(Received for publication, November 10, 1995; and in revised form, February 2, 1996)
From the
Very limited structural information is available concerning the
superfamily of G-protein-coupled receptors with their
seven-transmembrane segments. Recently a non-peptide antagonist site
was structurally and functionally replaced by a metal ion site in the
tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the
-opioid receptor by substituting two residues at the outer portion
of transmembrane V (TM-V), Asp
and Lys
, and
one residue at the top of TM-VI, Ala
, with histidyl
residues. The histidyl residues had no direct effect on the binding of
either the non-peptide antagonist
[
H]diprenorphine or the non-peptide agonist,
[
H]CI977, just as these mutations/substitutions
did not affect the apparent affinity of a series of other peptide and
non-peptide ligands when tested in competition binding experiments.
However, zinc ions in a dose-dependent manner prevented binding of both
agonist and antagonist ligands with an apparent affinity for the metal
ion, which gradually was built up to 10
M.
This represents an increase in affinity for the metal ion of about
1000-fold as compared with the wild-type
receptor and is specific
for Zn(II) as the affinity for e.g. Cu(II) was almost
unaffected. The direct transfer of this high affinity metal ion switch
between two only distantly related receptors indicates a common overall
arrangement of the seven-helix bundle among receptors of the rhodopsin
family.
G-protein-coupled receptors with their seven-transmembrane
segments (7TM) ()constitute a true superfamily of membrane
proteins with hundreds of individual members. Among the three major
families of 7TM receptors, the rhodopsin-like family is by far the
quantitatively dominating. All types of chemical messengers act on
these receptors, and they also serve as sensory molecules in our
olfactory system and obviously in the eye. However, our knowledge about
their molecular structure is quite limited. Rhodopsin has recently been
characterized by cryoelectron microscopy, and a seven-helix bundle was
observed(1, 2) . Yet, their resolution does not allow
for firm identification of which helix corresponds to which electron
density. Thus, in fact we do not even yet know the helical connectivity
for certain or whether the helical bundle is arranged in a clockwise or
anti-clockwise manner, even though the general model proposed by
Baldwin (3) has become widely accepted.
Although the mutations that affect ligand binding in different 7TM receptors appear to cluster on certain faces of the helices(4) , we do not have any hard evidence that the many different receptors adopt a similar conformation. Ligand binding sites as such have not been transferred between receptors, although chimeric constructs and single substitutions in some cases have conveyed an increased affinity for a particular ligand(5, 6, 7, 8) .
Recently we introduced a high affinity zinc-binding site in the
tachykinin NK1 receptor(9) . This site was introduced by
systematically replacing residues in the binding site for the prototype
non-peptide antagonist, CP96,345, with His residues. In the final
construct, the zinc ion appeared to be coordinated with a submicromolar
affinity by two histidyl residues placed at the top of TM-V and one at
the top of TM-VI(9) . In the present study we have attempted to
move this well defined zinc-binding site to the -opioid receptor,
which is only 30% identical to the NK-1 receptor in the transmembrane
domains. Amino acid residues located at the three equivalent positions
at the top of TM-V and -VI were mutated to histidyl residues in the
-opioid receptor. The 1000-fold increase in zinc affinity as
determined by the ability of the metal ion to inhibit the binding of
both radiolabeled opioid agonist and radiolabeled opioid antagonist
supported the notion that the helical arrangement is similar among 7TM
rhodopsin-like receptors.
Figure 3:
Model
of the D223H/K227H/A298H -opioid receptor. The receptor model was
built using rhodopsin as template with the connectivity of the
transmembrane as suggested by Baldwin(3) . The three-histidine
side chain (in blue) coordinating the zinc ion (in red) was orientated in accordance with a tetrahedral geometry
of metal ion chelation. The tetrahedral mode of coordination was most
optimal with the NE2 nitrogen as the chelating ligand in all of the
three histidine imidazole rings as also seen for most other
Zn(II)-binding sites(14) . The distances between the NE2
nitrogen and the zinc ion was close to 2.0 Å for all three
chelating ligands in accordance with observed distances in structurally
resolved metal-binding sites(14) .
