Exploring the unique pharmacology of a novel opioid receptor, ZFOR1, using molecular modeling and the `message–address' concept

Iain J. McFadyen,2, Thomas G. Metzger, M.Germana Paterlini and David M. Ferguson,1

Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55414, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies have probed the structural basis of ligand selectivity in the mu, delta and kappa opioid receptors through the application of molecular modeling techniques in concert with the `message–address' concept. Here, this approach was used in an attempt to rationalize the unique pharmacological profile of a recently cloned novel opioid receptor, ZFOR1 (ZebraFish Opioid Receptor 1). Specifically, a model of the transmembrane domains of ZFOR1 was constructed and used to explore the binding modes of various prototypical opioid ligands. The results show that the `message' portion of the binding pocket of ZFOR1 is highly conserved; hence, the binding modes of non-selective opioid ligands are well preserved. In contrast, a small number of variant residues at the extracellular end of the binding pocket, particularly Lys288 (VI:26) and Trp304 (VII:03), are shown to create adverse steric interactions with all delta and kappa selective ligands examined, thereby disrupting their binding modes. These results are consistent with, and serve as an explanation for, the observed pharmacology of this receptor, lending support to both the validity of the `message–address' concept itself and to the use of molecular modeling approaches in its application.

Keywords: message/address/modeling/opioid/structure-function/ZFOR1


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The opioid receptor family is classically considered to comprise three subtypes, namely mu, delta and kappa, as distinguished by differences in sequence, pharmacology, anatomical distribution and the physiological effects which they mediate (for reviews see Kieffer, 1995Go; Satoh and Minami, 1995Go; Dhawan et al., 1996Go). As members of the superfamily of G-protein coupled receptors (GPCRs), they share the characteristic structure of that protein class, namely a bundle of seven {alpha}-helical transmembrane (TM) domains connected by alternating intra- and extracellular loops (for review, see Kieffer, 1995Go). An incredibly diverse array of ligands is capable of interacting with the opioid receptors (for review, see Aldrich, 1996Go), including a variety of endogenous opioid peptides, the clinically important analgesic agent morphine and related alkaloids (termed `opiates') and synthetic ligands based on a number of different structural scaffolds. Perhaps not surprisingly given this structural diversity, opioid ligands cover the entire spectrum of possible selectivities for the mu, delta and kappa ligands. This has been rationalized in terms of a `message–address' concept (Takemori and Portoghese, 1992Go), whereby each ligand contains a common `message' portion which confers activity and a variable `address' moiety which imparts selectivity. By logical extension, the mu, delta and kappa receptors must share a well-preserved binding pocket for the ligand `message' whilst also possessing individual `address' recognition elements.

We have previously described molecular models of the TM domains of all three classical opioid receptors together with the proposed docking modes of certain prototypical selective and non-selective opioid ligands (Metzger et al., 1996Go; Paterlini et al., 1997Go; Subramanian et al., 1998Go, 2000Go; Podlogar et al., 2000Go). This has allowed us to extend the `message–address' concept by identifying the key regions and more specifically the individual residues of these receptors which comprise both the `message' and `address' recognition elements. For opiate ligands, a well-preserved binding pocket located in the extracellular half of the helical bundle and comprising residues from TMs 3, 5, 6 and 7 is implicated in binding the common epoxymorphinan `message'. Meanwhile, selectivity is conferred by a few key variable residues at the extracellular end of the helical bundle, most notably VI:26 and VII:03 (see Materials and methods for an explanation of the numbering scheme), through an exclusionary mechanism (Metzger and Ferguson, 1995Go; Metzger et al., 1996Go, 2001).

