Assembling NMR structures for the intracellular loops of the human thromboxane A2 receptor: implication of the G protein-coupling pocket

Abstract

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.

Figures

Figure 1

(A) The seven TM domain (TM1-TM7) model of the human TP receptor. This 3D structure was constructed using homology modeling within the Insight II program. The distances between the iLP2 termini (connecting the third and fourth TM domains) and between the iLP3 termini (connecting the fifth and sixth TM domains) are shown. (B) Topology model of the TP receptor. The heavy lines represent the regions that were emphasized for these studies. (C) The peptide mimicking the human TP receptor iLP2 (residues 1−23) and iLP3 (residues 1−29), is shown with homocysteine added at both ends of the loop.

Figure 2

Number of constraints per residue for the TP iLP2 (A) and TP iLP3 (B) peptides derived from NMR structural analyses.

Figure 3

NMR solution 3-D structures of the constrained TP iLP2 and TP iLP3 peptides. Superimposition of ten structures using the α-carbons of TP iLP2 (A) and TP iLP3 (C) after DGII calculations based on the NOE constraints and dihedral angles. Superimposition of ten structures using the α-carbons of TP iLP2 (B) and TP iLP3 (D) obtained from the energy refinement calculations.

Figure 4

Secondary structure analyses by Hα Chemical Shift Index for TP iLP1 (A) and TP iLP2 (B).

Figure 5

The arrangement of TP iLP2 (A, B) and TP iLP3 (C, D) in respect to TM3 and TM4 (A, B), and TM5 and TM6 (C, D) of the TP receptor working model. The distance between TM3 and TM4 is 12.99 Å (A) while the distance between TM5 and TM6 is 12.41 Å (C) The distances between the termini of the NMR structure of iLP2 and iLP3 are 9.14 Å and 9.59 Å, respectively (A, C). The NMR structure of the iLP2 was connected between the TM3 and TM4, and iLP3 structure was connected between TM5 and TM6 of the TP receptor working model and an energy minimization was performed for the connected molecule. The resultant distance between TM3 and TM4 changed to 10.95 Å (B) while for TM5 and TM6 it changed to 14.96 Å (D). The distance between the termini of the NMR structure of iLP2 and iLP3 also changed to 13.31 Å and 15.96 Å, respectively.

Figure 6

Structure model of the TP intracellular domain that was generated based on the NMR study, and a putative G protein-binding pocket, shown in three different schemes: (A) α-carbons only, (B) CPK mode with all the side chain and (C) potential energy surface. Some key residues, known to be important to the G protein-coupling from the mutagenesis studies are also shown.

Figure 7

Docking of the C-terminal domain of Gαq into the putative binding site on the TP intracellular domain. The first orientation is shown in stick render form (A) and potential energy surface mode (B). The second orientation is also shown in stick render form (C) and potential energy surface mode (D).

Figure 8

The docking model is shown in potential energy surface mode. Some additional residues that might also be important for G protein coupling are indicated.