Unité de Neuropharmacologie Moléculaire, Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, 205 route de Narbonne, 31077 Toulouse Cedex 4, France
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Abstract |
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Keywords: amino acid sequence cluster analysis/annotation of function/nociceptin/opioid and amine G protein-coupled receptors/rhodopsin molecular modelling/site-directed mutagenesis
Abbreviations: CEC, chloroethylclonidine GPCR, G-protein coupled receptor MTSEA, 2-aminoethylmethanethiosulphonate ORL1, opioid receptor like 1 PDB, Protein Data Bank r.m.s.d., root mean square distance TM, transmembrane
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Introduction |
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Site-directed mutagenesis techniques supported by molecular modelling are often used to identify GPCR ligand binding sites (Schwartz, 1994; van Rhee and Jacobson 1996
; Bikker et al., 1998
; Flower, 1999
). This approach has been used to establish the existence of a general cationic monoamine neurotransmitter binding pocket, comprising elements from the extracellular parts of TM helices III, (IV), V, VI and VII (see Bikker et al., 1998, for a review). This same TM region appears also to be used as an endogenous peptide agonist binding site by (structural) members of the opioid receptor family. Here again the evidence principally derives from mutagenesis studies (Surrat et al. 1994
; Befort et al., 1996a
,b
; Meng et al., 1996
, 1998
; Mollereau et al., 1996
; Mansour et al., 1997
; Li et al., 1999
; Moulédous et al., 2000
) in tandem with prior and posterior comparative modelling (Befort et al., 1996b
, 1999
; Paterlini et al., 1997
; Strahs and Weinstein, 1997
; Pogozheva et al., 1998
; Topham et al., 1998
; Filizola et al., 1999
).
We have described a molecular model of the complex formed between the (opioid receptor-like) ORL1 receptor and its endogenous heptadecapeptide agonist, nociceptin (Topham et al., 1998). Although the ORL1 receptor binds and responds poorly to opiate ligands (Butour et al., 1997
), in structural terms it clearly belongs to the opioid receptor family, the TM domain sharing ~61% sequence identity with the
-,
- and µ-receptors. Comparatively few TM mutational events are required to convert the ORL1 receptor into a functional opioid receptor (Meng et al., 1998
). Modelling studies of receptorligand complexes suggest that topologically equivalent TM binding pockets accommodate the N-terminal F1GGF tetrapeptide of nociceptin and the endogenous opioid peptide Y1GGF `message' sequence (Paterlini et al., 1997
; Pogozheva et al., 1998
; Topham et al., 1998
; Filizola et al., 1999
). The `vestigial' opioid binding pocket in the ORL1 receptor has been proposed to serve a locatory role (Topham et al., 1998
). Binding of the F1GGF part of nociceptin may thus act to promote other interactions with the receptor, in particular with the structurally variable second extracellular loop, shown to be essential for activation (Mollereau et al., 1999
). This view is reinforced by the identification in a combinatorial chemical library of a hairpin peptide antagonist of the ORL1 receptor that is an agonist of the three opioid receptor types (Becker et al., 1999
).
In order for comparative modelling of GPCRs to be either of predictive value or of assistance in the interpretation of ligand binding experiments, two minimum requirements need to be met. The first is the availability of a reliable structural template for modelling of the seven TM helix framework. The second is a knowledge of how the receptor amino acid sequence should be threaded onto the framework for modelling. Both remain problematic. On the one hand, conscription of high-resolution X-ray crystallographic structures of bacteriorhodopsin is precluded by the lack of significant shared sequence identity between archaebacterial bacteriorhodopsin and mammalian GPCRs. Technical difficulties (Ostermeier and Michel, 1997), on the other hand, continue to thwart the determination of the first high-resolution GPCR structure. However, major advances have been made towards the elucidation of vertebrate rhodopsin structures using electron cryomicroscopy (Schertler and Hargrave, 1995
; Unger et al., 1997
; Krebs et al., 1998
; Schertler, 1998
) in combination with genetic, biochemical and other biophysical information (Baldwin et al., 1997
; Herzyk and Hubbard, 1998
).
