Abstract
Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Here, we have identified the PSK binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog [(125)I]-[N(epsilon)-(4-azidosalicyl)Lys(5)]PSK with DcPSKR1 using UV irradiation and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel "on-column photoaffinity labeling" methodology that allows repeated incorporation of the photoaffinity label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies and identified a peptide fragment that corresponds to the 15-amino-acid Glu(503)-Lys(517) region of DcPSKR1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Deletion of Glu(503)-Lys(517) completely abolishes the ligand binding activity of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a ligand binding pocket that directly interacts with PSK.
Highlights
Grant 14COEA02) and by Grants-in-aid for Scientific Research for Priority Areas (14036214) and Young Scientists (18687003)
Overexpression of domain of carrot PSKR1 (DcPSKR1) in Tobacco BY-2 Cells—To obtain the relatively large amount of DcPSKR1 protein required for the direct identification of the PSK binding domain by photoaffinity labeling, we first overexpressed DcPSKR1 in BY-2 suspension cells, which grow rapidly and can be harvested weekly
Using positive mode lized DcPSKR1-⌬KD-His6 on nickel-chelating HiTrapTM HP- MALDI-TOF MS analysis, we identified two specific molecular Sepharose beads using a His6 tag and performed solid-phase ion peaks at m/z 1752.83 and 1880.87 in the eluate of the immuphotoaffinity labeling in a transparent narrow column by noaffinity column; these peaks were not detected in the control directly irradiating the column with UV light (365 nm)
Summary
Vector Construction and Transformation of Tobacco BY-2 Cells—For construction of plasmids pBI121-DcPSKR1-His and pBI121-DcPSKR1-⌬KD-His, PCR was performed using primers PSKR-5f (5Ј-GCtctagaATTTGCCTTGTTTTGTTGAGC-3Ј) and PSKR-3r (5Ј-CTAGTGGTGGTGGTGGTGGTGACTACTGACATCAATGTTTTCGAGCC-3Ј) for DcPSKR1His, using primers PSKR-5f and PSKR-3t (5Ј-CTAGTGGTGGTGGTGGTGGTGACTGCTAGTGGATTTCAAAATGTC3Ј) for DcPSKR1-⌬KD-His (His coding regions are underlined; restriction sites are in lowercase), and using DcPSKR1 cDNA as a template. 10 l of labeled DcPSKR1-⌬KD-His was treated with 100 pmol of TPCK-treated trypsin (Sigma) in buffer containing 20 mM Tris-HCl (pH 7.6), 10 mM CaCl2 and 10% CH3CN (v/v) at 37 °C for 16 h at a total volume of 20 l. For Asp-N digestion, 10 l of labeled DcPSKR1-⌬KD-His was treated with 100 pmol of endoproteinase Asp-N (TakaraBio, Shiga, Japan) in 50 mM sodium phosphate buffer (pH 8.0) at 37 °C for 16 h at a total volume of 20 l All of these samples were analyzed by SDSPAGE using the NuPage 12% BisTris gel and autoradiography. Column equilibration and chromatography were performed using buffer containing 20 mM HEPES-KOH (pH 7.0) and 150 mM NaCl. On-column tryptic digests of [125I]ASAPSK-labeled DcPSKR1-⌬KD-His (200,000 counts/min eq.) were applied to the column, and 1.0-ml fractions were analyzed for radioactivity using an autowell ␥ system (ALOKA). Blue dextran (Amersham Biosciences) and cyanocobalamin were used for molecular mass calibration
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