Recently, the novel G-protein coupled receptor ZFOR1 (ZebraFish Opioid Receptor 1) was cloned from the teleost Danio rerio (zebrafish) (Barrallo et al., 1998Go). ZFOR1 shares significantly higher sequence identity with the mu, kappa and especially the delta opioid receptors than with the next most closely related GPCRs, subtypes 1–5 of the somatostatin family (Figure 1Go, Table IGo). However, ZFOR1 displays a pharmacological profile distinct from all three mammalian opioid receptor subtypes (Table IIGo) (Rodriguez et al., 2000Go). Specifically, ZFOR1 binds certain non-selective opioid ligands with moderate affinities whilst exhibiting greatly reduced affinity for all mu, delta and kappa selective ligands tested to date, at least 1400-fold as compared with their preferred mammalian opioid receptor (Table IIGo). This presents a useful opportunity to test the validity of the `message–address' concept and the use of molecular modeling in its application, by attempting to provide a structural explanation for the unique pharmacological profile of this novel receptor. Here we describe the process of model building and refinement, ligand docking and sequence analysis which has allowed us to identify the structural elements underlying the pharmacology of ZFOR1.



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Fig. 1. Alignment of the sequences ZFOR1 and the µ, {delta} and {kappa} opioid receptors. The sequences used are (ID, accession number): Mu (OPRM_RAT, P33535); delta (OPRD_MOUSE, P32300); kappa (OPRK_RAT, P34975) (all from SwissProt) and ZFOR1 (DRAJ1596, AJ001596, from GenBank). Consensus key: *, fully conserved residue; :, strong conservation; ., weak conservation (as defined by ClustalW).

 

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Table I. Sequence identities
 

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Table II. Pharmacology of ZFOR1
 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Residue numbering

Two residue numbering schemes are used throughout. When referring to a residue from an individual receptor, the three-letter code and position in the primary sequence are used, e.g. Trp304. When comparing equivalent positions across multiple receptors, the generic GPCR numbering scheme of Baldwin (1993) is used, whereby roman numerals indicate TM helix and arabic numerals denote position from the N-terminal end of that helix. For example Trp304 in ZFOR1, Leu300 in delta, Trp320 in mu and Tyr312 in kappa all occupy the same relative position in the seventh TM region and are therefore all referred to as VII:03.

Sequence manipulation

Protein sequences were retrieved from either the Swiss-Prot (Bairoch and Apweiler, 2000Go) or GenBank (Benson et al., 1999Go) online databases. Similarity searching across databases was performed with BlastP (Alschul et al., 1997Go). Multiple sequence alignments were created using ClustalW (Thompson et al., 1994Go). All such procedures were carried out within the Biology Workbench 3.2 suite of online search and analysis tools (workbench.sdsc.edu), using default settings throughout.

Modeling

The model of the transmembrane regions of ZFOR1 was constructed as described previously (Subramanian et al., 2000Go). Briefly, a starting structure for ZFOR1 was generated from an existing delta opioid receptor model (Podlogar et al., 2000Go) using SCWRL (Bower et al., 1997Go) to determine initial side chain torsional angles by reference to a backbone-dependent rotamer library (Dunbrack and Karplus, 1993Go). In order to relax side chain packing fully, the starting structure of the helical bundle was subjected to energy minimization with strong positional constraints on backbone atoms only (5 kcal/Å2.mol) until the r.m.s.d. of the gradient reached 0.001 Å. This was followed by 400 ps of MD simulation (step size 1 fs) at 300 K wherein positional constraints on backbone atoms were gradually reduced to a final value of 0.05 kcal/Å2.mol. A final energy minimization was performed as above. The AMBER suite of programs (Pearlman et al., 1995Go; Case et al., 1999Go) with the Cornell et al. force field (Cornell et al., 1995Go) was used throughout, with a non-bonded cut-off distance of 12.0 Å and a distance-dependent dielectric of 4r. The structural quality of the model was assessed using PROCHECK (Laskowski et al., 1993Go), which showed 96.7% of the residues in the most favorable helical region and the remaining six residues in the additionally allowed helical region. Side chain {chi}1 and {chi}2 torsional angles were in the most favorable regions for all but five residues, which were in the additionally allowed regions and there were zero bad side chain contacts.

The putative ligand binding pocket of ZFOR1 was identified using the DOCK 3.5 suite of programs (Meng et al., 1992Go). Specifically, a solvent-accessible molecular surface of the interior of the receptor model (heavy atoms only) was created using the Connolly algorithm (Connolly, 1983Go) and then the interior of this surface was `filled' with spheres of radii 1.4–4.0 Å, using SPHGEN. Since this protocol tends to generate a large number of spheres, the sphere file was examined and trimmed manually, as described previously (Subramanian et al., 1998Go).

