Engineering a G protein-coupled receptor for structural studies: stabilization of the BLT1 receptor ground state.
Structural characterization of membrane proteins is hampered by their instability in detergent solutions. We modified here a G protein-coupled receptor, the BLT1 receptor of leukotriene B(4), to stabilize it in vitro. For this, we introduced a metal-binding site connecting the third and sixth transmembrane domains of the receptor. This modification was intended to restrain the activation-associated relative movement of these helices that results in a less stable packing in the isolated receptor. The modified receptor binds its agonist with low-affinity and can no longer trigger G protein activation, indicating that it is stabilized in its ground state conformation. Of importance, the modified BLT1 receptor displays an increased temperature-, detergent-, and time-dependent stability compared with the wild-type receptor. These data indicate that stabilizing the ground state of this GPCR by limiting the activation-associated movements of the transmembrane helices is a way to increase its stability in detergent solutions; this could represent a forward step on the way of its crystallization.
Martin
Aimée
A
Institut des Biomolécules Max Mousseron, UMR 5247 CNRS Universités Montpellier I et II, Faculté de Pharmacie, 15 Av. Ch. Flahault, BP14491, 34093 Montpellier Cedex 5, France.
Damian
Marjorie
M
Laguerre
Michel
M
Parello
Joseph
J
Pucci
Bernard
B
Serre
Laurence
L
Mary
Sophie
S
Marie
Jacky
J
Banères
Jean-Louis
JL
D000595:Amino Acid Sequence; D001665:Binding Sites; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D018102:Receptors, Leukotriene B4 / Q000819:agonists / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D015032:Zinc / Q000378:metabolism
D003902:Detergents; C513240:LTB4R protein, human; D008024:Ligands; D018102:Receptors, Leukotriene B4; D019204:GTP-Binding Proteins; D015032:Zinc