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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 E474A, (A), and K475A, (B) by SDS-PAGE after trypsin digestion in the dark (D) or under BL irradiation (L).Time courses of the digestion are indicated. nt indicates the sample without the trypsin-treatment. The four arrowheads indicate the bands of major proteolytic products.
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pone.0124284.g006: Peptide mapping of At phot1 LOV2-STK E474A, (A), and K475A, (B) by SDS-PAGE after trypsin digestion in the dark (D) or under BL irradiation (L).Time courses of the digestion are indicated. nt indicates the sample without the trypsin-treatment. The four arrowheads indicate the bands of major proteolytic products.

Mentions: To understand the involvement of these structural changes in the kinase activation by BL, peptide mapping was performed with E474A and K475A that impaired light activation of kinase activity. Both substitutes produced bands 1 and 2, which is similar to WT in the dark. This indicates that these amino acid substitutions did not alter the surface structure of WT. BL induced the degradation of band-1 into band-3 and 4 in the E474A, which is similar to WT (Fig 6A). In contrast, K475A exhibited the production of band-3 but not band-4 (Fig 6B). Because K475A lost the trypsin digestion site at Lys475, it is reasonable that band-4 could not be observed. These results demonstrated that BL induces similar structural changes in these substitutes to those of the WT in the A’α/Aβ gap as well as in the Jα-helix; however, it cannot transfer the signal to the later processes responsible for kinase activation by BL.


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 E474A, (A), and K475A, (B) by SDS-PAGE after trypsin digestion in the dark (D) or under BL irradiation (L).Time courses of the digestion are indicated. nt indicates the sample without the trypsin-treatment. The four arrowheads indicate the bands of major proteolytic products.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124284.g006: Peptide mapping of At phot1 LOV2-STK E474A, (A), and K475A, (B) by SDS-PAGE after trypsin digestion in the dark (D) or under BL irradiation (L).Time courses of the digestion are indicated. nt indicates the sample without the trypsin-treatment. The four arrowheads indicate the bands of major proteolytic products.
Mentions: To understand the involvement of these structural changes in the kinase activation by BL, peptide mapping was performed with E474A and K475A that impaired light activation of kinase activity. Both substitutes produced bands 1 and 2, which is similar to WT in the dark. This indicates that these amino acid substitutions did not alter the surface structure of WT. BL induced the degradation of band-1 into band-3 and 4 in the E474A, which is similar to WT (Fig 6A). In contrast, K475A exhibited the production of band-3 but not band-4 (Fig 6B). Because K475A lost the trypsin digestion site at Lys475, it is reasonable that band-4 could not be observed. These results demonstrated that BL induces similar structural changes in these substitutes to those of the WT in the A’α/Aβ gap as well as in the Jα-helix; however, it cannot transfer the signal to the later processes responsible for kinase activation by BL.

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.