Visualization of the docking modes of various ligands to ZFOR1 was performed by analogy with previously published models. Specifically, the complex of each ligand docked to its preferred receptor was aligned to the final structure of ZFOR1 by means of a least-squares fit across all backbone heavy atoms (r.m.s.d. of ZFOR1 to delta was 1.38 Å, to mu 1.46 Å and to kappa 1.58 Å). The mammalian receptor structures were removed, leaving the ligand of interest superimposed upon the ZFOR1 binding site. The ligand–ZFOR1 complexes were then subjected to energy minimization as described above, with strong positional constraints on backbone atoms (5 kcal/Å2.mol) but none on side chain or ligand atoms. Any atomic overlap between receptor and ligand was relieved through manual adjustments of side chain torsional angles in order to facilitate minimization. In some cases, where receptor–ligand overlap or steric clash was too extensive to be resolved by side chain rotation, manual adjustment of ligand position had to be used, taking care not to disrupt the initial binding mode significantly. In general, a ligand–receptor distance <50% of the combined van der Waals radii (for atom pairs) was used to define severe steric clash or overlap. If the final, minimized ligand–receptor complex was essentially unchanged from the initial complex and the key interactions characteristic of opioid ligand binding were present, we would predict the ligand to exhibit at least moderate affinity for ZFOR1. However, if energy minimization caused a dramatic shift in ligand position such that the key interactions with the receptor were comprehensively disrupted, we would predict the ligand to be incapable of binding to ZFOR1 with high affinity. The ligand–receptor complexes used were SNC80 (Podlogar et al., 2000Go) and naltrindole (NTI) at delta (Metzger et al., 1996Go), U69,593 (Subramanian et al., 1998Go) and norbinaltorphimine (norBNI) at kappa (Metzger et al., 1996Go) and fentanyl (Subramanian et al., 2000Go) and naloxone at mu (Metzger et al., 1996Go).

Midas Plus (Ferrin et al., 1988Go, 2000Go) was used throughout for visualization. SPDBV (Guex and Peitsch, 1997Go) and POV-Ray 3.1 (www.povray.org) were used to create the rendered images.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Similarities between ZFOR1 and mammalian opioid receptors

Approximately two-thirds of the residues in the transmembrane domains (130 of 205) are absolutely conserved across mu, delta, kappa and ZFOR1 (Table IGo and Figure 1Go). The binding pocket identified by DOCK lies within the extracellular portion of the helical bundle and is surrounded by 30 residues from TMs 3, 5, 6 and 7, defined as those residues with any side chain heavy atom within 5 Å of any sphere. Echoing the overall composition of the helical bundle, two-thirds of the residues in the binding pocket are absolutely conserved. The distribution of conserved and variant residues is distinctly non-random; the deepest portion of the binding pocket is completely preserved and the 10 variable positions cluster exclusively at or near the extracellular surface (Figure 2Go).



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Fig. 2. The proposed binding pocket of ZFOR1. View from within the membrane, looking into the binding pocket from a point outside TM2 (removed for clarity). TM helices are numbered and shown as gray ribbons. Residues within 5 Å of the pocket are colored green if conserved across ZFOR1 and all three mammalian opioid receptors or red if variable.

 
The predicted binding mode of naloxone to ZFOR1 lies almost entirely within the deepest, most conserved portion of the binding pocket (Figure 3Go). The ligand experiences no steric clashes with proximal side chains and minimization does not significantly alter the complex. At the atomic level all of the key interactions between the pharmacophore features of naloxone and their cognate residues are present in the final complex (Metzger et al., 1996Go). For example, the quaternary nitrogen interacts with Asp III:07 (131 in ZFOR1) and the tyrosyl ring with Tyr III:08 (132), Phe V:11 (226), Trp VI:16 (278) and His VI:20 (282).



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Fig. 3. The proposed docking mode of naloxone to ZFOR1. Naloxone is shown as a stick model colored by atom type. TM helices of ZFOR1 are shown as green ribbons and numbered. Residues shown in blue space filling representation (Asp131 III:08 in red) form a binding pocket common to the µ, {delta} and {Phi} and ZFOR1 receptors.

