Mutational Effects on Protein Structural Changes and Interdomain Interactions in the Blue‐light Sensing LOV Protein YtvA

Losi, Aba; Ghiraldelli, Elisa; Jansen, Sven; Gärtner, Wolfgang

doi:10.1562/2005-05-25-ra-541

Cited by 33 publications

(48 citation statements)

References 51 publications

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“…Consistent with the results presented here, mutational analysis has shown that the LOV-surface salt bridge of YtvA does not appreciably contribute to the overall secondary structure of the full-length sensor protein (33). Moreover, chromophore binding is not impaired upon disruption of the LOV-surface salt bridge in full-length YtvA (33). Yet, our mutational analysis with the isolated LOV2 of Arabidopsis phot1 domain clearly indicates that disruption of the Glu-Lys salt bridge results in an almost complete loss of chromophore binding without compromising protein secondary structure (Fig.…”

Section: Discussionsupporting

confidence: 91%

“…3). Consistent with the results presented here, mutational analysis has shown that the LOV-surface salt bridge of YtvA does not appreciably contribute to the overall secondary structure of the full-length sensor protein (33). Moreover, chromophore binding is not impaired upon disruption of the LOV-surface salt bridge in full-length YtvA (33).…”

Section: Discussionsupporting

confidence: 90%

“…FTIR spectroscopy studies indicate that light-driven formation of LOV 390 in YtvA gives rise to a conformational change in its C-terminal STAS (sulfate transporter/ anti-sigma factor) domain (32). Interestingly, a J␣-like helix has also been identified in YtvA (33) suggesting that this bacterial sensor protein, and possibly others, possesses a mode of photoactivation analogous to that operative for the phototropins.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Mutational Analysis of Phototropin 1 Provides Insights into the Mechanism Underlying LOV2 Signal Transmission

Jones

Feeney

Kelly

et al. 2007

Journal of Biological Chemistry

115

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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 light-induced structural changes within a conserved ␣-helix situated C-terminal to LOV2. Here 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 (Gln 575 ) 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 Gln 575 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.Light is critical in shaping the growth and development of plants. The effect of light on plant morphogenesis is mediated through a variety of photoreceptors with specific spectral properties. Genetic analysis using the model plant Arabidopsis thaliana has shown that the effects of blue (390 -500 nm) and UV-A light (320 -390 nm) are mediated by at least two classes of blue light receptors, cryptochromes and phototropins (1-4). Phototropins function to regulate a range of photoresponses, including phototropism, stomatal opening, and chloroplast movement, all of which serve to optimize the photosynthetic efficiency of plants (2). Phototropin activity has also been implicated in regulating extension-growth responses in Arabidopsis such as cotyledon expansion (5), leaf expansion (6), and growth promotion under weak light conditions (7).Arabidopsis contains two phototropins designated phot1 and phot2 (2). Phot1 and phot2 are flavoprotein photoreceptors whose protein structure can be divided into two segments as follows: a photosensory domain at the N terminus and a serine/ threonine kinase domain at the C terminus (Fig. 1A). The N-terminal photosensory domain of the phototropins contains a repeated motif of ϳ110 amino acids called LOV1 and LOV2, respectively (8 -10). LOV 2 domains are members of the large and diverse superfamily of Per, Arnt, Sim (PAS) domains associated with cofactor binding and mediating protein interactions (11). LOV domains are more closely related to...

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Mutational Analysis of Phototropin 1 Provides Insights into the Mechanism Underlying LOV2 Signal Transmission

Jones

Feeney

Kelly

et al. 2007

Journal of Biological Chemistry

115

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“…This salt bridge is located in the region of YtvA-LOV undergoing the most pronounced light-induced structural change (7), although the two residues themselves show only minor rearrangements. Disruption of the salt bridge in the E56Q mutant slows down the dark state recovery rate in vitro by a factor of 2 (39). However, in vivo, this mutant shows a similar extent of light activation of B as the wild type ( Fig.…”

Section: Resultsmentioning

confidence: 86%

In Vivo Mutational Analysis of YtvA from Bacillus subtilis

Ávila-Pérez¹,

Vreede

Tang

et al. 2009

Journal of Biological Chemistry

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LOV2 domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted J␣ (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8,9). Stress via the addition of salt or ethanol (for a review, see Ref.10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13,14). YtvA, which mediates light activation of B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16). When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9,14,17,18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180 o upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central ␤-sheet and the C-terminal linker region, the J␣-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavinbinding pocket, the ␤-sheet of the LOV domain, and the GTPbinding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approac...

