Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: implication of the G protein-coupling pocket.
It has been reported that the multiple intracellular loops (iLPs) of the thromboxane A(2) receptor (TP) are involved in the receptor G protein coupling. In this study, a high-resolution 2D NMR technique was used to determine the 3D structures of the first, second, and third iLPs of the TP using synthetic peptides constrained into the loop structures. 2D (1)H NMR spectra, TOCSY and NOESY were obtained for the two peptides from proton NMR experiments. The NMR data was processed and assigned through the Felix 2000 program. Standard methods were used to acquire sequence-specific assignments. Structure calculations were processed through DGII and NMR refinement programs within the Insight II program. We were able to calculate and use the NOE constraints to obtain the superimposed structure of 10 structures for each iLP peptide. The NMR-determined structures of the iLP peptides were used to refine a homology model of the TP. A 3D G-protein-binding cavity, formed by the three intracellular loops, was predicted by the docking of the C-terminal domain of the Galphaq. Based on the structural model and the previous mutagenesis studies, the residues, R130, R60, C223, F138, L360, V361, E358 and Y359, which are important for interaction with the G protein, were further highlighted. These results reveal the possibly important molecular mechanisms in TP signaling and provide structural information to characterize other prostanoid receptor signalings.
Wu
Jiaxin
J
The Center for Experimental Therapeutics and PharmacoInformatics, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Room 521 Science & Research Building 2, Houston, TX 77204-5037, USA.
Feng
Mary
M
Ruan
Ke-He
KH
D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D003461:Crystallography / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000648:ultrastructure*; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000737:chemistry* / Q000648:ultrastructure*
D043562:Receptors, G-Protein-Coupled; D044244:Receptors, Thromboxane A2, Prostaglandin H2