1 Non-classical amine recognition evolved in a large clade of olfactory receptors 2 3 Qian Li1, Yaw Tachie-Baffour1, Zhikai Liu1, Maude W. Baldwin2, Andrew C. Kruse3, and Stephen 4 D. Liberles1* 5 6 1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; 7 2Department of Organismic and Evolutionary Biology, Harvard University and the Museum of 8 Comparative Zoology, Cambridge, MA 02138, USA; 9 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 10 Boston, MA 02115, USA 11 12 *Correspondence to [email protected], 13 phone: (617) 432-7283, fax: (617) 432-7285 14 15 Competing interests 16 The authors declare that no competing interests exist. 17 18 19 20 Biogenic amines are important signaling molecules, and the structural basis for their 21 recognition by G Protein-Coupled Receptors (GPCRs) is well understood. Amines are 22 also potent odors, with some activating olfactory trace amine-associated receptors 23 (TAARs). Here, we report that teleost TAARs evolved a new way to recognize amines in a 24 non-classical orientation. Chemical screens de-orphaned eleven zebrafish TAARs, with 25 agonists including serotonin, histamine, tryptamine, 2-phenylethylamine, putrescine, and 26 agmatine. Receptors from different clades contact ligands through aspartates on 27 transmembrane α-helices III (canonical Asp3.32) or V (non-canonical Asp5.42), and diamine 28 receptors contain both aspartates. Non-classical monoamine recognition evolved in two 29 steps: an ancestral TAAR acquired Asp5.42, gaining diamine sensitivity, and subsequently 30 lost Asp3.32. Through this transformation, the fish olfactory system dramatically 31 expanded its capacity to detect amines, ecologically significant aquatic odors. The 32 evolution of a second, alternative solution for amine detection by olfactory receptors 33 highlights the tremendous structural versatility intrinsic to GPCRs. 34 35 2 36 INTRODUCTION 37 38 Biogenic amines are key intercellular signaling molecules that regulate physiology and behavior. 39 In vertebrates, biogenic amines include adrenaline, serotonin, acetylcholine, histamine, and 40 dopamine, and these biogenic amines can be detected by G Protein-Coupled Receptors 41 (GPCRs) expressed on the plasma membrane of target cells. The biochemical and structural 42 basis for biogenic amine recognition by GPCRs is well understood and involves an essential salt 43 bridge between the ligand amino group and a highly conserved aspartic acid on the third 44 transmembrane α-helix of the receptor (Asp3.32; Ballesteros-Weinstein indexing)1,2. Mutation of 45 Asp3.32 in adrenaline, serotonin, acetylcholine, histamine, and dopamine receptors eliminates 46 amine recognition2,3, and GPCRs that recognize amines through another motif have not been 47 identified. 48 49 Trace amine-associated receptors (TAARs) are a family of vertebrate GPCRs distantly related 50 to biogenic amine receptors4. There are 15 TAARs in mouse, 6 in human, and 112 in zebrafish, 51 and in these or related species, all TAARs except TAAR1 function as olfactory receptors5-7. All 52 human TAARs (6/6), most mouse TAARs (13/15), and some zebrafish TAARs (27/112) retain 53 Asp3.32, suggesting that many TAARs would recognize amines. High throughput chemical 54 screens yielded ligands for TAAR18-10 and many olfactory TAARs that retain Asp3.32, including 55 TAAR3, TAAR4, TAAR5, TAAR7s, TAAR8s, TAAR9, and TAAR13c7,11,12. Each of these 56 receptors detects different amines, some of which evoke innate olfactory behaviors12-16. In 57 mouse, TAAR4 detects the aversive carnivore odor 2-phenylethylamine14, while TAAR5 detects 58 the attractive and sexually dimorphic mouse odor trimethylamine7,15. Knockout mice lacking 59 TAAR4 and TAAR5 lose behavioral responses to TAAR ligands identified in cell culture 60 studies13,15. 61 3 62 The selectivity of olfactory TAARs for particular amine odors suggests structural variability within 63 the ligand-binding pocket across the TAAR family. One clade of TAARs (including TAAR1, 64 TAAR3, and TAAR4) detects primary amines derived by amino acid decarboxylation, while a 65 second clade (including TAAR5, TAAR7s, TAAR8s, and TAAR9) prefers tertiary amines11. 66 Structural models and mutagenesis studies of TAAR1, TAAR7e, and TAAR7f predicted that 67 ligand binding occurs in the membrane plane, Asp3.32 forms a salt bridge to the ligand amino 68 group, and other transmembrane residues provide a scaffold that supports ligand binding and 69 enables selectivity11,17. 70 71 A third TAAR clade (clade III TAARs) arose in fish, and includes the majority (87/112) of 72 zebrafish TAARs6. Most clade III TAARs lack Asp3.32 (85/87), and it has been proposed that 73 these receptors may not detect amines6,18. However, ligands have not been identified for any of 74 these 85 receptors. More generally, ligands are known for only one zebrafish TAAR, TAAR13c, 75 which retains Asp3.32 and detects the carrion odor cadaverine12. Structure-activity analysis 76 revealed that TAAR13c preferentially recognizes odd-chained, medium-sized diamines, and 77 likely contains two distinct cation recognition sites within the ligand-binding pocket12. However, 78 the structural basis for diamine recognition by TAAR13c was unknown. 