Copper-mediated thiol potentiation and mutagenesis-guided modeling suggest a highly conserved copper-binding motif in human OR2M3

Abstract

Sulfur-containing compounds within a physiological relevant, natural odor space, such as the key food odorants, typically constitute the group of volatiles with the lowest odor thresholds. The observation that certain metals, such as copper, potentiate the smell of sulfur-containing, metal-coordinating odorants led to the hypothesis that their cognate receptors are metalloproteins. However, experimental evidence is sparse-so far, only one human odorant receptor, OR2T11, and a few mouse receptors, have been reported to be activated by sulfur-containing odorants in a copper-dependent way, while the activation of other receptors by sulfur-containing odorants did not depend on the presence of metals. Here we identified an evolutionary conserved putative copper interaction motif CC/CSSH, comprising two copper-binding sites in TMH5 and TMH6, together with the binding pocket for 3-mercapto-2-methylpentan-1-ol in the narrowly tuned human receptor OR2M3. To characterize the copper-binding motif, we combined homology modeling, docking studies, site-directed mutagenesis, and functional expression of recombinant ORs in a cell-based, real-time luminescence assay. Ligand activation of OR2M3 was potentiated in the presence of copper. This effect of copper was mimicked by ionic and colloidal silver. In two broadly tuned receptors, OR1A1 and OR2W1, which did not reveal a putative copper interaction motif, activation by their most potent, sulfur-containing key food odorants did not depend on the presence of copper. Our results suggest a highly conserved putative copper-binding motif to be necessary for a copper-modulated and thiol-specific function of members from three subfamilies of family 2 ORs.

Bitter Taste and Olfactory Receptors: Beyond Chemical Sensing in the Tongue and the Nose

“…Hence, strategies aiming at enhancing the sampling of the conformational space of the receptor-ligand complex have been applied to overcome this limitation. These include flexible docking approaches and experimental data-driven model refinement (Di Pizio et al 2017 ; Nowak et al 2018 ; Bushdid et al 2018 ; Bushdid et al 2019 ; Di Pizio et al 2020a , b ), as well as molecular dynamics (MD) simulations (Gelis et al 2012 ; Charlier et al 2012 ; Charlier et al 2013 ; Topin et al 2014 ; Marchiori et al 2013 ; Sandal et al 2015 ; Li et al 2016 ; Fierro et al 2017 ; Ahmed et al 2018 ; Fierro et al 2019 ; Alfonso-Prieto et al 2019a , 2019b ; Haag et al, 2020 ; Schneider et al 2020 ).…”

The Crossroads between Host Copper Metabolism and Influenza Infection

“…These factors regulate the expression of several dozens of genes, including genes for which the products take part in the reprogramming of energy metabolism in tumor cells. Moreover, copper is required for the induction of Golgi-complex-independent secretion of interleukins and cytokines [ 61 ] as well as for odorant receptor activation [ 62 ]. It has also been shown that copper controls organogenesis and cell differentiation in embryo development [ 63 ].…”

“…Thus, IAV RNA-transcriptase activity is inhibited by copper chelators in vitro and in vivo [ 174 ]. Additionally, IAV, similar to SARS-CoV-2, causes loss of smell [ 175 , 176 ], and the co-activators of the odorant receptors are copper ions [ 62 ]. Moreover, IAV uses cell-signaling cascades for its reproduction, including copper-dependent signaling pathways [ 177 , 178 ].…”

Conserved C‐terminal motifs in odorant receptors instruct their cell surface expression and cAMP signaling

“…C-terminal numbering starts after Y7.53. 59 Moreover, the specific constitution of these motifs is class-specific, for example, the dominant "[+]x[+]" motif at C-term 3-5 , which occurs at high rates in either class, is predominantly a "KxK" in class-I ORs, but a "RxK" or "RxR" in class-II ORs, and has been shown previously to be highly conserved within ORs. 31,40,41 As the variables plot in Figure 1 shows, the major contributors to the differentiation of class-I and class-II ORs are the class-I-specific "[+] x[+]" motif at C-term 8-10 , and the class-II-specific "[+][+]" motif at C-term 12-13 (Figure 1A).…”