Structural basis of ligand recognition in 5-HT3 receptors

Abstract

The 5-HT(3) receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT(3) receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation-π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT(3) receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT(3) receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT(3) receptor.

Figure 1

Protein engineering of 5HTBP. (A) Schematic representation of the 5-HT₃ receptor. The ligand-binding site is located in the extracellular domain at the subunit interface. The inset illustrates the contribution of different binding site loops (A–F) in the acetylcholine-binding protein (AChBP). Loop numbers correspond to the number of residues that were substituted to the corresponding residues of the 5-HT₃ receptor. Granisetron is shown in magenta sticks and transparent spheres. Yellow are tolerated substitutions, red are mutations that show specific [³H]granisetron binding. (B) Sequence alignment of AChBP and 5-HT₃ receptors. Blue colouring indicates BLOSUM62 conservation score. Yellow and red colours in AChBP correspond to those in panel A. Boxed residues indicate positions that are critical for granisetron or serotonin binding in the 5-HT₃ receptor. (C) Example of a radioligand saturation experiment on 5HTBP (construct A1B2D1_WE1, see Table 1), which displayed high-affinity [³H]granisetron binding. AChBP, acetylcholine-binding protein; 5-HT₃R, 5-HT₃ receptor.

Figure 2

X-ray crystal structures of 5HTBP in complex with serotonin and granisetron. (A,B) 2F_o-F_c electron density contoured at 1.5σ for serotonin and granisetron, respectively. (C) Detailed interactions of serotonin in the ligand-binding site of construct A1B2D1_W, which contains mutations Y53W, S92E, V140L and K141T (underlined). The principal (+) subunit is shown in white, the complementary subunit (−) in blue and serotonin in yellow. On the principal face, the aminoethyl nitrogen of serotonin forms hydrogen bonds (shown as dashed lines) with W145 and Y91, as well as a cation–π interaction with W145. On the complementary face, the 5-hydroxy group of serotonin forms hydrogen bonds with a water molecule (shown as a red sphere) in loop E and the backbone atoms of I104 and I116. (D) Detailed interactions of granisetron in the ligand-binding site of construct D2, which contains mutations Y53W and Q55R (underlined). Granisetron forms a hydrogen bond with a stretch of water molecules (w1–w5) that extend into the vestibule of the ligand-binding domain. The tropane ring protrudes deep into the binding site and interacts with W53, whereas the indazole moiety points out of the pocket and is stabilized by interactions with R55.

Figure 3

Molecular blueprint for ligand recognition in the 5-HT₃R. (A,B) Structural determinants of serotonin and granisetron recognition, respectively. Boxed residues represent observed contacts in 5HTBP, grey residues are homologous residues in the 5-HT₃ receptor. Superscript letters indicate binding site loops A–F. Red dashed lines indicate hydrogen bonds, grey indicate hydrophobic interactions and blue indicate cation–π interactions. The key cation–π interaction between the indazole moiety of granisetron and R55 is shown in red. (C) Superposition of granisetron- and serotonin-bound structures shows side-chain reorientations and a conformational change in the C loop. Serotonin (yellow) adopts a binding pose that partially overlaps with, but is nearly perpendicular to granisetron (orange) and critically depends on interactions with loop E. 5-HT₃R, 5-HT₃ receptor.

Figure 4

A cation–π interaction probed in the human 5-HT₃R with chemically tuned granisetron analogues. (A) In the crystal structure of granisetron-bound 5HTBP, we observe a cation–π interaction between the indazole moiety of granisetron (shown in yellow) and the positively charged R55 (homologous to R92 in 5-HT₃R) of loop D in the binding site. (B) Chemical structures of gran-1F, -2F and -3F. (C) Typical concentration–response curves for displacement of [³H]granisetron binding by fluorinated granisetrons at the human 5-HT₃R. These plots were used to determine K_i values and subsequently create the fluorination plot shown (D; data=mean±s.e.m., n=4–6). Electrostatic potentials surfaces for indazole and fluorinated derivatives are shown near each average data point. Electrostatic potentials span a range of −100 (red) to +100 (blue) kJ/mol. 5-HT₃R, 5-HT₃ receptor.