Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1.
We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4). We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.
Baneres
Jean-Louis
JL
UMR 5074 CNRS, Chimie Biomoléculaire et Interactions Biologiques, Faculté de Pharmacie, 15 Avenue Ch. Flahault, BP 14491, 34093, Cedex 05, Montpellier, France. baneres@pharma.univ-montp1.fr
Martin
Aimée
A
Hullot
Pierre
P
Girard
Jean-Pierre
JP
Rossi
Jean-Claude
JC
Parello
Joseph
J
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007975:Leukotriene B4 / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018102:Receptors, Leukotriene B4 / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; D018102:Receptors, Leukotriene B4; D011994:Recombinant Proteins; D007975:Leukotriene B4; D014364:Tryptophan