Journal:JBSD:16

The extracellular subunit interface of the 5-HT3 receptors: a computational alanine scanning mutagenesis study

Francesca De Rienzo, Arménio J. Moura Barbosa, Marta A.S. Perez, Pedro A. Fernandes, Maria J. Ramos, Maria Cristina Menziani ^[1]

Molecular Tour
The serotonin type-3 receptor (5-HT3-R) is a cation selective transmembrane protein channel that belongs to the Cys–loop Ligand-Gated Ion Channel (LGIC) superfamily (http://www.ebi.ac.uk/compneur-srv/LGICdb/LGICdb.php), which also includes receptors for nicotinic acetylcholine (, PDB code 2bg9), γ-aminobutyric acid and glycine. 5-HT3-R is involved in signal transmission in the central and peripheral nervous system and its malfunctioning leads to neurodegenerative and psychiatric diseases, therefore it is an important target for drug design research. A few drugs active against 5-HT3-R are already on the market, such as, for example, palonosetron (http://en.wikipedia.org/wiki/Palonosetron) and granisetron (http://en.wikipedia.org/wiki/Granisetron). The 5-HT3R is made of five monomers assembled in a to form an ion channel permeable to small ions (Na+, K+); each subunit contains three domains: an (shown on the example of nAChR, 2bg9). To date, five different 5-HT3-R subunits have been identified, the 5-HT3 A, B, C, D and E; however, only subunits A and B have been extensively characterised experimentally. The of nAChR is located at the extracellular region, at the interface between two monomers (α-γ and α-δ; 2 identical α monomers, chains A and D, are colored in same color - lavender), called the principal and the complementary subunits. The 3D structure of 5-HT3-R has not been experimentally solved yet; however, it has been obtained computationally by means of homology modelling techniques. (http://salilab.org/modeller/) Thus, the extracellular region of the 5HT3 subunits A and B are modelled by homology with the 3D structure of the nAChR subunit A (2BG9-A) and are used to assemble receptor structures as pseudo-symmetric pentamers made either of five identical subunits A (homomeric 5-HT3A-R – homopentamer-aaaaa.pdb) or of both subunits A and B (heteromeric 5-HT3A/B-R in the BBABA arrangement –heteropentamer-bbaba.pdb) in a still debated arrangement.^[2] A complete characterization of the extracellular moiety of the dimer interface of the 5-HT3-R, is obtained by the Computational Alanine Scanning Mutagenesis (CASM) approach ^[3], which simulates the substitution, one by one, of all the amino acid residues at the subunit-subunit interfaces with an Ala, thus to assess the interface binding contribution of single residue side-chains. The most relevant residues for interface stabilization (Figure 3) are classified as “hot spots” that stabilize the interface by more than 4 kcal/mol and “warm spots” that contribute to interface stabilization by more than 2 kcal/mol. From this analysis the important aromatic cluster located at the interface core and formed by residues W178 (principal subunit), Y68, Y83, W85 and Y148 (complementary subunit) is highlighted (Figure 3).^[4] In addition, two important groups of interface residues are probably involved in the coupling of agonist/antagonist binding to channel activation/inactivation: W116-H180-L179-W178-E124-F125 (principal subunit) and Y136-Y138-Y148-W85-(P150) (complementary subunit), where W178 and Y148 appear to be critical residues for the binding/activation mechanism (Figure 5). (dimer-AA-ser1.pdb, dimer-AA-ser2.pdb, dimer-AA-pal1.pdb, dimer-AA-pal2.pdb) Finally, the comparison of the AA interface with the BB interface shows differences which could explain the reasons why the homopentamer 5-HT3B-R, if expressed, is not functional (Figura 6 and 7).^[5]