Gianluigi Caltabiano

Τhe muscarinic M2 acetylcholine receptor, one of the few G protein-coupled receptors that has not only been crystallized in both active and inactive conformations, but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14μs in total) to study conformational changes triggered by the inverse agonist R-(−)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 muscarinic (PBD id 4MQS) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY2119620 when bound simultaneously with the orthosteric agonist iperoxo and find that it restricts the conformation of Trp4227.35 in a position that modulates the orientation of the Tyr4267.39 at the orthosteric-binding pocket.

The dopamine D₃ receptor (D₃R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D₃R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D₃R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D₃R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D₃R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D₃R dimerization.

Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide–receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding.

Protein sequence alignments and database search methods use standard scoring matrices calculated from amino acid substitution frequencies in general sets of proteins. These general-purpose matrices are not optimal to align accurately sequences with marked compositional biases, such as hydrophobic transmembrane regions found in membrane proteins. In this work, an amino acid substitution matrix (GPCRtm) is calculated for the membrane spanning segments of the G protein-coupled receptor (GPCR) rhodopsin family; one of the largest transmembrane protein family in humans with great importance in health and disease. The GPCRtm matrix reveals the amino acid compositional bias distinctive of the GPCR rhodopsin family and differs from other standard substitution matrices. These membrane receptors, as expected, are characterized by a high content of hydrophobic residues with regard to globular proteins. On the other hand, the presence of polar and charged residues is higher than in average membrane proteins, displaying high frequencies of replacement within themselves. Analysis of amino acid frequencies and values obtained from the GPCRtm matrix reveals patterns of residue replacements different from other standard substitution matrices. GPCRs prioritize the reactivity properties of the amino acids over their bulkiness in the transmembrane regions. A distinctive role is that charged and polar residues seem to evolve at different rates than other amino acids. This observation is related to the role of the transmembrane bundle in the binding of ligands, that in many cases involve electrostatic and hydrogen bond interactions. This new matrix can be useful in database search and for the construction of more accurate sequence alignments of GPCRs.

G protein–coupled receptors, including the M₃ muscarinic acetylcholine receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved fluorescence resonance energy transfer studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI, and VII as well as intracellular helix VIII to the overall organization. Molecular modeling studies based on both these results and an X-ray structure of the inactive state of the M₃ receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M₃ receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally resolved, multiphoton fluorescence resonance energy transfer. Modeling studies furthermore suggest an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, which is consistent with the presence of cholesterol at key locations in many G protein–coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.

The finding that ergotamine binds serotonin receptors in a less conserved extended binding pocket close to the extracellular entrance, in addition to the orthosteric site, allowed us to obtain 5-HT₇R antagonist 6 endowed with high affinity (K_i = 0.7 nM) and significant 5-HT_1AR selectivity (ratio >1428). Compound 6 exhibits in vivo antidepressant-like effect (1 mg/kg, ip) mediated by the 5-HT₇R, which reveals its interest as a putative research tool or pharmaceutical in depression disorders.