title: 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 αI domain); the other at the βI domain. Mutagenesis of the α'1 helix showed that isoflurane binding sites at the βI 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 αI and βI domains allosteric to the ICAM-1 binding site, and that isoflurane binding stabilizes LFA-1 in the closed conformation.
authors: Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115, USA. koichi.yuki@childrens.harvard.edu
authors: Koichi
authors: K
authors: Yuki
authors: Weiming
authors: W
authors: Bu
authors: Jin
authors: J
authors: Xi
authors: Mehmet
authors: M
authors: Sen
authors: Motomu
authors: M
authors: Shimaoka
authors: Roderic G
authors: RG
authors: Eckenhoff
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D007530:Isoflurane / Q000737:chemistry / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; D016169:Lymphocyte Function-Associated Antigen-1; D007530:Isoflurane