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 (2BG9 –http://www.rcsb.org/pdb/explore.do?structureId=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 pseudo-symmetric pentameric shape to form an ion channel permeable to small ions (Na+, K+); each subunit contains three domains: an intracellular portion, a transmembrane domain and an extracellular region. (Figure1, left) 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 ligand binding site (Figure1, right) is located at the extracellular region, at the interface between two monomers, 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]

[1] A. J. M. Barbosa, F. de Rienzo, M. J. Ramos, M. C. Menziani Computational analysis of ligand recognition sites of homo and heteropentameric 5-HT3 Receptors European Journal of Medicinal Chemistry, 2010, 45, 4746-4760.

[2] I.S. Moreira, P.A. Fernandes, M.J. Ramos Computational alanine scanning mutagenesis - an improved methodological approach Journal of Computational Chememistry, 2007, 28, 644-654.

[3] F. De Rienzo, A. J. M. Barbosa, M.A.S. Perez, P. A. Fernandes, M. J. Ramos, M. C. Menziani The extracellular subunit interface of the 5-HT3 Receptors: a Computational Alanine Scanning Mutagenesis study Journal of Bimolecular Structure and Dynamics, 2012, 30, 280-298. DOI:10.1080/07391102.2012.680029

[4] F. De Rienzo , M. Del Cadia, M. C. Menziani, A first step towards the understanding of the 5-HT3 receptor subunit heterogeneity from a computational point of view. Physical Chemistry Chemical Physics, 2012, DOI: 10.1039/C2CP41028A