Computational model of the complex between GR113808 and the 5-HT4 receptor guided by site-directed mutagenesis and the crystal structure of rhodopsin

A computational model of the transmembrane domain of the human 5-HT4 receptor complexed with the GR113808 antagonist was constructed from the crystal structure of rhodopsin and the putative residues of the ligand-binding site, experimentally determined by site-directed mutagenesis. The recognition mode of GR113808 consist of: (i) the ionic interaction between the protonated amine and Asp(3.32); (ii) the hydrogen bond between the carbonylic oxygen and Ser(5.43); (iii) the hydrogen bond between the ether oxygen and Asn(6.55); (iv) the hydrogen bond between the C-H groups adjacent to the protonated piperidine nitrogen and the pi electrons of Phe(6.51); and (v) the pi-sigma aromatic-aromatic interaction between the indole ring and Phe(6.52). This computational model offers structural indications about the role of Asp(3.32), Ser(5.43), Phe(6.51), Phe(6.52), and Asn(6.55) in the experimental binding affinities. Asp(3.32)Asn mutation does not affect the binding of GR113808 because the loss of binding affinity from an ion pair to a charged hydrogen bond is compensated by the larger energetical penalty of Asp to disrupt its side chain environment in the ligand-free form, and the larger interaction between Phe(6.51) and the piperidine ring of the ligand in the mutant receptor. In the Phe(6.52)Val mutant the indole ring of the ligand replaces the interaction with Phe(6.52) by a similarly intense interaction with Tyr(5.38), with no significant effect in the binding of GR113808. The mutation of Asn(6.55) to Leu replaces the hydrogen bond of the ether oxygen of the ligand from Asn(6.55) to Cys(5.42), with a decrease of binding affinity that approximately equals the free energy difference between the SH...O and NH...O hydrogen bonds. Because these residues are also present in the other members of the neurotransmitter family of G protein-coupled receptors, these findings will also serve for our understanding of the binding of related ligands to their cognate receptors.