Our model of the ORL1 receptor (Topham et al., 1998) used the membrane spanning helix limit definitions of Baldwin (1993) and was largely based on the C
TM domain template of Herzyk and Hubbard (1995), in turn derived from the 9 Å bovine rhodopsin projection map of Schertler et al. (1993). Significant structural differences, most notably increases in the tilt angles of TM helices III and V, are evident in the refined rhodopsin C
template of Baldwin et al. (1997), built from the 7.5 Å frog projection map of Unger et al. (1997) using modified helix limits determined from a comprehensive analysis of GPCR sequences. However, construction of ORL1 (and opioid) receptor models on the basis of the Baldwin et al. (1997) template leaves the TM ligand binding site partly exposed to lipid as a consequence of the loose packing of the extracellular parts of helices V and VI. These helix sections are presumed to form the rear wall of the F1/Y1 and F4 sub-sites, in which aromatic side chains at positions 1 and 4 of nociceptin and the opioid peptides are proposed to bind.
In Topham et al. (1998), we drew attention to distance limits involving residues at the extracellular extremities of TM helices V and VI, deduced from correlated site-directed mutagenesis studies of the 1B-adrenergic receptor (Hwa et al., 1995
) and the engineering of a zinc ion binding site in the
-opioid receptor (Thirstrup et al., 1996
), that are poorly satisfied by the Baldwin et al. (1997) template. Here we present evidence, based on cluster analyses of a multiple sequence alignment of GPCR seven-helix folds, that the TM ligand binding sites in the opioid/ORL1 and amine receptor families are structurally well conserved. Using the
-opioid receptor TM bundle as a model system (see Figure 1
), we find that spatial constraints, deduced from biochemical studies of amine, opioid and rhodopsin receptors, are better met by the recent Herzyk and Hubbard (1998) rhodopsin template, based on the Schertler and Hargrave (1995) 6 Å resolution frog rhodopsin projection map, both as a consequence of the reduced physical separation of the extracellular parts of TM helices V and VI and differences in the rotational orientation of helix V within the transmembrane binding site. We describe a modification to the Baldwin et al. (1997) structure, in which helix V, the least clear feature in the Unger et al. (1997) rhodopsin projection map, has been rebuilt with an alternative proline kink, but similar overall tilt angle, such that the experimentally derived spatial constraints can be accommodated.
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Computational methods |
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The universal GPCR residue notation system of Baldwin (1993), retained by Baldwin et al. (1997), is used throughout, with additional references made to residue numbering in individual receptors to aid identification. Correspondence with residue numbers of the human -opioid receptor amino acid sequence (Mansson et al., 1994
) is indicated in Figure 1
.
Statistical analyses of a multiple GPCR amino acid sequence alignment
A discontinuous multiple sequence alignment, corresponding to 198 residue positions within the seven sets of helix boundary limits defined by Baldwin et al. (1997) (see Figure 1), was constructed and checked manually. The SWISS-PROT (Bairoch and Apweiler, 1999
) codes of the 167 rhodopsin-like class A GPCR sequences that were used, sorted according to the protein family classifications of Horn et al. (1998), are given in Table I
. Sequences of human receptors were chosen wherever possible or appropriate.
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where N is the number of residue types (20) and p(Ri|Lj) is the (conditional) probability of occurrence of residue type Ri at a given alignment position Lj. To avoid bias, probabilities were corrected for the normalized frequency of residue type occurrence in the whole population:
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where W'(Ri|Lj) is the frequency of occurrence of residue type Ri at position Lj,
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and W'(Ri) is the global frequency of occurrence of residue type Ri. The entropy may assume a maximum value of Hmax = lnN, corresponding to a uniform distribution, and a minimum value of zero, corresponding to a totally conserved residue position. For convenience, we use the term relative entropy, defined as H/Hmax, values of which lie in the range 01.
Sequence similarity over the seven TM helices and at 18 residue positions defining the TM ligand binding site in the opioid/ORL1 receptor structural family (see Table II) was investigated using principal coordinate analysis (classical scaling) techniques (Chatfield and Collins, 1980
). Elements in an nxn distance (dissimilarity) matrix, comprising the negative natural logarithm of pairwise sequence identities, were normalized in a driver routine and passed to the CMDS Fortran subroutine, distributed by Fionn Murtagh at the http://astro.u-strasbg.fr/~fmurtagh//mda-sw/ Web site. The program outputs the n 1 eigenvalues and projections of the amino acid sequences on the first seven principal components.
