title: Tyrosine replacement of PSGL-1 reduces association kinetics with P- and L-selectin on the cell membrane.
abstract: Binding of selectins to P-selectin glycoprotein ligand-1 (PSGL-1) mediates tethering and rolling of leukocytes on the endothelium during inflammation. Previous measurements obtained with a flow-chamber assay have shown that mutations of three tyrosines at the PSGL-1 N-terminus (Y46, Y48, and Y51) increase the reverse rates and their sensitivity to the force of bonds with P- and L-selectin. However, the effects of these mutations on the binding affinities and forward rates have not been studied. We quantified these effects by using an adhesion frequency assay to measure two-dimensional affinity and kinetic rates at zero force. Wild-type PSGL-1 has 2.2- to 8.5-fold higher binding affinities for P- and L-selectin than PSGL-1 mutants with two of three tyrosines substituted by phenylalanines, and 9.6- to 49-fold higher affinities than the PSGL-1 mutant with all three tyrosines replaced. In descending order, the affinity decreased from wild-type to Y48/51F, Y46/51F, Y46/48F, and Y46/48/51F. The affinity differences were attributed to major changes in the forward rate and minor changes in the reverse rate, suggesting that these tyrosines regulate the accessibility of PSGL-1 to P- and L-selectin via electrostatic interactions, which is supported by molecular-dynamics simulations. Our results provide insights into the structure-function relationship of receptor-ligand binding at a single-residue level.
authors.affiliation: Key Laboratory of Microgravity, Institute of Mechanics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Botao
authors.initials: B
authors.lastname: Xiao
authors.forename: Chunfang
authors.initials: C
authors.lastname: Tong
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Jia
authors.forename: Rui
authors.initials: R
authors.lastname: Guo
authors.forename: Shouqin
authors.initials: S
authors.lastname: Lü
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Rodger P
authors.initials: RP
authors.lastname: McEver
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhu
authors.forename: Mian
authors.initials: M
authors.lastname: Long
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D019041:L-Selectin / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D019007:P-Selectin / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D014443:Tyrosine
chemical_list: D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D019041:L-Selectin; D014443:Tyrosine