 
Differences between ZFOR1 and mammalian opioid receptors

Sequence identity between ZFOR1 and the individual mammalian opioid receptors is highest for delta, as mentioned previously, and significantly less for mu and kappa (Table IGo). However, when considering the transmembrane regions alone the rank order of identity becomes delta {approx} mu > kappa. Furthermore, in the critical area surrounding the binding pocket ZFOR1 actually resembles the mu receptor most closely of all and the rank order of identity is now mu > delta = kappa (Table IGo).

The binding pocket of ZFOR1 differs from those of the mammalian opioid receptors by at most six residues (Tables I and IIIGoGo). We have investigated the role of these variant residues in the binding modes of certain prototypical selective opioid ligands, namely naltrindole and SNC80 (delta selective), U69,593 and norBNI (kappa) and fentanyl (mu).


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Table III. Variant residues in the binding pocket of ZFOR1 and their interactions with ligands
 
The delta selective antagonist naltrindole is based on the same structural skeleton as naloxone. The `message' portion of the molecule aligns within the binding pocket in a similar manner to naloxone (Figure 4Go). However, naltrindole contains an additional indole moiety which aligns towards the extracellular end of the binding pocket proximal to four of the six residues which differ between ZFOR1 and delta (Table IIIGo). Two of these residues, Ile285 (VI:23, Val in delta) and Val307 (VII:06, Cys in delta), do not create any unfavorable steric interactions with the ligand. However, the side chains of Lys288 (VI:26) and Trp304 (VII:03) overlap so extensively with the indole moiety that the clashes cannot be relieved by side chain rotation. If the overlap is relieved by small adjustment to the ligand position, energy minimization subsequently leads to large changes in ligand position and the loss of all characteristic opioid ligand–receptor interactions (see above).



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Fig. 4. The proposed docking mode of naltrindole to ZFOR1. Naltrindole is shown as a stick model colored by atom type. TM helices of ZFOR1 are shown as green ribbons and numbered. Space-filling representation: Lys288 (VI:26) and Trp304 (VII:03) in blue, Asp131 (III:07) in red. Naltrindole shows steric overlap with both residues in blue.

 
In the case of SNC80, a delta selective agonist of the diarylmethylpiperazine class, the majority of the molecule aligns favorably within the conserved area of the binding pocket but the phenyldiethylamide ring overlaps extensively with the same two residues, Lys288 (VI:26) and Trp304 (VII:03).

The kappa selective antagonist norbinaltorphimine (norBNI) shares the epoxymorphinan skeleton common to naloxone and naltrindole. Again, the `message' portion of the ligand occupies the well conserved lower portion of the binding pocket. However, norBNI contains, as its `address' element, an entire second epoxymorphinan moiety which projects towards the extracellular end of the binding pocket where it contacts three of the six residues which vary between ZFOR1 and kappa (Figure 5Go). The sheer size of this moiety leads to extensive steric overlap with all three of these residues (Table IIIGo), namely Lys288 (VI:26, Glu297 in kappa), Val307 (VII:06, Cys315 in kappa) and Trp304 (VII:03, Tyr312 in kappa). It was not possible to adjust the ligand position manually to resolve these conflicts without also disrupting the binding mode. In addition, there is potential for an unfavorable electronic interaction between the positively charged 17' nitrogen of the ligand and the positively charged amine group of Lys288 (VI:26). This is in direct contrast to the kappa receptor where the equivalent Glu297 carries a negative charge complementary to the ligand.



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Fig. 5. The proposed docking mode of norBNI to ZFOR1. NorBNI is shown as a stick model colored by atom type. TM helices of ZFOR1 shown as green ribbons and numbered. Space-filling representation: Lys288 (VI:26) and Trp304 (VII:03) in blue, Asp131 (III:07) in red. NorBNI shows steric overlap with both residues in blue.

 
The kappa selective agonist U69,593 binds largely within the well preserved lower portion of the binding pocket. In addition, it possesses a much smaller structural skeleton than norBNI (arylacetamide versus fused epoxymorphinan) and consequently does not extend far enough towards the extracellular surface to interact with Lys288 (VI:26). However, the phenyl ring of U69,593 does clash with the side chains of Trp304 (VII:03, Glu297 in kappa) and Val307 (VII:06, Cys315 in kappa) (Table IIIGo), resulting in a disruption of the ligand binding mode upon minimization.

