(Received for publication, January 18, 1996; and in revised form, February 12, 1996)
From the
In the present study we examine the role of transmembrane
aromatic residues of the -opioid receptor in ligand recognition.
Three-dimensional computer modeling of the receptor allowed to identify
an aromatic pocket within the helices bundle which spans transmembrane
domains (Tms) III to VII and consists of tyrosine, phenylalanine, and
tryptophan residues. Their contribution to opioid binding was assessed
by single amino acid replacement: Y129F and Y129A (Tm III), W173A (Tm
IV), F218A and F222A (Tm V), W274A (Tm VI), and Y308F (Tm VII).
Scatchard analysis shows that mutant receptors, transfected into COS
cells, are expressed at levels comparable with that of the wild-type
receptor. Binding properties of a set of representative opioids were
examined. Mutations at position 129 most dramatically affected the
binding of all tested ligands (up to 430-fold decrease of deltorphin II
binding at Y129A), with distinct implication of the hydroxyl group and
the aromatic ring, depending on the ligand under study. Affinity of
most ligands was also reduced at Y308F mutant (up to 10-fold).
Tryptophan residues seemed implicated in the recognition of specific
ligand classes, with reduced binding for endogenous peptides at W173A
mutant (up to 40-fold) and for nonselective alkaloids at W274A mutant
(up to 65-fold). Phenylalanine residues in Tm V appeared poorly
involved in opioid binding as compared with other aromatic amino acids
examined. Generally, the binding of highly selective
ligands
(TIPP
, naltrindole, and BW373U86) was weakly modified by these
mutations. Noticeably, TIPP
binding was enhanced at W274A
receptor by 5-fold. Conclusions from our study are: (i) aromatic amino
acid residues identified by the model contribute to ligand recognition,
with a preponderant role of Y129; (ii) these residues, which are
conserved across opioid receptor subtypes, may be part of a general
opioid binding domain; (iii) each ligand-receptor interaction is
unique, as demonstrated by the specific binding pattern observed for
each tested opioid compound.
Opiates are strong analgesic compounds currently used in the
treatment of pain. They also have euphoriant action and strong
addictive properties. Opiates, as well as endogenous opioid peptides,
exert their biological action through three classes of membrane
receptors, known as µ, , and
. The recent cloning of
three genes encoding these receptors (for a review, see (1) )
has shown that opioid receptors belong to the G protein-coupled
receptors (GPRs) (
)superfamily with a seven transmembrane
domain topology and share high protein sequence identity, particularly
in transmembrane and intracellular regions. The availability of opioid
receptor clones allows to examine the mechanisms of action of opioid
ligands at the molecular level, and here we investigate the structural
basis of receptor-opiate interactions in the mouse
-opioid
receptor (mDOR, see (2) and (3) ).
It is believed that the seven putative transmembrane domains of GPRs are folded as helices and tightly associated within the membrane to form the receptor protein core. The binding site of GPRs which recognize small biogenic amines has been best characterized(4) . Three-dimensional computer modeling relying on bacteriorhodopsin structure as a scaffold and the analysis of amino acid conservation in sequence alignments were used in conjunction with site-directed mutagenesis data to identify residues which interact with the small nonpeptidic ligands. The binding site was shown to lie within the helical bundle, about 15 Å from the extracellular surface and spanning Tms II to VI, with a central role for Tm III. Other GPRs bind peptide ligands with sizes ranging from tripeptides (e.g. N-formyl peptide or thyrotropin-releasing hormone) to proteins (follicle-stimulating, luteinizing, and thyroid-stimulating hormones). Mutagenesis experiments demonstrated that, in addition to transmembrane residues, extracellular domains participate in ligand recognition (for reviews, see (5) and (6) ), indicating that the peptide binding site extends beyond the transmembrane pocket for these receptors.
Opioid
receptors are GPR family members endowed with an extremely large ligand
repertoire(7) . They bind a wide variety of structurally
diverse molecules, including small rigid alkaloid compounds, synthetic
peptides, as well as a family of endogenous peptides. Opioid receptors
are therefore expected to interact with their ligands at multiple
sites, both extracellular and intramembranous, an assumption which is
supported by initial point mutagenesis and chimeric
studies(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
We have focused on the role of transmembrane domains in the
receptor. In a previous study, we have demonstrated that the conserved
Asp residue in Tm III, although not interacting directly with the
positive charge of opioid ligands, is critically involved in
maintaining intact binding properties of the receptor(19) .
Other reports have shown implication of charged residues found in Tm II
and Tm VI in opioid recognition(8, 9) . In this study,
we have used our knowledge from studies of other GPRs and receptor
modeling to establish a putative topology of the receptor binding site.