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Interactive graphics manipulations were performed using the SYBYL (versions 6.3 and 6.4) software package (Tripos). Energy minimization was carried out using the SYBYL implementation of the Powell torsional gradient algorithm and the AMBER all-atom force field of Weiner et al. (1986). The electrostatic model comprised a distance-dependent dielectric constant with a non-bonded cut-off of 8 Å and an value of 4.
The -opioid receptor TM helix bundle (Figure 1
) was modelled using the rhodopsin templates of Baldwin et al. (1997) and Herzyk and Hubbard (1998). In the interests of brevity, the two models are respectively referred to throughout as B97 and HH98. Helix backbone geometry was generated from the C
coordinates using SYBYL. Side chains were initially placed in accordance with secondary structural rotamer preferences (McGregor et al., 1987
) and, where this resulted in severe steric clashes, manually adjusted to the next preferred combination. Further refinement was achieved by successive rounds of energy minimization, with helix backbone atoms alternately constrained or freed and manual side chain adjustment. The procedure was assisted by interim screening of side chain conformations against the August 1999 release of the Dunbrack rotamer libraries (Dunbrack and Karplus, 1993
; Dunbrack and Cohen, 1997
). The target energy convergence gradient was progressively reduced to a final value of 0.1 kcal/mol.Å in the absence of any constraints.
Our strategy for the remodelling of TM helix V in the Baldwin et al. (1997) C template on the basis of structural constraints, deduced from experimental studies of the opioid/ORL1 and amine receptor families and with due regard to the location of the most highly conserved residue positions in class A rhodopsin-like GPCRs, was to consider initially the N- and C-terminal sections either side of the proline at position V:14 as physically detached structural elements. Coordinated translations of the two helix segments of the B97 model in the XY plane, accompanied by clockwise rotations (as viewed from the extracellular surface) of the N-terminal fragment, provided a first construct that could be reannealed to restore real protein geometry. The composite helix was exhaustively energy minimized in isolation, redocked into the receptor and the process repeated. The helix was then completely rebuilt from its C
atom positions alone, minimized once more in isolation, redocked and a minimized receptor model bundle generated from which a new set C
coordinates for helix V, now repositioned and with a different proline kink, could be extracted. The final model, denoted B97mod5, was worked up from scratch in an identical manner to the B97 and H98 models, using the Baldwin et al. (1997) starting template to model the other six helices.
Surface area calculations
Inter-helical contact surface areas were computed using GRASP (Nicholls et al., 1991) from differences in the sum of solvent-accessible surface areas of isolated helices and the solvent-accessible surface area of the same helices when packed. Relative side chain solvent accessibilities were calculated from residue solvent-contact surfaces using PSA (
ali, 1991
). A spherical probe of radius 1.4 Å and the Chothia (1976) Van der Waals radii were employed in both sets of calculations.
In order to distinguish between membrane-exposed residue side chains and solvent-accessible side chains within receptor cavities, calculations of relative side chain solvent accessibilities were performed in vacuo and in the presence of an artificial membrane. Large differences in these two values indicate membrane-exposed positions along a helix, whereas peaks in relative side chain accessibility profiles for membrane-embedded models provide a direct readout of exposed residue positions within TM cavities. The artificial membrane consisted of a cubic lattice of methane molecules inter-spaced by 4.5 Å to a depth of 36 Å, centred on the Z axis of the Baldwin et al. (1997) coordinate system, taken to be perpendicular to the membrane plane. Minimized receptor molecules were docked into the membrane by superposition of selected C atom sets on to the (unminimized) Baldwin et al. (1997) C
rhodopsin equivalents. Superposition of all 198 C
atoms in the B97 model yielded an r.m.s.d. value of 0.77 Å. The C
atoms of helix V were omitted for the B97mod5 model and the r.m.s.d. corresponding to the remaining 168 topologically equivalent positions was 0.94 Å. The minimized H98 bundle superposed to 2.46 Å over 184 common positions in the seven helices (see caption to Figure 4
). Overlapping methanes within an inter-atomic contact distance of 1.3 Å were then automatically deleted and those remaining in receptor cavities were manually removed.
