Structural prerequisites for G-protein activation by the neurotensin receptor

Abstract

We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A(3.49), L310A(6.37), F358A(7.42)) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358(7.42) causes the conserved W321(6.48) to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na(+) ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310(6.37) side chain dictates the position of R167(3.50) of the highly conserved D/ERY motif. These residues, together with the presence of E166(3.49) provide determinants for G-protein activation by NTSR1.

Figure 1. Mutational analysis of NTSR1 for activation of G protein.

Agonist-stimulated activation of Gq: GDP/[³⁵S]GTPγS exchange assays contained purified Gq protein, [³⁵S]GTPγS, insect cell membranes with NTSR1 and saturating concentrations of NTS (20 μM). Fold stimulation of the exchange of GDP for [³⁵S]GTPγS at Gq in the presence of NTS is compared with the nucleotide exchange in the absence of ligand (number of independent experiments: wild-type NTSR1 n=7; NTSR1-GW5 n=1; NTSR1-E n=4; NTSR1-L n=5; NTSR1-F n=4; NTSR1-EL n=5; NTSR1-EF n=4; NTSR1-LF n=7; NTSR1-ELF n=6). A value of 1 (dotted line) indicates the absence of receptor-catalysed nucleotide exchange. All G-protein activation experiments were conducted with NTSR1 constructs (containing the wild-type ICL3, not T4L) in urea-washed P2 insect cell membranes. The identity of the NTSR1 constructs is given in Supplementary Table 1. Error bars correspond to s.e.m.

Figure 2. Overview of NTSR1 structures bound to the peptide agonist NTS_8–13.

Cartoon representation of NTSR1-ELF-T4L (blue; NTS_8–13 in purple), NTSR1-LF-T4L (green) and NTSR1-GW5-T4L (grey, NTS_8–13 in orange, PDB code 4GRV). NTS_8–13 is depicted as a stick model. (a) Side view of NTSR1-ELF-T4L. Residues E166^3.49, L310^6.37 and F358^7.42 are shown as cyan spheres; residues D113^2.50, W321^6.48 and R167^3.50 are depicted in red. (b) Extracellular view. An arrow indicates ECL3, which is shifted towards the receptor core in NTSR1-ELF-T4L. (c,d) Intracellular view. Arrows indicate the position shift of the intracellular ends of TM3, TM5, TM6 and TM7 of NTSR1-ELF-T4L (c) and NTSR1-LF-T4L (d) compared with NTSR1-GW5-T4L. ICL1 is disordered in NTSR1-ELF-T4L. In contrast to NTSR1-GW5-T4L, NTSR1-ELF-T4L and NTSR1-LF-T4L have a short helix H8. T4L has been omitted from the intracellular view for clarity.

Figure 3. TM7 and helix 8.

(a) NTSR1-LF-T4L is coloured green with mesh outlining the electron density of H8. (b) NTSR1-ELF-T4L in blue. (c) NTSR1 mutant TM86V-ΔIC3A in cyan (PDB code 3ZEV18). The partial unwinding of TM7 in NTSR1-ELF-T4L and NTSR1-LF-T4L (indicated by the pink colour) after the conserved NP^7.50xxY motif allows F376^8.50 to adopt its current position. The aromatic ring of F376^8.50 is weakly anchored between TM1 and TM7 in NTSR1-LF-T4L, but not in NTSR1-ELF-T4L. The 2mFo-DFc sigma-A weighted maps are contoured at 1σ. (d,e) Comparison of active-like NTSR1-ELF-T4L (blue) with TM86V-ΔIC3A (cyan). TM3–TM6 have been omitted for clarity.

Figure 4. A hydrogen bond and van der Waals system links the agonist peptide with the hydrophobic core of NTSR1-LF and NTSR1-ELF.

NTSR1-GW5-T4L (a) (PDB code 4GRV), NTSR1-LF-T4L (b) and NTSR1-ELF-T4L (c) are shown in grey, green and blue, respectively. Individual residues are shown as a stick model and are labelled. L13 of NTS_8–13 (yellow) is connected to R327^6.54, R328^6.55 and Y324^6.51 via a hydrogen bond network. Water molecule W9 is labelled. A close-by water molecule W10 that is bonded to L13 of NTS_8–13 in NTSR1-LF-T4L has been omitted for clarity. No corresponding water molecules were detected in NTSR1-ELF-T4L, likely because of the lower resolution of the structure. A series of stacking interactions relate Y324^6.51 to F358^7.42, W321^6.48 and the hydrophobic core residue F317^6.44. (d) Position of the F^6.44 side chain in NTSR1-ELF-T4L (blue), the NTSR1 mutant TM86V-ΔIC3A (cyan, PDB code 3ZEV) and the inactive (purple, PDB code 2RH1) and active β₂AR (orange, PDB code 3SN6), viewed from the intracellular side.

Figure 5. The collapsed Na⁺ ion-binding pocket.

(a) NTSR1-GW5-T4L (grey, PDB code 4GRV), (b) NTSR1-LF-T4L (green), (c) NTSR1-ELF-T4L (blue). The conserved D113^2.50 residue (yellow), which has been assigned a crucial role in the Na⁺ ion sensitivity of agonist binding, engages in hydrogen bond interactions with T156^3.39, S362^7.46 and N365^7.49 preventing the coordination of a Na⁺ ion. Other polar interactions between nearby residues are also shown. Residues of TM5 have been removed from the cartoons for clarity. (d) Inactive δ-opioid receptor (purple, PDB code 4N6H25) with a Na⁺ ion coordinated by residues D95^2.50, N131^3.35 S135^3.39 and two water molecules. (e) NTSR1 mutant TM86V-ΔIC3A (cyan, PDB code 3ZEV). Polar interactions are shown as dashed cyan lines. No electron density for water molecules or a Na⁺ ion has been reported.

Figure 6. Effect of L310^6.37 on the positioning of the R167^3.50 side chain.

(a,b) Comparison of NTSR1-LF-T4L (green) and NTSR1-ELF-T4L (blue) with NTSR1-GW5-T4L (grey, PDB code 4GRV). Hydrogen bonds are indicated by dashed lines, and water molecules are represented as spheres. In NTSR1-GW5-T4L, the R167^3.50 side chain is stabilized by a hydrogen bond network to N257^5.58, S164^3.47 and G306^6.33, facilitated by the L310A mutation. The presence of the larger side chain of L310^6.37 in NTSR1-LF-T4L and NTSR1-ELF-T4L is sterically incompatible with such an arrangement and the R167^3.50 side chain interactions with N257^5.58 and S164^3.47 are lost. (c) The R167 side chain now adopts a conformation similar to that seen in metarhodopsin II (ref. ; purple, PDB code 3PQR), the β₂AR–Gs complex (orange, PDB code 3SN6) and the active M2 receptor (red, PDB code 4MQS).