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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.


Peptide mapping of At phot1 LOV2-STK WT (A) and (B), and C512A (C), by SDS-PAGE after Trypsin digestion in the dark (D) or under BL irradiation (BL).nt indicates the sample without the trypsin-treatment. The gels were stained with CBB. (A) The mapping after BL irradiation with the different fluence rates for 15 min. (B) and (C) show the time courses of the digestion. The four arrowheads indicate the bands of major proteolytic products. (D) Schematic diagram for the positions of the cleavage sites for the four major bands. The black and white bars indicate the proteolytic products obtained in the dark or under BL irradiation, respectively.
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pone.0124284.g005: Peptide mapping of At phot1 LOV2-STK WT (A) and (B), and C512A (C), by SDS-PAGE after Trypsin digestion in the dark (D) or under BL irradiation (BL).nt indicates the sample without the trypsin-treatment. The gels were stained with CBB. (A) The mapping after BL irradiation with the different fluence rates for 15 min. (B) and (C) show the time courses of the digestion. The four arrowheads indicate the bands of major proteolytic products. (D) Schematic diagram for the positions of the cleavage sites for the four major bands. The black and white bars indicate the proteolytic products obtained in the dark or under BL irradiation, respectively.

Mentions: To see the BL-induced structural changes in the At phot1 LOV2-STK, peptide mapping was performed by limited proteolysis with trypsin. Trypsin digested the WT into two major polypeptides band-1 and band-2 in the dark (Fig 5A). Considering Mass spectrometry and the amino acid sequence of LOV2-STK, we assigned the band-1 and 2 as 463–631 and 835–996, respectively. Band-1 consists of half of the A’α, A’α/Aβ gap, LOV2 and half of the linker between LOV2 and STK including an entire Jα-helix. Band-2 includes the C-terminal half of STK and the C-terminal end part (Fig 5A and 5D, S2 Fig and S2 Table). The two digestion sites, Lys631 in the linker region and Lys835 in the activation loop of STK, do not form a tight structure to protect against trypsin attack in the dark. Under BL, band-1 was degraded further into band-3, 463–603 and band-4, 475–603 (Fig 5A, 5B and 5D). Trypsin digested the substrate in proportion to the BL fluence and the digestion time. To assure the involvement of the photoreaction of LOV2 in these degradations, peptide mapping was performed with a C512A substitute lacking the S390 formation. C512A exhibited a similar SDS-PAGE band pattern to that of WT in the dark, indicating that the Cys512 to Ala substitution does not alter the surface structure of WT in terms of trypsin accessibility (Fig 5). In contrast to WT, C512A did not show marked degradations of band-1 into band-3 and band-4 under BL. Thus, it can be concluded that the formation of S390 causes the conformational changes detectable by trypsin digestion. Because Lys603 is located in the middle of the Jα-helix and Lys475 in the A’α/Aβ gap, these results clearly revealed BL-induced structural changes in the gap as well as in the Jα-helix.


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)

Peptide mapping of At phot1 LOV2-STK WT (A) and (B), and C512A (C), by SDS-PAGE after Trypsin digestion in the dark (D) or under BL irradiation (BL).nt indicates the sample without the trypsin-treatment. The gels were stained with CBB. (A) The mapping after BL irradiation with the different fluence rates for 15 min. (B) and (C) show the time courses of the digestion. The four arrowheads indicate the bands of major proteolytic products. (D) Schematic diagram for the positions of the cleavage sites for the four major bands. The black and white bars indicate the proteolytic products obtained in the dark or under BL irradiation, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124284.g005: Peptide mapping of At phot1 LOV2-STK WT (A) and (B), and C512A (C), by SDS-PAGE after Trypsin digestion in the dark (D) or under BL irradiation (BL).nt indicates the sample without the trypsin-treatment. The gels were stained with CBB. (A) The mapping after BL irradiation with the different fluence rates for 15 min. (B) and (C) show the time courses of the digestion. The four arrowheads indicate the bands of major proteolytic products. (D) Schematic diagram for the positions of the cleavage sites for the four major bands. The black and white bars indicate the proteolytic products obtained in the dark or under BL irradiation, respectively.
Mentions: To see the BL-induced structural changes in the At phot1 LOV2-STK, peptide mapping was performed by limited proteolysis with trypsin. Trypsin digested the WT into two major polypeptides band-1 and band-2 in the dark (Fig 5A). Considering Mass spectrometry and the amino acid sequence of LOV2-STK, we assigned the band-1 and 2 as 463–631 and 835–996, respectively. Band-1 consists of half of the A’α, A’α/Aβ gap, LOV2 and half of the linker between LOV2 and STK including an entire Jα-helix. Band-2 includes the C-terminal half of STK and the C-terminal end part (Fig 5A and 5D, S2 Fig and S2 Table). The two digestion sites, Lys631 in the linker region and Lys835 in the activation loop of STK, do not form a tight structure to protect against trypsin attack in the dark. Under BL, band-1 was degraded further into band-3, 463–603 and band-4, 475–603 (Fig 5A, 5B and 5D). Trypsin digested the substrate in proportion to the BL fluence and the digestion time. To assure the involvement of the photoreaction of LOV2 in these degradations, peptide mapping was performed with a C512A substitute lacking the S390 formation. C512A exhibited a similar SDS-PAGE band pattern to that of WT in the dark, indicating that the Cys512 to Ala substitution does not alter the surface structure of WT in terms of trypsin accessibility (Fig 5). In contrast to WT, C512A did not show marked degradations of band-1 into band-3 and band-4 under BL. Thus, it can be concluded that the formation of S390 causes the conformational changes detectable by trypsin digestion. Because Lys603 is located in the middle of the Jα-helix and Lys475 in the A’α/Aβ gap, these results clearly revealed BL-induced structural changes in the gap as well as in the Jα-helix.

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.