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, 100 (6), 3035-9

Is the Olfactory Receptor a Metalloprotein?

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Is the Olfactory Receptor a Metalloprotein?

Jiangyun Wang et al. Proc Natl Acad Sci U S A.

Abstract

The sense of smell is arguably our most primal faculty and also the least understood. Even our own olfactorily impaired species is capable of detecting approximately 10,000 distinct scents [Buck, L. & Axel, R. (1991) Cell 65, 175-187]. To achieve that amazing diversity, mammals have approximately 1,000 olfactory genes, which accounts for approximately 3% of their entire genome [Mombaerts, P. (1999) Science 286, 707-711]. The olfactory receptors (ORs) are believed to be seven-helix transmembrane proteins, with an odorant-binding site on the periplasmic domain and a G protein-binding site on the cytoplasmic domain. Odorants first bind to an OR, which then undergoes some structural change that triggers the G protein activation and the following cascade of events leading to nerve cell activity. The structural details of ORs, however, remain to be determined. In this paper, we will describe a hypothesis in which metal ions play an important role for odorant recognition. We analyze the predicted structure and consensus sequence of the ORs and propose a metal-binding site in the loop between fourth and fifth helix (4-5 loop). We have prepared synthetically a pentapeptide that contains this putative binding site and find that it not only has high affinity for binding Cu(II) and Zn(II) ions, but that it also undergoes a dramatic transition to an alpha-helical structure upon metal ion binding. Based on these observations, we propose a "shuttlecock" mechanism for the possible structural change in ORs upon odorant binding. This mechanism involves membrane penetration of the 4-5 loop after residue charge neutralization by metal ion binding.

Figures

Figure 1
Figure 1
Human olfactory thresholds. Shown are gas phase concentrations at threshold of detection (3); vapors of liquids have been normalized (5) by analyte vapor pressure at 298 K (6).
Figure 2
Figure 2
(A) Secondary structure prediction of hOR o2d2 using the HMM for the native sequence. At the top, the thick bars indicate predicted TM helices and the higher and lower thin bars designate periplasmic and cytoplasmic loops, respectively. (B) Secondary structure prediction of hOR o2d2 using HMM for the charge-neutralized structure (as modeled by Glu-180→Val mutation). (C) Secondary structure prediction with amino acid aligned to the TM helix and loop regions. Coding: hydrophobic residues in black, polar residue in green, positively charged in blue, and negatively charged in red. The consensus metal ion-binding site (conserved in nearly three-quarters of known sequences) is highlighted in orange on blue.
Figure 3
Figure 3
Far-UV CD spectrum of HAKCE peptide, in the absence of metal ions (solid line) and in the presence of 1.0 equivalent of Cu(II) added (dotted line); 20 mM peptide, pH 7.4 potassium phosphate buffer, 298 K.
Figure 4
Figure 4
The homology models of the hOR o2d2 are constructed by using the program MODELER and BIOPOLYMER in INSIGHT-II (Accelrys). (Left) A model of the nonmetalated apoprotein; the predicted hOR transmembrane helix is aligned to the transmembrane helix of bovine rhodopsin (PDB ID code 1F88). (Right) Proposed structure after binding a metal ion (e.g., Cu2+ or Zn2+); the alignment shifts to reflect the speculated 4–5 loop insertion and ejection of the fourth helix. (Inset) Predicted coordination geometry to the metal ion from the glutamate, histidine, and cysteine residues of the consensus sequence HXXC[DE] in the 4–5 loop.
Figure 5
Figure 5
The proposed mechanism of olfactory response via a transmembrane shuttlecock. In the absence of odorant binding, the stable state is the embedded loop conformation (Upper Right). Upon odorant binding, the primary structural response is loop ejection (Lower Left). The G protein activation is a kinetic phenomenon in this mechanism; full equilibration of the OR with odorant leads to no further G protein activation, which may, in part, account for the process of olfactory adaptation (i.e., loss of response after initial exposure to a constant odorant concentration).

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