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Essential role of the A'α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Kashojiya S, Okajima K, Shimada T, Tokutomi S - PLoS ONE (2015)

Bottom Line: Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation.These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK.The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Osaka Prefecture University, Sakai, Osaka, Japan.

ABSTRACT
Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

No MeSH data available.


(A) Kinase activity of WT, D788A, and ΔA’α of At phot1 LOV2-STK on P1Nt in the dark (D) or under BL irradiation (L). The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively. (B) Absorption spectra and light minus dark absorption difference spectrum (inset) of ΔA’α in a solution containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C. The black and the gray line were measured after dark adaptation and BL irradiation, respectively.
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pone.0124284.g002: (A) Kinase activity of WT, D788A, and ΔA’α of At phot1 LOV2-STK on P1Nt in the dark (D) or under BL irradiation (L). The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively. (B) Absorption spectra and light minus dark absorption difference spectrum (inset) of ΔA’α in a solution containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C. The black and the gray line were measured after dark adaptation and BL irradiation, respectively.

Mentions: To elucidate the function of A’α-helix, the effect of A’α-helix truncation on the kinase activity was studied (Fig 2A). ΔA’α prepared using the E. coli expression system was not stable and easily formed aggregates in a 100 mM NaCl buffer solution; therefore, the NaCl concentration was increased to 500 mM to protect against aggregation. The kinase activities of WT, its kinase-dead D788A substitute and ΔA’α were measured using P1Nt as an artificial substrate. In the dark, WT exhibited a faint phosphorylation band compared to D788A whose kinase activity was undetectable. ΔA’α had a similar phosphorylation level to WT in the dark. BL strongly activated the kinase of WT that was impaired by the amino acid substitution at Asp788 to Ala in accordance with previous results [20,38]. In contrast, activation did not occur in ΔA’α indicating that A’α including Glu474 is an essential element for activation. We have previously reported that the kinase activity of At phot1 LOV2-STK correlates with the lifetime of S390 in LOV2 and that shortening of the S390 lifetime reduced the kinase activity [20]. Therefore, the kinetics of the photoreaction in ΔA’α was studied (Fig 2B). The ground state absorption spectra of WT and ΔA’α had a similar ratio of the height at 450 nm to 280 nm indicating that they bound a similar amount of FMN. ΔA’α exhibited a characteristic absorption spectral change with a reversible formation of S390 (Fig 2B inset). Its half decay time was calculated as 89 s at 20°C (Table 1). The decay time was almost the same as that of WT in the buffer containing 500 mM NaCl (91 s at 20°C), and the absorption peaks did not shift, indicating that the presence of the A’α-helix does not affect LOV photochemistry. Taken together, these results indicate that the A’α-helix is essential for the intramolecular signaling from LOV2 to the kinase, while it is not involved in the photochemical properties of LOV2 in the LOV2-STK of At phot1.


Essential role of the A'α/Aβ gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Kashojiya S, Okajima K, Shimada T, Tokutomi S - PLoS ONE (2015)

(A) Kinase activity of WT, D788A, and ΔA’α of At phot1 LOV2-STK on P1Nt in the dark (D) or under BL irradiation (L). The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively. (B) Absorption spectra and light minus dark absorption difference spectrum (inset) of ΔA’α in a solution containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C. The black and the gray line were measured after dark adaptation and BL irradiation, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401697&req=5

pone.0124284.g002: (A) Kinase activity of WT, D788A, and ΔA’α of At phot1 LOV2-STK on P1Nt in the dark (D) or under BL irradiation (L). The upper and lower panels indicate autoradiogram and CBB staining of SDS-PAGE gels, respectively. The arrow and the arrowhead indicate the position of LOV2-STK and P1Nt, respectively. (B) Absorption spectra and light minus dark absorption difference spectrum (inset) of ΔA’α in a solution containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl, 10% (w/v) glycerol, and 1 mM Na2EGTA at 20°C. The black and the gray line were measured after dark adaptation and BL irradiation, respectively.
Mentions: To elucidate the function of A’α-helix, the effect of A’α-helix truncation on the kinase activity was studied (Fig 2A). ΔA’α prepared using the E. coli expression system was not stable and easily formed aggregates in a 100 mM NaCl buffer solution; therefore, the NaCl concentration was increased to 500 mM to protect against aggregation. The kinase activities of WT, its kinase-dead D788A substitute and ΔA’α were measured using P1Nt as an artificial substrate. In the dark, WT exhibited a faint phosphorylation band compared to D788A whose kinase activity was undetectable. ΔA’α had a similar phosphorylation level to WT in the dark. BL strongly activated the kinase of WT that was impaired by the amino acid substitution at Asp788 to Ala in accordance with previous results [20,38]. In contrast, activation did not occur in ΔA’α indicating that A’α including Glu474 is an essential element for activation. We have previously reported that the kinase activity of At phot1 LOV2-STK correlates with the lifetime of S390 in LOV2 and that shortening of the S390 lifetime reduced the kinase activity [20]. Therefore, the kinetics of the photoreaction in ΔA’α was studied (Fig 2B). The ground state absorption spectra of WT and ΔA’α had a similar ratio of the height at 450 nm to 280 nm indicating that they bound a similar amount of FMN. ΔA’α exhibited a characteristic absorption spectral change with a reversible formation of S390 (Fig 2B inset). Its half decay time was calculated as 89 s at 20°C (Table 1). The decay time was almost the same as that of WT in the buffer containing 500 mM NaCl (91 s at 20°C), and the absorption peaks did not shift, indicating that the presence of the A’α-helix does not affect LOV photochemistry. Taken together, these results indicate that the A’α-helix is essential for the intramolecular signaling from LOV2 to the kinase, while it is not involved in the photochemical properties of LOV2 in the LOV2-STK of At phot1.

Bottom Line: Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation.These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK.The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Osaka Prefecture University, Sakai, Osaka, Japan.

ABSTRACT
Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aβ strand of LOV2 (A'α/Aβ gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aβ gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

No MeSH data available.