We have identified a number of aromatic amino acid residues as
candidates for opiate recognition and we have examined the contribution
of their side chains to the binding of a large set of structurally
distinct opiate molecules, using a site-directed mutagenesis approach.
We provide evidence that a cluster of aromatic residues may represent a
major area of interaction for most ligands. In addition our results
demonstrate a distinct mode of interaction for each ligand under
investigation, ruling out the possibility of a unique opioid binding
site.
Opioids are cationic compounds with strong hydrophobic
character. Structure-function studies have shown that a positively
charged nitrogen atom located at a fixed distance from a phenolic ring,
a common structure between alkaloids and opioid peptides, represents
the active or ``message'' part of opioid molecules (see (22) ). The positive charge being an absolute requirement for
ligand binding, the existence of a salt bridge has long been postulated
to be the major interaction between the ligand and its receptor site.
In preliminary work(19) , we tested the hypothesis that this
interaction occurs at Asp in mDOR. This negatively
charged residue is present in Tm III of all GPRs that bind small
cationic neurotransmitters and is known to interact directly with
catecholamines in the
2-adrenergic receptor. Our results showed
that, although this Asp residue is a structural component of the
binding site, it does not interact directly with opioid ligands.
Another study has shown that Asp
(Tm II), the only other
anionic residue present in transmembrane domains, does not represent a
common attachment point for opioids(8) . The lack of evidence
for a main electrostatic interaction between opioids and the
receptor strengthens the hypothesis that non-ionic interactions are
predominantly responsible for ligand chelation in the binding site.
Figure 1:
Positioning of investigated aromatic
residues in a model of the mouse -opioid receptor. A view is shown
from the membrane (A) and from the extracellular space (B). The seven-transmembrane helical backbone is shown in blue and side chains of Tyr
(Tm III),
Trp
(Tm IV), Phe
(Tm V), Phe
(Tm V), Trp
(Tm VI), and Tyr
(Tm VII)
residues in yellow. The side chain of the critical D128
residue in Tm III (19) is represented in red. Residues
found at position 173, 218, 222, 274, and 308 are clustered in the
central pore of the helical bundle, where they seem to form an aromatic
pocket.
Residue Tyr, which is adjacent to the critical
Asp 128 residue, does not face the binding pocket in this model, but
rather participates in reinforcing helical III intrinsic stability
through aromatic-aromatic interactions occurring between
Tyr
, Phe
, and Phe
.
Interestingly, in another model of mDOR derived from bovine opsin
crystallographic data, (
)the side chain of Tyr
seems more likely to be located inside the pore due to a
different position of Tm III relative to adjacent helices. In addition,
this tyrosine residue is present in muscarinic m3 and TRH receptors and
was shown to play a critical role in ligand
recognition(23, 24) . We therefore have included
examination of the role of Tyr
in our study despite its
outside positioning in our model.
Figure 2: Site-directed mutagenesis of aromatic amino acid residues of mDOR. A schematic representation of the putative seven transmembrane domain topology of the receptor is shown and residues mutated in this study highlighted. The single-letter amino acid code is used. The numbers next to mutated amino acids refer to their positions within the mDOR sequence.
Figure 3:
Structure of opioid ligands under study.
Amino acid sequences of peptide ligands and chemical structures of
alkaloids are shown. TIPP, TIPP, naltrindole, naloxone, and
diprenorphine are antagonists. Altogether these compounds constitute a
representative set of opioid ligands.
Figure 4:
Binding profile of mutant receptors. The
abbreviations used are: -end,
-endorphin; Dyn
A, dynorphin A, Leu-enk and Met-enk, Leu- and
Met-enkephalin, respectively; Delt II, deltorphin II; BW, BW373U86; NTI, naltrindole; Brem,
bremazocine; and Nalox, naloxone. Histograms represent effect
of the mutation on ligand binding with black bars showing the
ratio of K
values at mutant and WT
receptors. Value 1 means that the mutation does not modify
ligand binding. Results are presented using the same scale for all
mutants to underscore the relative contribution of each amino acid
residue under study.
Three-dimensional modeling has allowed us to identify
transmembrane aromatic amino acid residues as possible candidates for
opioid binding at the receptor, and we have investigated their
role by site-directed mutagenesis. It is important to note that the
mutations under study generally affect the binding of some, but not all
investigated opioid ligands. We can therefore assume that structural
modifications, that would dramatically change the general conformation
of the receptor binding site, do not take place in the mutated
receptors. Consequently, our results indicate that amino acid residues
Tyr
, Trp
, Phe
,
Phe
, Trp
, and Tyr
indeed
participate in ligand recognition. Whether alterations of ligand
binding observed in this study arise from the disruption of direct
ligand-receptor interactions or rather from subtle effects on helical
packing is not known.
