Introduction:
Olfaction is a major neurosensory function by which mammals learn about
the external environment. The initial step involved in olfaction is the interaction of
the odorant with the olfactory receptors (ORs).
To recognize odors
corresponding to different odorants, a combinatorial approach has been
developed in mammals.
There are 100’s of different ORs in each mammal.
Each odorant binds to multiple ORs and each OR binds to multiple odorants to
generate different activation patterns for different odorants (Malnic, et. al., 1999,
Firestein, 2001). ORs make up the largest family of genes in the human genome
and belong to a superfamily of GTP-protein binding coupled receptors (GPCRs)
(Mombaerts, 1999). Most enzyme families have developed specificity towards
certain ligands and hence the binding sites in these families display a high
degree of conservation.
However, as the ORs have to recognize numerous
diverse odorants, a large repertoire of proteins has been developed with binding
site sequence variability (Pilpel & Lancet, 1999). Such variability in the sequence
profiles is also known to occur in immunoglobulins, T cell receptors and major
histocompatibility complex proteins.
It has also been hypothesized that the
odorant binding sites are present in transmembrane segments 3, 4 and 5 where
the highest sequence variability is found. It has been postulated that since most
good ligands for metal ions form better odorants (methylthiol binds to some OR >
1 million times stronger than the OR that corresponds to methanol), they must be
coordinated to a metal ion bound in an OR (Wang, et. al., 2003).
Knowledge about the three dimensional structural structure of ORs is
crucial for understanding how they function and also for drug design. However,
as membrane proteins are difficult to crystallize, it has been difficult to gain any
structural information about the ORs yet. GPCRs are membrane bound proteins
that consist of seven transmembrane helices. Here, we have used the structure
of bovine rhodopsin, a GPCR, and other homologous proteins such as
bacteriorhodopsin, sensory rhodopsin and halorhodopsin to generate models of
the structure for a olfactory receptor, O2D2. Previous reports in the literature
have used just one of the above structures to generate models for the olfactory
receptor but the presence of other templates could improve the model (Floriano,
et. al., 2000, Vaidehl, et. al., 2002).
Modeling Methods:
Prediction of Transmembrane helices:
Prediction of transmembrane helices of the olfactory receptor O2D2 was
done using TMHMM v. 2.0 (Sonnhammer, et. al., 1998). It was predicted to be a
seven helix bundle similar to GPCR as shown in Fig. 1A. It is interesting to note
that the putative metal binding domain is predicted to be in the loop between
helices 4 and 5. This is because of the presence of the charged residue E180 in
O2D2. However, upon metal binding, this region becomes neutral. On creating
a mutation E180V corresponding to the metal bound neutral residue, there are 8
putative transmembrane helices (Wang, et. al., 2003) as seen in Figure 1b. As
the templates used in this study have 7 transmembrane helices, it is expected
that the model will also have 7 transmembrane helices. The above analysis
indicated that the olfactory receptor might form an eight-helix bundle after metal
binding.
Alignment of O2D2 to rhodopsin:
The templates used for modeling were 1L9H (bovine rhodopsin) (Okada,
et. al., 2002), 1E12 (halorhodopsin) (Kolbe, et. al., 2000) and 1JGJ (sensory
rhodopsin) (Luecke, et. al., 2001). It has been suspected for the GPCRs, there
will not be much difference in the placement and angle of the transmembrane
helices and hence these are good templates for our modeling. These templates
were chosen on the basis of best resolution and least residues missing in the
structure. The structure of bacteriorhodopsin was not used in this study because
there were 20 residues missing in its high-resolution structures. The structural
alignment, in which all the structurally similar regions are aligned, of these three
structures was done using STAMP v.4.2 (Russel & Barton, 1992). Structural
alignment based methods are better than the sequence alignment based
methods. In Figure 2, we can see that the transmembrane helices of the three
structures align well though the helices in bovine rhodopsin are larger than those
in both the archaea rhodopsins.
Based on the alignment from STAMP, a profile for this family was created
using HMMER v.2.1 (Eddy, 1998). HMMER creates a hidden Markov model of
the sequence and can be used to search sequences from a database that belong
Figure 1A
Figure 1B
Figure 1: Secondary structure prediction of the human olfactory receptor O2D2 using TMHMM v. 2 for A.
the native sequence and B. the mutant E180V. On the top, the thick red bars indicate predicted
transmembrane helices, the thin blue lines indicate cytoplasmic region and the think pink line indicates
extracellular region.
Figure 2: top: The primary sequence and secondary structure alignment of structural alignment rhodopsin
family. Here, cylinders indicate helical regions in that structure and arrows indicate beta sheet region.
Bottom: The alignment of the structures in ribbon representation. Here green represents 1JGJ, brown
represents 1E12 and white represents 1L9H. Figure was developed using VMD (Humphrey, et. al., 1996).
to a particular family. To check how good this profile was, we searched for
proteins belonging to this class using this profile in the SWISSPROT database
(Boeckmann, et. al., 2003). The profile recognized more than 400 proteins (data
Figure 3: The profile of the O2D2 olfactory receptor family.
not shown) from the SWISSPROT database, which belonged to the GPCR
superfamily and had no false positives, which shows the profile was able to
differentiate between proteins belonging to the GPCR superfamily from other
superfamilies.
