domains, LOV1 and LOV2, each bind a flavin mononucleotide (FMN) chromophore (?max = 447 nm, referred as D447) and form the light-sensitive domains, of which LOV2 is principal. Blue-light absorption produces a covalent adduct between a very
S227
conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well characterised LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by three orders of magnitude in the sub-molar concentration range, via a base-catalysed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cystein. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethylpyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)−1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contribute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)−1, leading to a net adduct decay time constant of 30 s at pH 8. doi:10.1016/j.cbpa.2007.01.500
P2.9 Green light control of plant form and function K. Folta, S. Maruhnich, A. Dhingra, D. Kumar, (University of Florida, United States) Plants rely on signals from the environment to shape their growth and development. Of the suite of signals sensed, light influences both acute and long-term biological processes. Various aspects of the light signal, such as quantity (fluence and/or fluence rate), quality (wavelength) and duration (photoperiod) are integrated to generate appropriate biological response to best match ambient conditions. Light signals are sensed via a series of photosensors and integrated by their attendant signaling pathways. Of these, the phytochromes, cryptochromes, phototropins and other LOV-domain proteins have been well described, and are activated to wavebands similar to those that optimally stimulate photosynthesis. However, other wavelengths (such as far-red) certainly contain information that dramatically remodels gene expression and plant stature. Similarly, recent efforts have expanded on reports from the classical literature and examined the role of green light (500– 550 nm). Through the use of homemade narrow-bandwidth LED light arrays we have shown that green light induces a series of responses that are contrary to those typically associated with normal photomorphogenic development. Whereas red, blue and far-red light cause a decrease in stem elongation rate, green light induces an increase in stem elongation. Green light irradiation causes an acute down-regulation of specific chloroplast
P2.7 Structure-function analysis of phototropin blue-light receptors J. Christie, M. Jones, S. Sullivan, C. Thomson, (University of Glasgow, United Kingdom) Phototropins (phot1 and phot2) are blue light-activated serine/ threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis, and is necessary to activate phot1 kinase activity through lightinduced structural changes within a conserved Éø-helix situated C-terminal to LOV2. We have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln575) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN-binding. These findings, in combination with double mutant analyses, indicate that Gln575 plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain. Additional structure-function analysis of Arabidopsis phot1 will also be presented. doi:10.1016/j.cbpa.2007.01.499
P2.8 A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain J. Kennis, M. Alexandre, J. Arents, R. van Grondelle, (Vrije Universiteit (Vrije University), The Netherlands); K. Hellingwerf, (University of Amsterdam, The Netherlands) Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the Cterminal part of Avena sativa phototropin 1. The N-terminal part contains two Light, Oxygen or Voltage (LOV) sensing domains, LOV1 and LOV2, each bind a flavin mononucleotide (FMN) chromophore (?max = 447 nm, referred as D447) and form the light-sensitive domains, of which LOV2 is principal. Blue-light absorption produces a covalent adduct between a very
S227
conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well characterised LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by three orders of magnitude in the sub-molar concentration range, via a base-catalysed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cystein. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethylpyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)−1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contribute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)−1, leading to a net adduct decay time constant of 30 s at pH 8. doi:10.1016/j.cbpa.2007.01.500
P2.9 Green light control of plant form and function K. Folta, S. Maruhnich, A. Dhingra, D. Kumar, (University of Florida, United States) Plants rely on signals from the environment to shape their growth and development. Of the suite of signals sensed, light influences both acute and long-term biological processes. Various aspects of the light signal, such as quantity (fluence and/or fluence rate), quality (wavelength) and duration (photoperiod) are integrated to generate appropriate biological response to best match ambient conditions. Light signals are sensed via a series of photosensors and integrated by their attendant signaling pathways. Of these, the phytochromes, cryptochromes, phototropins and other LOV-domain proteins have been well described, and are activated to wavebands similar to those that optimally stimulate photosynthesis. However, other wavelengths (such as far-red) certainly contain information that dramatically remodels gene expression and plant stature. Similarly, recent efforts have expanded on reports from the classical literature and examined the role of green light (500– 550 nm). Through the use of homemade narrow-bandwidth LED light arrays we have shown that green light induces a series of responses that are contrary to those typically associated with normal photomorphogenic development. Whereas red, blue and far-red light cause a decrease in stem elongation rate, green light induces an increase in stem elongation. Green light irradiation causes an acute down-regulation of specific chloroplast