Phenylalanine 35 and tyrosine 82 are involved in the uptake and release of ligand by porcine odorant-binding protein.
Structural and molecular dynamics studies have pointed out the role of aromatic residues in the uptake of ligand by porcine odorant-binding protein (pOBP). The shift of Tyr82 from its position during the opening of the binding cavity has been shown, and was supposed to participate in the entrance of the ligand. Several Phe residues in the vicinity of Tyr82 could also participate in the binding process. To clarify their involvement, we performed molecular dynamics studies to simulate the dissociation of undecanal, a ligand previously co-crystallized with pOBP. The results confirmed the key-role of Tyr82 and pointed out the participation of Phe35 in controlling the reorientation of undecanal towards the exit. To bring experimental support to both published (binding) and present simulations (dissociation), we have mutated these two residues and over expressed the wild type pOBP, the two single mutants and the double mutant in the yeast Pichia pastoris. As fluorescence spectroscopy implies the uptake of the fluorescent probe and release in displacement experiments, we monitored the binding ability of the four proteins for 1-aminoanthracene (1-AMA). The experimental results indicated that both residues are involved in the uptake of ligand as the three mutated proteins were unable to bind 1-AMA, contrary to the wild type recombinant pOBP that bound 1-AMA with the expected affinity.
Nagnan-Le Meillour
Patricia
P
INRA, UMR 8576 CNRS/USTL, Unité de Glycobiologie Structurale et Fonctionnelle, Bâtiment C9, Villeneuve d'Ascq, France. patricia.le-meillour@univ-lille1.fr
Lagant
Philippe
P
Cornard
Jean-Paul
JP
Brimau
Fanny
F
Le Danvic
Chrystelle
C
Vergoten
Gérard
G
Michalski
Jean-Claude
JC
D000595:Amino Acid Sequence; D000818:Animals; D000873:Anthracenes / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D010449:Peptide Mapping; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D018035:Receptors, Odorant / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013552:Swine; D014443:Tyrosine / Q000737:chemistry*
D000873:Anthracenes; D005456:Fluorescent Dyes; D008024:Ligands; D018035:Receptors, Odorant; C045648:odorant-binding protein; D014443:Tyrosine; D010649:Phenylalanine; C049062:1-aminoanthracene