Figure 5:
Binding fingerprint of each investigated
ligand on a schematic representation of the -opioid receptor
binding site. Scheme of the receptor derives from the top view of the
three-dimensional model (Fig. 1B), and the side chains
of mutated amino acid residues are colored. Each color represents the
extend of affinity decrease due to the mutation. Results from the
previously studied D128N mDOR mutant receptor (19) are also
represented in the figure.
The significant contribution of Tyr in ligand binding
seems to be in contradiction with its outward orientation in the
proposed model (see Fig. 1B). One hypothesis may be
that the possible implication of Tyr
in aromatic stacking
suggested by the model (see ``Results'') is important to
maintain helix III conformation and that Tyr
plays a
structural role. This interpretation, however, is not consistent with
the observation that the aromatic ring of Tyr
does not
seem to be involved in the binding of some ligands (
-endorphin,
dynorphin A, TIPP
, BW373U86, and naltrindole), which rather
interact with the OH group of the tyrosine residue. A more likely
possibility to explain the decreased binding potency of opioid ligands
at Y129F and Y129A mutants is that the amino acid side chain faces the
inner core of the helices bundle and act as an anchor point for these
ligands. Refinement of the model is now required to assess this
hypothesis. In particular, revision of the alignment procedure and
consideration of mutagenesis data on
2 receptor may lead to
repositioning of helix III.
Although less drastically involved in
receptor binding properties, Tyr (Tm VII) contributes
significantly to the binding of most ligands tested in this study. This
residue is also found in TRH receptor, bradykinin receptor, and in
amine binding GPRs and was shown important for agonist and antagonist
binding in m3 muscarinic receptor (23) . Also a histidine
residue is present at homologous position in adenosine A1 receptor and
mutation into leucine demonstrated its critical role in ligand
recognition(25) . Finally, position 308 in mDOR corresponds to
that of a lysine residue in the opsin family, where it represents the
attachment point of retinal, the covalently bound ligand of this
receptor family. Thus, the OH group of Tyr
, which seems
to be generally involved in opioid binding at the
receptor,
appears to play a role similar to that of homologous residues in other
GPRs.
Mutations of phenylalanine residues of Tm V poorly affect
ligand binding compared with other mutations examined in this study.
Particularly, F218A represents the only mutation in this study that
barely affects ligand binding. A phenylalanine residue is also present
at this position in other GPRs. At present a role for this amino acid
has not been documented, but the involvement of a serine residue, which
is found at homologous position in receptors that bind small biogenic
amines (Ser in
2-adrenergic, Ser
in D1
dopamine, Ser
in
2-adrenergic), has been clearly
demonstrated. The serine amino acid residue was suggested to interact
with the catechol moiety of the ligand, both from three-dimensional
modeling studies (4) and mutagenesis experiments where a
25-fold affinity decrease is described following serine to alanine
substitution (29, 30) . Our results indicate that
Phe
in the
receptor does not participate to ligand
binding to the same extent as the homologous serine residue in
catecholamine receptors. Modification of Phe
into alanine
alters binding potency of a larger number of ligands compared with
F218A mutation. Taken together, results from mutations of the two
phenylalanine residues in Tm V suggest that Phe
is
located closer to the binding site than Phe
, which is
consistent with our model (Fig. 1A). Furthermore the
overall weak effect of both F218A and F222A mutations, relative to the
important alterations of ligand binding arising from modifications at
Tms III, IV, and VI, indicates that opioid ligands interact weakly with
Tm V and bind more centrally relative to the putative binding pocket.
Our study does not provide evidence for the existence of distinct agonist and antagonist binding sites in the region embedded between Tms III and VII, that we have investigated. Each amino acid substitution affects the binding of at least one agonist and one antagonist, suggesting largely overlapping sites for both ligand types in the transmembrane aromatic domain.
Altogether these observations suggest
that the set of aromatic residues proposed by our model is not involved
in agonist/antagonist- or selectivity, but rather constitute a
general binding domain for opioids. Consistent with this idea is the
fact that all six aromatic amino acids, identified by three-dimensional
modeling as potential key residues, are conserved across
-, µ-
and
-opioid receptors. We might therefore expect these residues to
play similar roles in µ and
receptors, and further
mutagenesis experiments may confirm this assumption. Finally, the
suggestion that transmembrane aromatic residues examined here are part
of a general binding domain raise the possibility that these residues
interact with the ``message'' part of opioid
molecules(22) , which is common to all tested compounds.
Further ligand-docking studies on a refined three-dimensional model
should provide precise indications in support of this hypothesis.