Critical role for polar residues in coupling leukotriene B4 binding to signal transduction in BLT1.
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.
Basu
Sudeep
S
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202; Departments of Microbiology and Immunology, Louisville, Kentucky 40202.
Jala
Venkatakrishna R
VR
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202.
Mathis
Steven
S
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202; Departments of Microbiology and Immunology, Louisville, Kentucky 40202.
Rajagopal
Soujanya T
ST
Departments of Microbiology and Immunology, Louisville, Kentucky 40202.
Del Prete
Annalisa
A
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202.
Maturu
Paramahamsa
P
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202.
Trent
John O
JO
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202; Department of Medicine, University of Louisville Health Sciences, Louisville, Kentucky 40202. Electronic address: john.trent@louisville.edu.
Haribabu
Bodduluri
B
Tumor Immunobiology Program, James Graham Brown Cancer Center, Louisville, Kentucky 40202; Departments of Microbiology and Immunology, Louisville, Kentucky 40202. Electronic address: H0bodd01@gwise.louisville.edu.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D006801:Humans; D007975:Leukotriene B4 / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D018102:Receptors, Leukotriene B4 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
D000596:Amino Acids; C513240:LTB4R protein, human; D008024:Ligands; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4