Fig 1.
Multiple sequence alignment of representative aminergic receptors and selected TAARs from different organisms.
Positions that are at least 30% or 95% conserved are highlighted in gray and black, respectively. Highly conserved residues in the class A GPCR family are indicated by the Ballesteros-Weinstein numbering (X.50 of helix X), as well as the conserved cysteines involved in disulfide bridges (in yellow). Residues at position 3.32 and 5.42/5.43 are highlighted in red. Non-conserved N-, C- terminal and ICL3 amino acid sequences are omitted from the figure. Acronyms: h5HT1B (human 5-hydroxytryptamine receptor 1B), hH1R (human histamine receptor H1), hD3R (human dopamine receptor D3), hADRB2 (human β2-adrenergic receptor), hTAAR6 (human trace-amine associated receptor 6), hTAAR8 (human trace-amine associated receptor 8), mTAAR6 (mouse trace-amine associated receptor 6), mTAAR8b (mouse trace-amine associated receptor 8b), rTAAR6 (rat trace-amine associated receptor 6), rTAAR8a (rat trace-amine associated receptor 8a), zTAAR13c (zebrafish trace-amine associated receptor 13c) and zTAAR13d (zebrafish trace-amine associated receptor 13d).
Fig 2.
The orthosteric ligand-binding pocket of human TAAR6 and TAAR8.
Surface representation of the molecular models of hTAAR6 (A and C) and hTAAR8 (B and D) in the active- (top panels) and inactive-like (bottom panels) conformations. Extracellular view of the identified ligand binding cavities with molecular surfaces colored by the electrostatic potential calculated using the program APBS with nonlinear Poisson-Boltzmann equation and contoured at ±10 kT/e (negatively and positively charged surface areas in red and blue, respectively). Residues contributing to the electronegative potential of the binding pocket are represented in sticks and numbered according to the receptor type (Ballesteros-Weinstein scheme in parenthesis). Calculated distances between carboxyl moieties of Asp3.32 and Asp5.43 are shown for each molecular structure (yellow dashed lines). Protein backbones are shown in cylinders except ECL2 conformations (here omitted for clarity).
Fig 3.
Molecular interactions of PUT and CAD with human TAAR6 and TAAR8.
(A) Key features of the full agonist adrenaline (ADR, blue sticks) in the binding pocket of the ADRB2 active structure (PDB ID:4LDO; region comprising the TMs 3-5-6-7 in green ribbons). (B) Superposition of molecular docking of putrescine (PUT, yellow sticks), and (C) cadaverine (CAD, orange sticks), in the active-like TAAR6 (light-gray ribbons) and TAAR8 (light-blue ribbons) molecular models. Contact residues at a distance < 3.5 Å from ligands are shown in sticks and numbered according to Ballesteros-Weinstein numbering. Predicted (ligand–receptor) hydrogen bonds and salt bridge interactions are shown in dashed lines. The 2D chemical structure of PUT and CAD protonated at physiological pH, with estimated pKa1 and pKa2 for each amino group are indicated at the bottom of panels B and C, respectively.
Fig 4.
Stability of interactions between PUT and CAD and human TAAR6 and TAAR8.
Time evolution (x-axis) of intermolecular distances (y-axis) between Asp3.32/5.43 (-COO-) and PUT/CAD (-NH3+) in 1.0 μs unbiased MD simulations. Each plot corresponds to one of the eight simulated ligand-receptor molecular complexes: hTAAR6active-like/PUT (A), hTAAR6active-like/CAD (B), hTAAR8active-like/PUT (C), hTAAR8active-like/CAD (D), hTAAR6inactive-like/PUT (E), hTAAR6inactive-like/CAD (F), hTAAR8inactive-like/PUT (G), hTAAR8inactive-like/CAD (H). Continuous and dotted lines correspond to distances between N1 and N2 atoms of the ligands with Asp3.32 (red) and Asp5.43 (blue) carboxyl groups, respectively. Black arrows at the bottom indicate the flip-transitions (180° rotation) of PUT and CAD in the binding pocket of the inactive-like models.
Fig 5.
Effect of PUT and CAD on the ‘transmission switch’ amino acids.
(A) Structural attributes of the ‘transmission switch’ residues 3.40, 5.50 and 6.44 (in sticks), in TMs 3-5-6 (in cylinders), on the ADRB2 in agonist bound active- (PDB ID: 3SN6, in green) and inverse agonist bound inactive- conformation (2RH1, in red). Arrows represent the observed movement of the helices in the transition from the inactive to the active state of the receptor. (B) Distribution of L3.40, P5.50 and F6.44 Cβ atoms positions (dots) in the TAAR6 and (C) V3.40, P5.50 and F6.44 Cβ atoms in the TAAR8 during simulations of active-like PUT/CAD bound in green/light green and inactive-like PUT/CAD bound in red/light red. Numbers correspond to the standard deviation (SD) of the Cβ atoms positions from the centroid of 100 evenly spaced snapshots extracted from the 1.0 μs of unbiased MD simulations.
Fig 6.
Cladogram representing the presence of TAARs in a consensus phylogeny of different vertebrates.
The total number of functional TAAR genes is shown in parenthesis for each organism. Teleost TAAR13c/TAAR13d with proven affinity for CAD/PUT, respectively and therian-specific TAAR6/TAAR8 with the conserved tandem of aspartates in the TM3 and TM5 appear in bold (corresponding to black silhouettes in the species of origin). Approximate divergence times between species (million years ago; MYA) are shown in the internal nodes.