79 80 Here, we find that TAAR13c recognizes cadaverine through two remote transmembrane 81 aspartic acids, Asp3.32 and Asp5.42. A TAAR13c variant with a charge-neutralizing mutation at 82 Asp5.42 has an altered ligand preference for monoamines rather than diamines. We propose that 83 Asp3.32 and Asp5.42 together form a general di-cation recognition motif, and note this motif to be 84 present in several zebrafish, mouse, and human TAARs. Surprisingly, Asp5.42 is retained in 84 85 out of 85 zebrafish TAARs that lack Asp3.32, raising the possibility that these receptors recognize 86 amines positioned in a non-canonical inverted orientation with the amino group pointing towards 87 transmembrane α-helix V. Using a high throughput chemical screening approach, we identified 4 88 ligands for eleven additional zebrafish TAARs, and found that different receptors detect amines 89 through salt bridge contacts at Asp3.32 and/or Asp5.42. TAAR10a, TAAR10b, TAAR12h, and 90 TAAR12i are Asp3.32-containing clade I TAARs that detect serotonin, tryptamine, 2- 91 phenylethylamine, and 3-methoxytyramine, while TAAR16c, TAAR16e, and TAAR16f are 92 Asp5.42-containing clade III TAARs that detect N-methylpiperidine, N,N- 93 dimethylcyclohexylamine, and isoamylamine. TAAR13a, TAAR13d, TAAR13e, and TAAR14d 94 have both Asp3.32 and Asp5.42, and recognize the di-cationic molecules putrescine, histamine, 95 and agmatine. 96 97 Based on these findings, we propose that a large clade of olfactory GPCRs evolved a novel way 98 to detect amines that involves a non-classical salt bridge to transmembrane α-helix V. 99 Furthermore, new TAAR ligands include several biogenic amines for which other receptors were 100 identified, including serotonin, histamine, and 2-phenylethylamine. These findings illustrate that 101 different members of the GPCR superfamily can evolve divergent ligand binding pockets for 102 recognition of the same agonist. 103 104 RESULTS 105 106 Two remote amine recognition sites in the cadaverine receptor 107 108 To gain structural insights into diamine recognition by the cadaverine receptor TAAR13c, we 109 generated a TAAR13c homology model (Figure 1A) based on the X-ray crystal structure of 19 110 agonist-bound β2 adrenergic receptor (Protein Data Bank Entry 4LDE) . The β2 adrenergic 111 receptor is the closest homolog (33% amino acid identity) of TAAR13c for which an active-state 112 structure is currently available. The homology model of TAAR13c indicated a canonical fold of 5 113 seven transmembrane α-helices with ligand bound at the core. Using the homology model, we 114 performed computational docking of cadaverine to the orthosteric binding site. One cadaverine 115 amino group of the docked ligand was located near Asp1123.32, which corresponds to the highly 116 conserved amine-contact site in biogenic amine receptors. The other cadaverine amino group 117 was positioned at the opposing end of the ligand-binding pocket, 3 Å away from another 118 aspartate, Asp2025.42. The second amino group was also within 5 Å of side chains from 119 Thr2035.43 and Ser2766.55, Leu192 and Phe194 on extracellular loop 2, and the backbone of 120 Ser1995.39 (additional nearby residues are depicted in Figure 1-figure supplement 1). Based on 121 its proximity and charge, Asp2025.42 was deemed a prime candidate to function as a counterion 122 that forms a salt bridge to the second cadaverine amino group. Position 5.42 in several other 123 GPCRs directly contacts ligands through hydrogen bonds, salt bridges, or van der Waals 124 interactions3, and our homology model suggests a role for this amino acid in ligand recognition 125 by TAAR13c. 126 127 We used an established reporter gene assay7,11 to query the response properties of TAAR13c 128 variants with charge-neutralizing mutations at Asp3.32 (TAAR13cD112A) and Asp5.42 129 (TAAR13cD202A). HEK-293 cells were co-transfected with plasmids encoding TAARs and a 130 cAMP-dependent reporter gene encoding secreted alkaline phosphatase (Cre-Seap). 131 Transfected cells were exposed to test ligands, and assayed for reporter activity using a 132 fluorescent phosphatase substrate. As previously published with this paradigm12, cadaverine 133 robustly activated TAAR13c, with a half maximal response occurring at ~10 μM. In contrast, 134 cadaverine failed to activate either the TAAR13cD112A or TAAR13cD202A mutant at any 135 concentration tested, up to 1 mM (Figure 1, Figure 1-figure supplement 1). 136 6 137 To ensure that mutation of Asp2025.42 did not impair protein folding or G protein coupling, we 138 asked whether the mutant receptor could be activated by other ligands. We tested 50 chemicals 139 for the ability to activate TAAR13cD202A, and identified the monoamine 3-methoxytyramine as an 140 agonist (Figure 1D). 3-methoxytyramine failed to activate wild type TAAR13c at any 141 concentration tested.