Test of the Binding Threshold Hypothesis for olfactory receptors: Explanation of the differential binding of ketones to the mouse and human orthologs of olfactory receptor 912-93

There continues to be uncertainty as to what aspect of an odorant is detected by an olfactory receptor (OR). Early models assumed that the shape of the odorant was most important (Amoore 1952). Experimental results reported by Buck and Axel (1991) showed that each OR recognizes several odors and each odor is recognized by several ORs. This raises the issue of what aspects of the molecule can provide this selectivity. Turin (1996) made the novel proposal that ORs detect the intramolecular vibrations of odors. But recently Keller and Vosshall (2004) provided three distinct experimental tests of the Turin model, all of which gave no evidence to support it. This leaves unresolved exactly what it is about an odorant that activates an OR. We recently reported first-principles computations that examine details of odorant binding to ORs (Floriano et al. 2000, 2004a; Hall et al. 2004). All results obtained thus far Reprint requests to: William A. Goddard III, Materials and Process Simulation Center (MC 139-74), California Institute of Technology, Pasadena, CA 91125, USA; e-mail: ; fax: (626) 585- 0918. Article and publication date are at 10.1110/ps.. Protein Science (2005), 14:703–710. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society 703 are consistent with the hypothesis that the binding strength of each odorant dominates the activation profile of each OR, and that each OR has some energy threshold, EBindThresh , below which there is no activation. This leads to The Binding Threshold Hypothesis (BTH) for activation of olfactory receptors (ORs): To activate an OR, the odorant must bind to the OR with binding energy above some threshold, EBindThresh . To further test the BTH we report here results for the binding of ketones to the mouse and human orthologs of OR 912-93 (denoted mOR912-93 and hOR912-93, respectively). mOR912-93 is activated by ketones while hOR912-93 is not (Gaillard et al. 2002). Background on olfaction In the last two decades there have been tremendous advances in understanding olfaction. The detection of odorants in human and mouse is mediated by hundreds of ORs belonging to the large superfamily of seven-helical transmembrane (TM) G protein-coupled receptors (GPCRs) (Buck and Axel 1991; Mombaerts 1999). There are ∼2.5 times as many functional OR genes in mouse (∼873) (Godfrey et al. 2004) as in human (∼347) (Malnic et al. 2004), as humans possess a significantly higher percentage of OR pseudogenes (Zhang and Firestein 2002), possibly explaining the less refined olfactory acuity in human. To obtain information about the molecular basis of olfaction, experimental studies of structure recognition of odorants by ORs have been conducted by several research groups (Araneda et al. 2000; Krautwurst et al. 1998; Zhao et al. 1998; Kajiya et al. 2001; Bozza et al. 2002). In particular, systematic experimental studies exposing a variety of small molecules to the mouse and rat I7 ORs provide information on which structural changes in the odorant molecule affect activation of ORs (Krautwurst et al. 1998; Arnaeda et al. 2000; Bozza et al. 2002). The 3D protein structures of these systems were predicted from first principles (MembStruk) by Hall et al. (2004). Hall et al. also used first-principles methods (HierDock) to predict binding sites and binding energies for the 56 odorants studied by Bozza, and obtained activation profiles in excellent agreement with experiment. Malnic et al. (1999) demonstrated that mouse ORs can be activated by multiple odorants and that each odorant elicits a response from a variety of different ORs. Floriano et al. (2000, 2004a) used MembStruk to predict the 3D protein structures of six of these mouse ORs, and used HierDock to predict binding sites and binding energies for the 24 odorants studied by Malnic. They also found activation profiles in excellent agreement with experiment. These computational studies found that different odorants bind to the same OR in the same binding regions, but sometimes with different binding conformations (Floriano et al. 2000, 2004a). These predictions could be directly tested by mutation experiments on ORs, but no such experiments have been reported. Gaillard et al. (2002) studied the closely related mouse and human orthologs of OR912-93 to gain an understanding of how protein sequence affects the function of these ORs. hOR912-93 has a single nonsense point mutation in the region corresponding to the N terminus of the protein (Rouquier et al. 1998), but mOR912-93 does not contain such a mutation (Rouquier et al. 1999). More recently it was found that correcting the nonsense mutation in hOR912-93 does not restore function (Gaillard et al. 2002). Exposure to straight-chain ketone odorants with 4–10 carbons and a carbonyl group in the second or third position results in a rise in intracellular [Ca+2] for mouse. No such response is observed in human, even after correcting the nonsense mutation. Gaillard et al. (2002) suggested that this indicates that pseudogenes may not be the sole reason for the relatively poor sense of smell in humans. Rather, the presence of other deleterious mutations in the human olfactory subgenome may have weakened the combinatorial code of human odor receptors over the course of evolution. To determine whether this is the case it would be useful to understand which mutations cause hOR912-93 to become inactive to ketones. To answer such questions we applied the MembStruk first-pri