Abstract
Phytosulfokine (PSK), 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. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand-binding pocket. Here, we identified the PSK-binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK derivatives with DcPSKR1, and mapped the cross-linked region by fragmentation. We also established a novel “on-column photoaffinity labeling” methodology that allows to increase the cross-linking efficiency. We purified a labeled DcPSKR1 tryptic fragment and identified a peptide fragment that corresponds to the 15-amino-acid Glu503-Lys517 region of DcPSKR1 by MALDI-TOF MS. Deletion of Glu503-Lys517 completely abolishes the ligand-binding activity of DcPSKR1. This region is in the island domain flanked by LRRs, indicating that this domain forms a ligand-binding pocket.