In contrast to the unfavorable steric interactions seen with all of the delta and kappa selective ligands discussed above, the proposed binding mode of the mu selective agonist fentanyl to ZFOR1 is remarkably well preserved (not shown). The majority of the binding pocket for fentanyl is identical with that seen in the mu receptor. Indeed, the ligand interacts with only two variant residues, namely Val307 (VII:06, Cys321 in mu) and Ile285 (VI:23, Val300 in mu), neither of which disrupts the ligand (Table IIIGo). Furthermore, the binding mode is essentially unchanged by minimization.


    Discussion
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 Materials and methods
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 References
 
The transmembrane regions of ZFOR1 have been modeled by homology to the closely related delta receptor and after extensive molecular dynamics the resulting model has high structural quality by a number of measures. The model contains a binding pocket qualitatively similar to those seen in the mu, delta and kappa (Metzger et al., 1996Go) receptors. The deepest portion of the pocket is strictly conserved, whilst the variable residues cluster exclusively at the extracellular surface.

In terms of the `message–address' concept, non-selective ligands such as naloxone and the structurally similar naltrexone can be considered to comprise entirely `message' with no `address'. Previous modeling studies have proposed that naltrexone binds to all three mammalian opioid receptors in a common manner, within the lower, conserved portion of the binding pocket (Metzger et al., 1996Go). This `message' recognition site is strictly conserved in ZFOR1 and as a result the common binding mode of naloxone and by extension, other small non-selective opiate ligands, is also well preserved. This is consistent with the moderate to high affinity for ZFOR1 displayed by naloxone, diprenorphine, bremazocine and EKC.

However, the affinities of these ligands for ZFOR1 are in most cases somewhat reduced compared with their affinities for the mammalian opioid receptors. There are several possible explanations for this. First, the topology of the binding site of ZFOR1 may in fact be less well preserved than is predicted by the model. Indeed, homology models of GPCRs based on low-resolution rhodopsin structures are themselves, necessarily, of low `resolution' and it is therefore dangerous to over-interpret results gained from them. However, the model presented here is able to explain adequately the majority of the observed pharmacology for ZFOR1, indicating that it is valid in the qualitative context in which it is used here. Alternatively, variant residues located at the edge of the `message' binding pocket may have a subtle disruptive effect on the binding modes of ligands which is not apparent from the model. One candidate is Val307 (VII:06), the only position close to the pocket which is conserved in all three mammalian opioid receptors (Cys321 in mu, Cys303 in delta, Cys315 in kappa) but variant in ZFOR1. However, it has been shown that the Cys303Ser delta receptor mutant retains unaltered ligand binding affinity for both the non-selective naloxone and the delta selective naltrindole (Ehrlich et al., 1998Go). The only other suitably positioned variant residues are Ile285 (VI:23) and Ile286 (VI:24). Again, however, a double mutant at the equivalent positions in delta (V281I/I282L) retained unchanged affinity for bremazocine, naltrindole and BW373,U86 (Meng et al., 1996Go). Although these results are gained from mutants of the mammalian opioid receptors, they provide circumstantial evidence against a role in ligand binding for the equivalent residues in ZFOR1. A series of suitable mutants of ZFOR1 would, of course, be required to settle the issue. Finally, the extracellular loops (EL), especially EL2 and EL3 which vary considerably in ZFOR1 from those found in any of the mammalian opioid receptors, may partially restrict access of all ligands to the binding pocket through a mechanism of exclusion. A similar mechanism has previously been suggested to underlie the role of these loops in the mammalian opioid receptors in conferring selectivity against non-favored ligands (Metzger and Ferguson, 1995Go).

