Temperature-dependent variations of ligand-receptor contact points in hAT(1).
Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT(1) has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT(1) receptor and two representative Met mutants (H256M-hAT(1) and F293M-hAT(1)) from the iterative mutagenesis study were photolabelled with the benzophenone-ligand (125)I-[Sar(1), Bpa(8)]AngII at temperatures ranging from - 15 degrees C to 37 degrees C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT(1) and F293M-hAT(1) across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT(1) was observed. At - 15 degrees C, H256M labelling was identical to that of hAT(1), indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37 degrees C labelling occurred almost exclusively at mutated residue H256M-hAT(1) in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures.
Arsenault
J
J
Département de pharmacologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada.
Renaud
M P La Haye
MP
Clément
M
M
Fillion
D
D
Guillemette
G
G
Leduc
R
R
Lavigne
P
P
Escher
E
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels; D017434:Protein Structure, Tertiary; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D008024:Ligands; D019920:Photoaffinity Labels; D044140:Receptor, Angiotensin, Type 1; D011994:Recombinant Proteins