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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) 3D structure of At phot1 LOV2-Jα (pdb ID: 4HHD). Key side chains of amino acid residues for intramolecular signaling are indicated with a stick model. FMN is indicated with a space-filling model. A’α-helix and Jα-helix are colored pink and cyan, respectively. The trypsin-digested site at Lys603 is colored orange. (B) Schematic illustration for the hypothetic intramolecular interactions involved in the BL signaling from FMN to STK of At phot1. Blue arrows indicate the intramolecular signaling. For details, see the “Discussion”.
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pone.0124284.g008: (A) 3D structure of At phot1 LOV2-Jα (pdb ID: 4HHD). Key side chains of amino acid residues for intramolecular signaling are indicated with a stick model. FMN is indicated with a space-filling model. A’α-helix and Jα-helix are colored pink and cyan, respectively. The trypsin-digested site at Lys603 is colored orange. (B) Schematic illustration for the hypothetic intramolecular interactions involved in the BL signaling from FMN to STK of At phot1. Blue arrows indicate the intramolecular signaling. For details, see the “Discussion”.

Mentions: After digestion in the Jα-helix under BL irradiation, trypsin digested at Lys475 in the A’α/Aβ gap that connects the A’α-helix and LOV2 and is located 9 Å apart from Lys603 [38] (Fig 8A). In the As phot1 LOV2-Jα, chymotrypsin and trypsin digested at Leu408 and Arg410 corresponding to Leu470 and Arg472 in At phot1 in the dark were also enhanced under light irradiation suggesting conformational changes in the C-terminal region of the A’α-helix (Fig 1). The lengths of the A’α-helix differ between the At and the As polypeptides; however, these results suggest that the protein structure is changed by BL in the region from the C-terminal region of the A’α-helix to the A’α/Aβ gap.


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) 3D structure of At phot1 LOV2-Jα (pdb ID: 4HHD). Key side chains of amino acid residues for intramolecular signaling are indicated with a stick model. FMN is indicated with a space-filling model. A’α-helix and Jα-helix are colored pink and cyan, respectively. The trypsin-digested site at Lys603 is colored orange. (B) Schematic illustration for the hypothetic intramolecular interactions involved in the BL signaling from FMN to STK of At phot1. Blue arrows indicate the intramolecular signaling. For details, see the “Discussion”.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124284.g008: (A) 3D structure of At phot1 LOV2-Jα (pdb ID: 4HHD). Key side chains of amino acid residues for intramolecular signaling are indicated with a stick model. FMN is indicated with a space-filling model. A’α-helix and Jα-helix are colored pink and cyan, respectively. The trypsin-digested site at Lys603 is colored orange. (B) Schematic illustration for the hypothetic intramolecular interactions involved in the BL signaling from FMN to STK of At phot1. Blue arrows indicate the intramolecular signaling. For details, see the “Discussion”.
Mentions: After digestion in the Jα-helix under BL irradiation, trypsin digested at Lys475 in the A’α/Aβ gap that connects the A’α-helix and LOV2 and is located 9 Å apart from Lys603 [38] (Fig 8A). In the As phot1 LOV2-Jα, chymotrypsin and trypsin digested at Leu408 and Arg410 corresponding to Leu470 and Arg472 in At phot1 in the dark were also enhanced under light irradiation suggesting conformational changes in the C-terminal region of the A’α-helix (Fig 1). The lengths of the A’α-helix differ between the At and the As polypeptides; however, these results suggest that the protein structure is changed by BL in the region from the C-terminal region of the A’α-helix to the A’α/Aβ gap.

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.