Abstract
Leukotriene B(4) (LTB(4)) mediates a variety of inflammatory diseases such as asthma, arthritis, atherosclerosis, and cancer through activation of the G-protein-coupled receptor, BLT1. Using in silico molecular dynamics simulations combined with site-directed mutagenesis we characterized the ligand binding site and activation mechanism for BLT1. Mutation of residues predicted as potential ligand contact points in transmembrane domains (TMs) III (H94A and Y102A), V (E185A), and VI (N241A) resulted in reduced binding affinity. Analysis of arginines in extracellular loop 2 revealed that mutating arginine 156 but not arginine 171 or 178 to alanine resulted in complete loss of LTB(4) binding to BLT1. Structural models for the ligand-free and ligand-bound states of BLT1 revealed an activation core formed around Asp-64, displaying multiple dynamic interactions with Asn-36, Ser-100, and Asn-281 and a triad of serines, Ser-276, Ser-277, and Ser-278. Mutagenesis of many of these residues in BLT1 resulted in loss of signaling capacity while retaining normal LTB(4) binding function. Thus, polar residues within TMs III, V, and VI and extracellular loop 2 are critical for ligand binding, whereas polar residues in TMs II, III, and VII play a central role in transducing the ligand-induced conformational change to activation. The delineation of a validated binding site and activation mechanism should facilitate structure-based design of inhibitors targeting BLT1.
Highlights
The use of multiple docking protocols and molecular dynamics of the binding pocket allowed the delineation of the Leukotriene B4 (LTB4) binding site in BLT1
The ligand is encompassed by transmembrane domains III, V, and VI, whereas ECL2 forms a lid over the pocket
The N241A, Y102A, E185A, and H94A mutants and the Y102A/N241A double mutant each attained the wild-type maximum levels of signaling in functional assays, including chemotaxis, calcium flux, and receptor phosphorylation, albeit at a much higher concentration of LTB4 as compared with BLT1. This suggests that, these residues located in transmembrane domains (TMs) III, V, and VI contribute to the binding affinity by countering the hydroxyl groups at 5th and 12th positions on LTB4, they are not absolutely critical for LTB4 binding or ligand-induced conformational changes associated with receptor activation
Summary
Homology Modeling and Identification of LTB4 Binding Site in BLT1—The multiple alignment of the human LTB4 receptors, BLT1 and BLT2, with bovine rhodopsin was generated using ClustalW [24]. The cells (0.5 ϫ 106 per assay) were incubated with 2.5 nM [3H]LTB4 (0.25 nM for BLT1) (163 Ci/mmol, PerkinElmer Life Sciences) along with increasing concentrations of cold ligand (Cayman Chemicals, Detroit, MI) in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM NaCl, 0.05% bovine serum albumin (fatty acid-free Fraction V, Sigma A8806)). These mixtures were incubated on ice with gentle agitation for 2 h followed by rapid filtration through GF/C filters (Whatman 1822-025) using manifold-vacuum setup and washed with 3 ml of ice-cold binding buffer. Real-time Fluorescence Microscopy—RBL-2H3 cells were cotransfected with -arrestin-GFP and either with wild-type or mutant receptor tagged with RFP (monomer), and images were captured as described previously [18, 34]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
