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Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1.

ABSTRACT: We previously demonstrated that isoflurane targets lymphocyte function-associated antigen-1 (LFA-1), a critical adhesion molecule for leukocyte arrest. However, it remains to be determined how isoflurane interacts with the full ectodomain LFA-1 and modulates its conformation and function. Isoflurane binding sites on the full ectodomain LFA-1 were probed by photolabeling using photoactivatable isoflurane (azi-isoflurane). The adducted residues were determined by liquid chromatography/mass spectrometry analysis. Separately, docking simulations were performed to predict binding sites. Point mutations were introduced around isoflurane binding sites. The significance of isoflurane's effect was assessed in both intracellular adhesion molecule-1 (ICAM-1) binding assays and epitope mapping of activation-sensitive antibodies using flow cytometry. Two isoflurane binding sites were identified using photolabeling and were further validated by the docking simulation: one at the hydrophobic pocket in the ICAM-1 binding domain (the ?I domain); the other at the ?I domain. Mutagenesis of the ?'1 helix showed that isoflurane binding sites at the ?I domain were significantly important in modulating LFA-1 function and conformation. Epitope mapping using activation-sensitive antibodies suggested that isoflurane stabilized LFA-1 in the closed conformation. This study suggested that isoflurane binds to both the ?I and ?I domains allosteric to the ICAM-1 binding site, and that isoflurane binding stabilizes LFA-1 in the closed conformation.

SUBMITTER: Yuki K

PROVIDER: S-EPMC3475260 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1.

Yuki Koichi K Bu Weiming W Xi Jin J Sen Mehmet M Shimaoka Motomu M Eckenhoff Roderic G RG

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20120719 11

We previously demonstrated that isoflurane targets lymphocyte function-associated antigen-1 (LFA-1), a critical adhesion molecule for leukocyte arrest. However, it remains to be determined how isoflurane interacts with the full ectodomain LFA-1 and modulates its conformation and function. Isoflurane binding sites on the full ectodomain LFA-1 were probed by photolabeling using photoactivatable isoflurane (azi-isoflurane). The adducted residues were determined by liquid chromatography/mass spectro ...[more]

PMID: 22815384

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Project description:We previously demonstrated that propofol interacted with the leukocyte adhesion molecule leukocyte function-associated antigen-1 (LFA-1) and inhibited the production of interleukin-2 via LFA-1 in a dependent manner. However, the binding site(s) of propofol on LFA-1 remains unknown.First, the inhibition of LFA-1's ligand binding by propofol was confirmed in an enzyme-linked immunosorbent assay (ELISA) ELISA-type assay. The binding site of propofol on LFA-1 was probed with a photolabeling experiment using a photoactivatable propofol analog called azi-propofol-m. The adducted residues of LFA-1 by this compound were determined using liquid chromatography-mass spectrometry. In addition, the binding of propofol to the ligand-binding domain of LFA-1 was examined using 1-aminoanthracene (1-AMA) displacement assay. Furthermore, the binding site(s) of 1-AMA and propofol on LFA-1 was studied using the docking program GLIDE.We demonstrated that propofol impaired the binding of LFA-1 to its ligand intercellular adhesion molecule-1. The photolabeling experiment demonstrated that the adducted residues were localized in the allosteric cavity of the ligand-binding domain of LFA-1 called "lovastatin site." The shift of fluorescence spectra was observed when 1-AMA was coincubated with the low-affinity conformer of LFA-1 ligand-binding domain (wild-type [WT] ?L I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT ?L I domain. In the 1-AMA displacement assay, propofol decreased 1-AMA fluorescence signal (at 520 nm), suggesting that propofol competed with 1-AMA and bound to the WT ?L I domain. The docking simulation demonstrated that both 1-AMA and propofol bound to the lovastatin site, which agreed with the photolabeling experiment.We demonstrated that propofol bound to the lovastatin site in LFA-1. Previously we showed that the volatile anesthetics isoflurane and sevoflurane bound to this site. Taken together, the lovastatin site is an example of the common binding sites for anesthetics currently used clinically.

Dataset Information

Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1.

Publications

Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1.

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