Propofol shares the binding site with isoflurane and sevoflurane on leukocyte function-associated antigen-1

Abstract

Background: 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.

Methods: 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.

Results: 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] αL I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT αL 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 αL I domain. The docking simulation demonstrated that both 1-AMA and propofol bound to the lovastatin site, which agreed with the photolabeling experiment.

Conclusions: 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.

Figure 1

Structure of leukocyte integrin. A, Global structure of leukocyte integrin. The structure shown is from protein data bank (PDB) 3K6S of other leukocyte integrin αXβ2. Integrin molecule leukocyte function–associated antigen-1 (LFA-1) (αLβ2) and αXβ2 share the β subunit in common and their α subunits (αL and αX) are highly homologous. Both bind to the intercellular adhesion molecule-1 (ICAM-1). The α subunit is shown in blue, and the β subunit is in green. The ligand-binding domain called the α I domain is shown in the red circle. The metal ion–dependent adhesion site (MIDAS) is shown in the gold sphere. ICAM-1 binds to the MIDAS. B, The surface of the α I domain of the LFA-1 is shown (PDB, 1ZOO). The allosteric pocket underneath the α7 helix called the “lovastatin site ” is shown in an arrow. The MIDAS is not seen in this figure.

Figure 2

Propofol inhibits intercellular adhesion molecule-1 (ICAM-1) binding to molecule leukocyte function–associated antigen-1 (LFA-1). A, ELISA-type ICAM-1–LFA-1 binding assay was performed in the presence of propofol or propofol analog (2,6-di-tert-butylphenol [DTBP]) at various concentrations. Both propofol and DTBP inhibited ICAM-1–LFA-1 binding. The data represent mean ± SD of triplicates. Each data point represents a unique replication. Statistical analysis was performed using 1-way analysis of variance with Tukey post hoc pairwise comparisons. *P < 0.01 versus mock-treated sample (no propofol, no DTBP). B, The competition assay of competitive LFA-1 antagonist TS1/22 antibody with propofol at various concentrations was performed. Soluble LFA-1 was used for coating at the concentration of 10 μg/mL, and TS1/22 was 5 μg/mL. The data represent mean ± SD of triplicates. Each data point represents a unique replication. Statistical analysis was performed using 1-way analysis of variance with Tukey post hoc pairwise comparisons. Although TS1/22 demonstrates a trend of some competition with propofol at 100 μM, statistically no difference was observed with narrow 99% 2-sided confidence intervals.

Figure 3

Propofol binding site(s) on molecule leukocyte function–associated antigen-1 suggested by photolabeling experiment and docking calculation. Azi-propofol-m (azi-Pm) photolabeling experiments were performed twice as shown in Table 1. The sequence coverage was significantly better in experiment 1, and we used experiment 1 as a representative. Notably, the adducted residues by azi-Pm were located at the allosteric pocket underneath the C-terminal α 7 helix of the αL I domain. A and B, The docked propofol on the α I domain (protein data bank 1ZOO) is shown. Residues photolabeled by azi-Pm are shown in blue. The blowup of the docking site is shown in panel B. The metal ion–dependent adhesion site (MIDAS) is shown in the gold sphere. In propofol: red, oxygen; green, carbon. C, Amino acid residues of the α I domain. Sequenced residues by mass spectrometry in experiment 1 are underlined. Adducted residues by azi-Pm are shown in asterisk. Residues with 4 Å from docked propofol are in red.

Figure 4

1-Aminoanthracene (1-AMA) directly interacts with the wild-type α I domain. A, Fluorescence assay measuring the binding of 1-AMA (10 μM) to the wild-type (WT) α I domain and high-affinity mutant (HA; 200 nM). Samples were excited at 380 nm. The representative fluorescence spectra of 2 experiments are shown in the figure. B, The docked 1-AMA on the α I domain (protein data bank [PDB] 1ZOO) is shown. The metal ion–dependent adhesion site (MIDAS) is shown in gold sphere. In 1-AMA: blue, oxygen; green, carbon.

Figure 5

Propofol binds to the wild-type (WT) α I domain. A, The binding of propofol to the α I domain was determined using 1-aminoanthracene (1-AMA) displacement assay. Fluorescence emission spectra after the excitation of samples at 380 nm are shown. Downward arrow indicates that 1-AMA is displaced by propofol titration. The WT αL I domain contributes little to fluorescence at 520 nm. The figure is representative of 3 experiments. B, The shift of fluorescence intensity at 520 nm from 1-AMA alone + the WT α I domain alone is plotted against different concentration of propofol. Data represent mean ± SD of triplicates. Each data point represents a unique replication. Statistical analysis was performed using 1-way analysis of variance using Tukey post hoc pairwise comparisons. * P < 0.01 versus mock-treated sample (no propofol).