The His residues were introduced in three stages in the
receptor at positions corresponding to those previously identified in
the NK-1 receptor (Fig. 1). First, a His residue was introduced
at position 24 in TM-VI, A298H (concerning the generic
nomenclature-numbering system for 7TM receptors; see Fig. 1and (4) ). Second, a bis-His site was introduced at positions 1 and
5 in TM-V, D223H/K227H. Finally, the His residues in the two constructs
were combined in the triple His construct, D223H/K227H/A298H.
Figure 1:
Diagrams of the -opioid receptor
with the mutated amino acids in white on a black background. The so called ``fingerprint'' amino acids
conserved in most G-protein-coupled receptors are indicated in black on a gray background(4) . At the top is
shown a helical wheel diagram based on the connectivity of the helices
as suggested by Baldwin(3) . A standard serpentine diagram is
shown at the bottom with the putative seven-transmembrane
helices.
As
shown in Table 1, although two of the substitutions could be
considered to be rather dramatic alterations of the side chain in both
size and charge, His for Ala and His for Asp, the introduction of the
three His residues only had a minor effect on the binding affinity of
both peptide agonists, dynorphin A and DAKLI, and non-peptide
antagonists, naloxone and nor-BNI. In fact, the affinity of the
non-peptide agonist CI977 was improved 6-7-fold in these
constructs compared with the wild-type receptor (Table 1).
The affinity for Zn(II) was monitored indirectly through its effect
on the binding of radiolabeled antagonist,
[H]DIP, and agonist,
[
H]CI977, respectively. The apparent affinity for
Zn(II) on the wild-type
receptor was 1.2
10
M, when using [
H]DIP as
radioligand ( Table 1and Fig. 2A). Introduction
of the single His residue at position VI:24, A298H, increased the
affinity for zinc ions 8-fold whereas in the construct with the bis-His
substituents at the top of TM-V the affinity for Zn(II) was increased
160-fold to 7.5
10
M. When all
three His residues were combined the affinity for Zn(II) was 1.2
10
M corresponding to a 1,000-fold
increase in apparent affinity ( Table 1and Fig. 2A). In the wild-type receptor and in the mono-
and bis-His constructs the results, obtained with the radiolabeled
agonist, [
H]CI977, were very similar to the
results obtained with the radiolabeled antagonist,
[
H]DIP ( Table 1and Fig. 2B). However, in the mutant receptor with the
triple-His construct the competition curve for Zn(II) was not
monophasic when using the radiolabeled agonist,
[
H]CI977. Analysis of the competition curve using
a two-site model gave a distribution of binding between a high affinity
site with an IC
of 0.6
10
M and a low affinity site with an IC
of 310
10
M.
Figure 2:
Gain of Zn(II) binding following
introduction of His residues at positions 223, 227, and 298. A, competition binding with ZnCl using
radiolabeled antagonist [
H]DIP in wild-type
-opioid receptor, A298H
, D223H/K227H
, and
D223H/K227H/A298H
. B, competition binding with
ZnCl
using radiolabeled agonist
[
H]CI977. Whole cell binding experiments were
performed in transiently transfected COS-7
cells.
We next used the information from
this study to construct a molecular model of the -opioid receptor
based on the rhodopsin template(1, 3) (Fig. 3). The orientation of transmembranes V and VI
could now be assigned to a more detailed level. Using the known
geometries of other structurally resolved metal-binding
sites(14, 15) the imidazole side chains were fixed at
preferential angles based on a rotamer library for preferential amino
acid side chain angles (16) to accommodate the binding of a
zinc ion in a tetrahedral coordination. This coordination of the zinc
ion was chosen as it is its usual mode of being chelated by histidyl
residues(14, 17) . The fourth chelating ligand is
expected to be a water molecule as observed in other systems (18) . The degrees of freedom for this system are limited, and
it was found that only one other arrangement of the two helices could
accommodate the binding of the zinc ion. A vertical movement of the two
histidines in TM-V four amino acids down along the C
scaffold
could thus also result in a histidyl arrangement enabling a tetrahedral
chelating metal-binding site. However, we do not favor this possibility
as this would turn Phe and Ala in position V:09 and V:12 away from the
hydrophilic binding pocket and toward the lipid bilayer by 40°.
These two amino acids correspond to the two serines in the
-adrenergic receptor, which have been proposed to
interact with monoamine ligands(19) . Therefore we chose the
orientation of TM-V and TM-VI as shown in Fig. 1and Fig. 3.