ZFOR1 exhibits vastly reduced affinity for all of the selective ligands tested to date, including both agonist and antagonist ligands of several different structural classes (Rodriguez et al., 2000Go). In all cases, the reduction is >1400-fold relative to affinity at the preferred mammalian receptor, a much greater reduction than that seen for any of the non-selective ligands tested (Table IIGo). We propose that delta and kappa selective ligands suffer from unfavorable steric and/or electronic interactions with a few key variant residues at the extracellular end of the binding pocket, thereby impairing their ability to bind to ZFOR1. Specifically, the bulky side chains of Lys288 (VI:26) and/or Trp304 (VII:03) clearly disrupt the proposed binding modes of naltrindole, SNC80, norBNI and U69,593 through unfavorable interactions. This is consistent with the known binding affinities for these compounds (Table IIGo).

However, although the binding affinity of naltrindole is reduced by over 1400-fold relative to the delta receptor, it is still moderate at ~28 nM (Table IIGo). Since the steric overlap that naltrindole experiences cannot be resolved without radically altering the binding mode upon subsequent minimization, we would expect the reduction in affinity to be even greater than is observed. The reasons for this discrepancy are unclear.

Note that the actual binding affinity of norBNI to ZFOR1 is unknown. We would predict that norBNI would display greatly reduced affinity for ZFOR1 compared with the kappa receptor owing to unfavorable steric and electronic interactions of the `address' moiety with Lys288 (VI:26) and Trp304 (VII:03).

In the case of opiate ligands such as naltrindole and norBNI, there is excellent evidence from previous mutagenesis studies of the mammalian opioid receptors that residues VI:26 and VII:03 play a key role in determining selectivity for/against delta and kappa receptors, respectively. For example, mutation of Trp284 (VI:26) of the delta receptor to the Lys residue found at the equivalent position of ZFOR1 causes no change in affinity for the non-selective opioid ligand bremazocine but at least a 10-fold decrease in affinity for a variety of delta selective ligands including SNC-80, [D-Pen2,D-Pen5] enkephalin, deltorphin II and naltrindole (Valiquette et al., 1996Go). Mutation of the same position in the kappa receptor, Glu297 (VI:26), to the ZFOR1 equivalent Lys causes a similar decrease in affinity for kappa selective ligands (Hjorth et al., 1995Go; Jones et al., 1998Go). Meanwhile, all members of a large library of delta receptor multiple point mutants in which residue VII:03 was changed to Trp, as seen in both mu and ZFOR1, were unable to bind any delta ligand with high affinity. Similar mutants in which VII:03 reverted to the original delta residue (Leu) largely regained affinity for delta ligands (Pepin et al., 1997Go).

Thus, mutation of either VI:26 or VII:03 in the delta or kappa opioid receptor to the Lys and Trp residues found in equivalent positions in ZFOR1 generally results in significant decreases in affinity for the appropriate selective ligands. The converse has also been shown to be true; that is, mutation of either VI:26 or VII:03 in one receptor to the corresponding residue from another imparts increased affinity for the appropriate selective ligands (Metzger et al., 2001Go). In terms of the model presented here, the most telling results from that study were those gained from mutants of the mu receptor at positions VI:26 and VII:03, which are identical in mu and ZFOR1. Replacement of Lys303 at position VI:26 of the mu receptor with the kappa-equivalent Glu resulted in increased affinity for all kappa ligands tested (Jones et al., 1998Go; Metzger et al., 2001Go). The mu mutant Trp318Ala (VII:03) showed almost wild-type affinities for both delta and kappa selective ligands, presumably through removal of steric bulk (Metzger et al., 2001Go). Therefore, a large body of evidence from mutagenesis studies in the mammalian opioid receptors provides strong, although indirect, support for the findings presented here that Lys288 (VI:26) and Trp304 (VII:03) act as critical determinants of selectivity against selective delta and kappa ligands in the novel receptor ZFOR1.

