Identification of ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling.
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.
Shinohara
Hidefumi
H
Graduate School of Bio-agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Ogawa
Mari
M
Sakagami
Youji
Y
Matsubayashi
Yoshikatsu
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018552:Daucus carota; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008027:Light; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana; D014357:Trypsin / Q000737:chemistry
D003432:Cross-Linking Reagents; D008024:Ligands; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C409473:phytosulfokine-alpha receptor, plant; D014357:Trypsin; D007930:Leucine