It has been reported that it is better to align a profile of the sequence to
the structural profile for GPCRs because the regions that are conserved over the
sequence profile give a stronger signal than the individual sequence. The profile
for O2D2 was made using CLUSTAL W from homologous non-redundant
sequence in the SWISSPROT database. In figure 3, we have the sequence
profile of O2D2.
Figure 4: The alignment in the profile of O2D2 family with the structural profile of the rhodpsin
family.
Before we carry out the modeling of O2D2, we have to make sure that the
alignment we get is correct. There are some conserved sequence motifs that
help to identify the position of the transmembrane segments in the GPCR
subfamilies (Mirzadegan, et. al., 2003). It is seen that the O2D2 subfamily also
has these elements (Pilpel, 1999, Zozulya, et. al., 2001, Fuchs, et. al., 2001) and
hence these motifs can be used to align the transmembrane segments in O2D2
Figure 5: A: Ribbon representation of the model structure of the olfactory receptor O2D2. B: The
space filling model of the model with polar residues shown in green, positively charged residues
in blue and negatively charged residues in red.
to those in the rhodopsin structures. We have used HMMER to align the O2D2
profile to the structural profile created from the rhodopsin structures. In helix 1 of
GPCR families, the highly conserved G20 and N21 (numbering starts form
beginning of helix) are also conserved in the O2D2 family – G41 and N42 (the
position refers to the alignment position of the family). Similarly in helix 2, D70 of
O2D2 aligns with the highly conserved D13 of helix 2 in the GPCR superfamily.
For helix 3, the DRY motif starting at 121 is highly conserved starting from 28 th
position of the 3rd helix. Also to be noted that C97 is conserved in all these
proteins because it is suspected that C97 forms a disulfide bond with C179
present in the previously reported extracellular loop.
In helix 4, the fully
conserved W158 of O2D2 was aligned to 11th position of the 4th helix. HMMER
aligned the first four helices satisfying the above criteria.
HMMER aligned the earlier reported extracellular loop between TM4 and
TM5 to TM5 of the rhodopsin family. However, as already stated above, it has
been suspected that O2D2 could be an eight-helix bundle when the HFFCE motif
is bound to metal ion forming the 5th helix. This would mean that the previously
reported TM5, TM6 and TM7 of O2D2 now form the 6th, 7th and 8th helices
respectively. On analysis of the alignment created by HMMer in Figure 4, it can
be seen that this is indeed the case. However, as the rhodopsin family has only 7
helices, there is no corresponding structural element for the 8th transmembrane
helix to align to with the rhodopsin family as shown in Figure 4.
Modeling of O2D2:
Modeling of O2D2 with the 3 templates was carried out using MODELLER
v.6.2 (Sali & Blundell, 1993) using restraints on the secondary structural
elements and also high refine level for ab initio structure prediction of loop
regions. The model of O2D2 that we obtained is shown in Figure 5A. The
space-filling model of the structure is shown in Figure 5B. It is analyzed to check
whether the residues are in the proper environment. In particular we check
whether any charged residues are exposed to the lipid. On similar analysis with
the rhodopsin family, it was observed that no charged residues are exposed to
the lipid bilayer towards the central region of the helix. Similarly, none of the
charged residues are exposed to the lipid in the middle of the membrane in the
model of O2D2.
Conclusions:
A 3-dimensional model of the structure of O2D2 was developed with three
templates. The loops and the C terminal region have to be refined further. It has
been believed that the olfactory receptors have 7 transmembrane helices.
However, our work suggests that O2D2 might have eight membrane spanning
domains. However, it could be that O2D2 starts of as a seven transmembrane
helix and then on binding the metal ion, the EC2 loop (2 nd extracellular loop)
enters in to the membrane in the place of TM4.
It has already been stated based on the variability profiles of sequence in
different regions of the protein that EC2 and TM3-5 are involved in odorant
recognition and binding and this model could be used for those studies (Pilpel &
Lancet, 1999). Hence the binding region of the olfactory receptors is very similar
to that of bacteriorhodopsin which also uses TM3-5 and EC2 for retinol binding. It
has also been shown that the amino acids involved in odorant binding for O2D2
are the ones which point towards each other as shown in Figure 6.
Future work would involve trying to model O2D2 with HFFCE in EC2 and
also in TM4 and TM5 positions with TM4 and TM5 as the loops on the
intracellular side. After this, a steered molecular dynamics study could help in
identifying how tightly the protein binds to the membrane and this will help in
knowing the stable conformation of O2D2 and hence some studies on the
function of the protein can be done after this.
Figure 6: 2-dimensional representation of the OR seven TM segments. Conserved residues are shown. In
addition, OR positions that align with ligand contact residues in other GPCRs are colored green; OR variable
positions that donot align with such residues are colored red. Circles are OR variable positions, while
squares are OR conserved positions.The conserved regions are shown in squares and the variable regions
in green.
Experimental work could involve knowing the exact role of EC2 in the
functioning of the protein and to see whether metal ions bind to the olfactory
receptor both in proteins such as wildtype O2D2 and mutant O2D2 with the
mutation E180V. Further, the exact position of the transmembrane helices could
help in improving the model. This can be done by labeling studies.
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