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.
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Authors | Botao Xiao, Chunfang Tong, Xiaoling Jia, Rui Guo, Shouqin Lü, Yan Zhang, Rodger P McEver, Cheng Zhu, Mian Long |
Journal | Biophysical journal
(Biophys J)
Vol. 103
Issue 4
Pg. 777-85
(Aug 22 2012)
ISSN: 1542-0086 [Electronic] United States |
PMID | 22947939
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Copyright | Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved. |
Chemical References |
- Membrane Glycoproteins
- P-Selectin
- P-selectin ligand protein
- L-Selectin
- Tyrosine
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Topics |
- Amino Acid Substitution
- Animals
- CHO Cells
- Cell Membrane
(metabolism)
- Cricetinae
- Cricetulus
- Humans
- Kinetics
- L-Selectin
(metabolism)
- Membrane Glycoproteins
(chemistry, genetics, metabolism)
- Molecular Dynamics Simulation
- P-Selectin
(metabolism)
- Protein Binding
- Protein Structure, Tertiary
- Static Electricity
- Tyrosine
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