The transfer of the triple-His zinc site from the NK-1
receptor to the -opioid receptor, which results in similar, high
affinity metal ion affinity in both receptors, indicates that the
relative orientation of the seven-helix bundle or at least of TM-V and
-VI is similar in these two proteins. Obviously we cannot exclude the
possibility that the presence of the metal ion is able to distort two
rather dissimilar receptor structures to form two equally high affinity
complexes. However, we find it more likely that the almost identical,
high affinity for zinc ions introduced by the three His residues in the
two different receptors indicate that these residues are placed
relatively similarly in relation to each other and in a rather similar
environment in the two proteins. Since only 30% of the residues are
identical in the NK-1 and the
receptor in the transmembrane
domains, the direct transfer of this zinc site supports the notion that
despite the low degree of primary sequence conservation, rhodopsin-like
receptors do share a common seven-helix bundle structure in the
membrane. The high affinity coordination of a metal ion in a 7TM
receptor and its ability to prevent ligand binding raises both
structural and functional issues.
Construction of metal-binding sites has been used extensively in the last few years in protein engineering, for example in de novo designed proteins(22, 23) , in modulating the enzymatic activity of proteins by metal ion switches(24) , and in modulating the activity of monoclonal antibodies(25, 26) . However, this has mainly been performed in proteins where a high resolution x-ray structure was available or in de novo designed proteins. Not until recently have metal ion sites started to be used in the structural and functional analysis of membrane proteins where basically no structural information is available(9, 27) . In the lactose permease a bis-His Mn(II) site was introduced on the basis of site-directed excimer fluorescence information of the spatial proximity of the two side chains in TM-VIII and -X(27) . In our case the mutational mapping of residues in the presumed binding pocket for a non-peptide antagonist was the starting point(8, 9, 28, 29) . Also it has recently been shown that introducing cysteine residues at these positions in rhodopsin can enable the formation of disulfide bridges(30) .
The introduction of the histidyl
residues between TM-V and TM-VI in the -opioid receptor could be
considered to be a rather dramatic alteration of the presumed binding
pocket. However, from previous studies (33, 34, 35, 36) it could be
anticipated that the histidyl residues introduced at the top of TM-V
and TM-VI might only have a minimal effect on the binding of the
ligands, and this was also found to be true (Table 1). For
instance, using chimeric receptors between the
- and
-opioid
receptor, it has been found that the selectivity of non-peptide
agonist is mainly achieved by interactions with structural elements
within TM-I to TM-IV and the second extracellular loop(36) ,
whereas the C-terminal portion of TM-IV and the second extracellular
loop are important for peptide agonist
binding(33, 34, 36) . In contrast, the region
responsible for the selectivity of the nonpeptide antagonist, nor-BNI,
has been located in and around the third extracellular
loop(34, 35) . From these studies it appears that TM-V
and TM-VI are not directly involved in the binding of the various
ligands as also found in the study presented here (Table 1).
The unchanged affinity for both agonist and antagonist binding in
the triple-His construct and the small size of the metal ion, in our
opinion, support an allosteric mechanism for the function of the zinc
ions on the mutated receptors. Thus, Zn(II) may exclude the other
ligands from binding by constraining the receptor in a conformation
that excludes their binding, i.e. stabilize a non-permissive
conformation(9, 32) . It cannot be excluded that
Zn(II) prevents agonist and antagonist binding by a volume exclusion
effect, as suggested for the molecular function of non-peptide versus peptide ligands(37) . However, the small size
of the metal ion and the presumed localization of ligand-binding
sites to other positions within the receptor make this rather unlikely.
Interestingly, a clear biphasic competition curve is observed for
Zn(II) when [H]CI977, the agonist, is used as a
radioligand as opposed to the classical monophasic competition curve
found with [
H]DIP, the antagonist. This indicates
that there is an important difference in the binding mode between
antagonists and agonists. The two-component Zn(II) curve observed with
the agonist tracer suggests the occurrence of two different states of
the receptor in the presence of bound agonist, at least in the presence
of Zn(II). On the other hand the displacement curve for antagonist
tracer reflects a single high affinity zinc-binding site. It could then
be envisioned that the antagonist keeps the receptor in a single
stringent conformation.