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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.


Schematic drawing of At phot1 (upper panel) and the secondary structure around A’α-helix region.Alignment of the amino acid sequences of phot in the A’α-helix regions using ClustalX. Arabidopsis thaliana, At; Avena sativa, As; Oryza sativa, Os; Adiantum capillus-veneris, Ac; Marchantia, polymorpha, Mp; Chlamydomonas reinhardtii, Cr. Filled and open arrow heads indicate the N-ends of LOV2-STK and ΔA’α constructs, respectively. Amino acid residues substituted in this study are highlighted.
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pone.0124284.g001: Schematic drawing of At phot1 (upper panel) and the secondary structure around A’α-helix region.Alignment of the amino acid sequences of phot in the A’α-helix regions using ClustalX. Arabidopsis thaliana, At; Avena sativa, As; Oryza sativa, Os; Adiantum capillus-veneris, Ac; Marchantia, polymorpha, Mp; Chlamydomonas reinhardtii, Cr. Filled and open arrow heads indicate the N-ends of LOV2-STK and ΔA’α constructs, respectively. Amino acid residues substituted in this study are highlighted.

Mentions: In addition to the Jα-helix, recent studies have identified the involvement of another α-helix named A’ in intramolecular signaling. A’α-helix is located upstream of the N-terminus of LOV2. In green algae Chlamydomonas reinhardtii (Cr) phot, amino acid mutations in this helix affected the regulation of STK activity [32]. The amino acid sequences for the A’α-helix are conserved among higher plant phots (Fig 1) suggesting that the helix may function in the intramolecular signal transduction from LOV2 to STK. In fact, an amino acid mutation in the A’α-helix region impaired in vivo phot1 signaling in the tomato [33]. The As phot1 LOV2-Jα polypeptide used in the previous crystal structure determination contained 7 amino acid residues in the A’α-helix region that forms a short 4 amino acid helix [28]. Based on this structure, molecular dynamics (MD) calculations proposed that the A’α-helix plays a role in intramolecular light signaling with the Jα-helix [34,35]. Recently, a crystal structure was determined with At phot1 LOV2-Jα with a larger number, 21, of amino acid residues in the A’α-helix region [36]. In contrast to the previous monomeric As phot1 LOV2-Jα with the short A’α-helix, At phot1 LOV2-Jα forms a dimer and each subunit has a longer A’α-helix. The N-terminal extension serves as the dimer interface by configuring a short α-helical coiled coil with a scissor-like shape. The Jα-helix attaches on the surface of the β-sheet of the LOV2 in a similar fashion as the previous As phot1 LOV2-Jα. Both helices in a subunit orient in a similar direction and interact with each other at their edges. All of the LOV2-containing polypeptides of At phot1 used so far include the longer A’α-helix. A TG study on the At phot1 LOV2-Jα polypeptide showed that the conformational change in the Jα-helix has a faster reaction rate than that of the A’α-helix [37]. The signaling process from FMN to A’α-helix and its communication with the signaling to the Jα-helix is obscure. Furthermore, the involvement of these processes in the signaling from LOV2 to STK is unknown. Thus, the function of the A’α-helix in these intramolecular BL signaling processes is to be examined in detail.


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)

Schematic drawing of At phot1 (upper panel) and the secondary structure around A’α-helix region.Alignment of the amino acid sequences of phot in the A’α-helix regions using ClustalX. Arabidopsis thaliana, At; Avena sativa, As; Oryza sativa, Os; Adiantum capillus-veneris, Ac; Marchantia, polymorpha, Mp; Chlamydomonas reinhardtii, Cr. Filled and open arrow heads indicate the N-ends of LOV2-STK and ΔA’α constructs, respectively. Amino acid residues substituted in this study are highlighted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401697&req=5

pone.0124284.g001: Schematic drawing of At phot1 (upper panel) and the secondary structure around A’α-helix region.Alignment of the amino acid sequences of phot in the A’α-helix regions using ClustalX. Arabidopsis thaliana, At; Avena sativa, As; Oryza sativa, Os; Adiantum capillus-veneris, Ac; Marchantia, polymorpha, Mp; Chlamydomonas reinhardtii, Cr. Filled and open arrow heads indicate the N-ends of LOV2-STK and ΔA’α constructs, respectively. Amino acid residues substituted in this study are highlighted.
Mentions: In addition to the Jα-helix, recent studies have identified the involvement of another α-helix named A’ in intramolecular signaling. A’α-helix is located upstream of the N-terminus of LOV2. In green algae Chlamydomonas reinhardtii (Cr) phot, amino acid mutations in this helix affected the regulation of STK activity [32]. The amino acid sequences for the A’α-helix are conserved among higher plant phots (Fig 1) suggesting that the helix may function in the intramolecular signal transduction from LOV2 to STK. In fact, an amino acid mutation in the A’α-helix region impaired in vivo phot1 signaling in the tomato [33]. The As phot1 LOV2-Jα polypeptide used in the previous crystal structure determination contained 7 amino acid residues in the A’α-helix region that forms a short 4 amino acid helix [28]. Based on this structure, molecular dynamics (MD) calculations proposed that the A’α-helix plays a role in intramolecular light signaling with the Jα-helix [34,35]. Recently, a crystal structure was determined with At phot1 LOV2-Jα with a larger number, 21, of amino acid residues in the A’α-helix region [36]. In contrast to the previous monomeric As phot1 LOV2-Jα with the short A’α-helix, At phot1 LOV2-Jα forms a dimer and each subunit has a longer A’α-helix. The N-terminal extension serves as the dimer interface by configuring a short α-helical coiled coil with a scissor-like shape. The Jα-helix attaches on the surface of the β-sheet of the LOV2 in a similar fashion as the previous As phot1 LOV2-Jα. Both helices in a subunit orient in a similar direction and interact with each other at their edges. All of the LOV2-containing polypeptides of At phot1 used so far include the longer A’α-helix. A TG study on the At phot1 LOV2-Jα polypeptide showed that the conformational change in the Jα-helix has a faster reaction rate than that of the A’α-helix [37]. The signaling process from FMN to A’α-helix and its communication with the signaling to the Jα-helix is obscure. Furthermore, the involvement of these processes in the signaling from LOV2 to STK is unknown. Thus, the function of the A’α-helix in these intramolecular BL signaling processes is to be examined in detail.

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.