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“…25 LOV-domain b-sheet rearrangement also appears to be required for interdomain interactions in the bacterial photosensor YtvA from Bacillus subtilis. 26 Further studies will determine whether a signal transmission mechanism involving the central b-sheet is common to other LOV-sensor proteins. Interestingly, molecular dynamic simulations indicate that the consequences of LOV1 photoexcitation differ to that of LOV2 and involve stabilizing a conserved surface salt bridge.…”

Section: Lov2 Signal Transmissionmentioning

confidence: 99%

Phototropin Receptor Kinase Activation by Blue Light

Jones

Christie

2008

Plant Signaling & Behavior

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Phototropins (phot1 and phot2) are blue light-activated serine/ threonine protein kinases that function to mediate a variety of adaptive processes that serve to optimize the photosynthetic efficiency of plants and thereby promote their growth. Light sensing by the phototropins is mediated by a repeated motif located within the N-terminal region of the protein designated the LOV domain. Although phototropins possess two LOV photosensors (LOV1 and LOV2), recent biophysical and structure-function analyses clearly indicate that the LOV2 domain plays a predominant role in regulating phototropin kinase activity owing to specific protein changes that occur in response to LOV2 photoexcitation. In particular, the central b-sheet scaffold plays a role in propagating the photochemical signal generated from within LOV2 to protein changes at the surface that are necessary for kinase activation. Light-Induced Activation of Phot1The primary amino acid structures of plant phototropins (phot1 and phot2) can be separated into two distinct regions: a N-terminal photosensory region linked to a C-terminal effector domain that includes a classic serine/threonine kinase motif. [1][2][3] The N-terminal photosensory region comprises two LOV domains each of which binds the blue light absorbing cofactor flavin mononucleotide (FMN) as chromophore. 4,5 LOV domains exhibit significant homology to motifs found in a diverse range of eukaryotic and prokaryotic proteins involved in sensing Light, Oxygen or Voltage, hence the acronym LOV. 6 Bacterially expressed LOV domains are photochemically reactive as monitored by absorbance spectroscopy. 7 In the dark, LOV domains bind FMN non-covalently forming a spectral species, LOV 447 , which absorbs maximally near 447 nm. 7,8 Irradiation of the domain induces the formation of a covalent bond between the C(4a) carbon of the FMN and the sulfur atom of a nearby, conserved cysteine residue within the domain. Cysteinyl adduct formation occurs within microseconds of illumination and produces a spectral species, LOV 390 , that absorbs maximally near 390 nm. 8 Mutation of the afore-mentioned active-site cysteine to either alanine or serine results in a loss of photochemical reactivity of the LOV domain. 7,8 Moreover, this mutation has been used successfully to probe the roles of LOV1 and LOV2 in regulating phototropin activity and function. [9][10][11][12] As reported previously (ref. 13), the Cys → Ala mutation within LOV2 of Arabidopsis thaliana phot1 (C517A) results in a loss of light-induced receptor autophosphorylation when expressed in insect cells (Fig. 1A), demonstrating that LOV2 is the principle photosensor regulating phot1 kinase activity. A similar mode of action has been reported for the LOV2 domain of Arabidopsis phot2. 12,14,15 The LOV1 domain, by contrast, is proposed to mediate receptor dimerization 16 and/or modulate the photoreactivity of LOV2. 14 Substitution of the conserved active-site cysteine with methionine has been shown to result in the formation of a unique photoproduct species abso...

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Mutational Effects on Protein Structural Changes and Interdomain Interactions in the Blue‐light Sensing LOV Protein YtvA

Cited by 33 publications

References 51 publications

Mutational Analysis of Phototropin 1 Provides Insights into the Mechanism Underlying LOV2 Signal Transmission

Mutational Analysis of Phototropin 1 Provides Insights into the Mechanism Underlying LOV2 Signal Transmission

In Vivo Mutational Analysis of YtvA from Bacillus subtilis

Phototropin Receptor Kinase Activation by Blue Light

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