The binding pocket of ZFOR1 is almost identical with that of the mu receptor, including both of the key positions discussed above, VI:26 (Lys) and VII:03 (Trp). Despite this similarity, affinity of the prototypical selective agonist DAMGO for ZFOR1 is reduced by >6000-fold relative to mu. However, extensive mutagenesis studies on the mammalian opioid receptors have shown that there are additional structural elements which contribute to selectivity for or against DAMGO binding. For example, the area surrounding EL1 is an important locus for discrimination by DAMGO between the mu and delta receptor (Fukuda et al., 1995aGo; Minami et al., 1995Go; Onogi et al., 1995Go; Wang et al., 1995Go). Indeed, a single residue at the boundary between TM2 and EL1, Lys108 (II:27), is largely responsible for the lack of DAMGO binding to the delta receptor (Fukuda et al., 1995bGo; Minami et al., 1996Go). The EL1 loop of ZFOR1 is almost identical with that of delta (Figure 1Go) and residue II:27 is Lys in both. Thus, we propose that DAMGO exhibits low affinity for ZFOR1 despite the presence of a mu-like binding pocket due to a mechanism of exclusion by a delta-like EL1 and more specifically Lys111 (II:27). However, these residues are not known to influence the binding of non-peptide mu selective ligands, which are not large enough to interact simultaneously with the binding pocket and the extracellular loops. Indeed, the proposed docking mode of fentanyl to ZFOR1 is remarkably free of steric clashes with the side chains of the receptor and we would predict that fentanyl would exhibit moderate to high affinity for ZFOR1.

Molecular modeling of the mu, delta and kappa receptors and docking of various prototypical opiate ligands has identified the areas and individual residues which are proposed to interact with the `message' and `address' portions of these ligands (Metzger et al., 1996Go). Here, this approach has successfully been transferred to ZFOR1 in an attempt to explain its unique pharmacology. The moderate to high affinity of non-selective ligands is consistent with a well-preserved `message' binding site, whilst the greatly reduced affinity of ZFOR1 for all selective ligands tested to date is proposed to be the result of a few key sequence differences. In particular, residues Lys288 (VI:26) and Trp304 (VII:03) at the extracellular end of the binding pocket are shown to cause unfavorable steric interactions with a variety of delta and kappa selective ligands. This is in close agreement with previous modeling and mutagenesis studies which have shown that the same residues are critical mediators of ligand selectivity in the mammalian opioid receptors. Unfortunately, we were unable to test the hypotheses suggested here regarding the role of these key residues or the binding affinity of norBNI and fentanyl to ZFOR1, because we could not obtain cDNA for ZFOR1 from Rodriguez et al. (Rodriguez et al., 2000Go).

ZFOR1 was originally assigned to the opioid receptor family (Barrallo et al., 1998Go) since it shares higher sequence identity with the mu, delta and kappa receptors than with the next most closely related proteins, the somatostatin receptors (Table IGo). Indeed, ZFOR1 has since been shown to exhibit at least moderate affinity for all non-selective ligands tested to date (Rodriguez et al., 2000Go). ZFOR1 was further tentatively classified as a delta receptor homolog on the basis that it shares the highest sequence identity with that subtype: 65% over the full sequence, 83% across the transmembrane domains (Barrallo et al., 1998Go). However, species homologs of the mu, delta and kappa opioid receptors cloned from a variety of mammalian species (for reviews, see Kieffer, 1995Go; Satoh and Minami, 1995Go) and one fish (Darlison et al., 1997Go) all share >89% and >96% sequence identity across the full sequence and the transmembrane domains, respectively. As a consequence, all known species homologs of the opioid receptors display almost identical pharmacology. In contrast, ZFOR1 displays a pharmacological profile very different from delta receptor homologs. The model presented here proposes that this unique binding profile has an equally distinct structural basis. Thus ZFOR1 may need to be considered as a sub-division of the opioid receptor family in its own right, separate from the classical mammalian mu, delta and kappa subtypes.

In summary, the molecular model presented here is able to rationalize adequately the known pharmacology of ZFOR1 in terms of the `message–address' concept, providing further evidence for the validity of this concept and for the usefulness of applying molecular modeling approaches in the investigation of ligand selectivities in the opioid receptor family. Furthermore, ZFOR1 should provide a novel background for mutagenesis studies aiming to explore the role of those residues highlighted in this study and previous studies of the mammalian opioid receptors.


    Notes
 
1 To whom correspondence should be addressed. Back

2 Current address: Wyeth-Ayerst Research, 85 Bolton Street, Cambridge, MA 02140, USA Back


    Acknowledgments
 
The authors acknowledge the NIH/NIDA for financial support to D.M.F. and M.G.P.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received November 27, 2000; revised July 30, 2001; accepted August 1, 2001.