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.-Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., Eckenhof, R.G. Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1.
- SourceAvailable from: harvard.eduEuropean Journal of Immunology 05/1979; 9(4):301-6. · 4.97 Impact Factor
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ABSTRACT: We used integrin alphaLbeta2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the alphaL I domain and beta2 I-like domain inhibit adhesion of wild-type alphaLbeta2 to intercellular adhesion molecule-1. However, with alphaLbeta2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, alphaLbeta2 containing a locked open I domain was completely resistant to inhibition by mAbs to the beta2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type alphaLbeta2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn(2+), as measured with mAb m24, which we map here to the beta2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn(2+)-induced exposure of the KIM127 epitope in the beta2 stalk region. Furthermore, locked open I domains, in alphaLbeta2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg(2+) and Mn(2+). These results suggest that Mn(2+) activates alphaLbeta2 by binding to a site other than the I domain, most likely the I-like domain of beta2.Proceedings of the National Academy of Sciences 01/2001; 98(5):2393-2398. · 9.74 Impact Factor
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ABSTRACT: The function of some multidomain proteins is regulated by interdomain communication. We use second-site suppressor cysteine mutations to test a hypothesis on how the inserted (I)-like domain in the integrin beta-subunit regulates ligand binding by the neighboring I domain in the integrin alpha-subunit [Huth, J. R., Olejniczak, E. T., Mendoza, R., Liang, H., Harris, E. A., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5231-5236; and Alonso, J. L., Essafi, M., Xiong, J. P., Stehle, T. & Arnaout, M. A. (2002) Curr. Biol. 12, R340-R342]. The hypothesis is that an interaction between the beta I-like metal ion-dependent adhesion site (MIDAS) and an intrinsic ligand in the linker following the alpha I domain, Glu-310, exerts a pull that activates the alpha I domain. Individual mutation of alpha(L) linker residue Glu-310 or beta(2) MIDAS residues Ala-210 or Tyr-115 to cysteine abolishes I domain activation, whereas the double mutation of alpha(L)-E310C with either beta(2)-A210C or beta(2)-Y115C forms a disulfide bond that constitutively activates ligand binding. The disulfide-bonded mutant is resistant to small molecule antagonists that bind to the beta I-like domain near its interface with the alpha I domain and inhibit communication between these domains but remains susceptible to small molecule antagonists that bind underneath the I domain alpha 7-helix and certain allosteric antagonistic antibodies. Thus, the alpha 7-helix and its linker are better modeled as a pull spring than a bell rope. The results suggest that alpha(L) residue Glu-310, which is universally conserved in all I domain-containing integrins, functions as an intrinsic ligand for the beta I-like domain, and that when integrins are activated, the beta I-like MIDAS binds to Glu-310, pulls the spring, and thereby activates the alpha I domain.Proceedings of the National Academy of Sciences 04/2004; 101(9):2906-11. · 9.74 Impact Factor