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Linear helix axes were calculated using P-CURVES v3.1 (Sklenar et al., 1989). For each peptide plane in a helix, the program returns the vectorial direction of the (local) helical axis, as a normalized eigenvector (U) and its reference point (P). In the case a straight-line helical axis, the direction vectors are identical and four parameters serve to define the axis in space. Two spherical polar angles describe its orientation; the polar angle (
), which is the angle between the Z axis and the helical axis, and the azimuthal angle (
), which is the angle made between the X axis and the projection of the helical axis in the XY plane. These were calculated from the unit vectors U according to standard trigonometric equations (Barrett and Mackay, 1987
). The positional parameters x0 and y0 are the orthogonal coordinates at which the helix axis intersects the membrane plane (z = 0) and were determined as the solutions to two simultaneous equations obtained from the direction vector at an arbitrary reference point. Proline kinks in helices V and VI are described by a bend angle (
) and
, the value of z at which the bend occurs. The bend angle was calculated as the scalar product of the direction vectors of the flanking helical axes and
was taken as the z value at the minimum inter-helical axis separation, determined iteratively from calculations of the distance between the two (non-intersecting) straight line axes (Bowyer and Woodwark, 1983
) as a function of z.
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Results |
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Residue codes following Baldwin (1993) for 18 amino acid positions operationally defining the opioid/ORL1 receptor transmembrane binding site are given in Table II. They were identified from an examination of the `loss' and `gain of function' site-directed mutagenesis literature and from modelling studies of the inter-residue contacts of the N-terminal tetrapeptide sequences of dynorphin A and nociceptin in complexes with their respective parent
-opioid (Paterlini et al., 1997
) and ORL1 (Topham et al., 1998
) receptors and of 5 Å contacts of
-naltrexone and
-etorphine with the
-opioid receptor (Filizola et al., 1999
), plus the interactions made by various alkaloid and small peptide ligands with the
-, µ- and
-opioid receptors, reported by Pogozheva et al. (1998). Also recorded in Table II
are side chain solvent accessibility mappings at equivalent residue positions in two amine receptors, determined using the substituted-cysteine accessibility method (Javitch et al., 1995
, 1998
; Fu et al., 1996
; Marjamäki et al., 1999
). There is a close correlation between residues contacted in the modelled receptorligand complexes and their inferred solvent accessibilities. The apparent exception (at position VII:9) in the nociceptinORL1 receptor complex is accounted for by a contact with the main chain of Leu307, the side chain itself being orientated towards the membrane.
Cluster analyses of a multiple GPCR amino acid sequence alignment
Figure 2A shows a representation in two dimensions of the distance relationships among 167 class A rhodopsin-like GPCR sequences at 198 aligned positions in the transmembrane seven-helix bundle (Figure 1
). Values of the first two principal components cluster according the receptor family classifications of Horn et al. (1998), annotated in Table I
. The vertebrate rhodopsin family forms an isolated cluster at the apex of a main triangular feature, whilst other GPCR family clusters are organized in a blunt arrow-shaped formation, the tail of which is made by the other triangle apexes. These distance relationships are consistent with the early divergence of rhodopsin and non-rhodopsin families and suggest that shifts in the relative positions and orientations of equivalent helices may have occurred. Differences in helixhelix distances and angles are well documented in all-
-helical globular protein structures sharing a common architecture, such as the globin family (Lesk and Chothia, 1980
). The bulk of the non-rhodopsin receptor seven-helix cores (78%), including those of the opioid and ORL1 receptors, are 2026% identical with that of human rhodopsin. Their main chain atoms can be estimated to superpose with an r.m.s.d. in the range 1.61.8 Å, according to the Chothia and Lesk (1986) relation that holds for globular proteins.
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Molecular modelling of the -opioid receptor
In order to examine the effect of helix geometry differences in the latest rhodopsin templates on the structure of the opioid/ORL1 receptor transmembrane ligand binding pocket, we have constructed three minimized models of the -opioid receptor TM helix bundle (Figure 1
). Two models, abbreviated as B97 and HH98, were built directly from the Baldwin et al. (1997) and Herzyk and Hubbard (1998) rhodopsin templates, respectively. The third modelled structure, a variant of the B97 model, in which helix V has been rebuilt with a different proline kink at position V:14, is referred to as B97mod5 and is described in more detail below. The backbone geometry of each receptor model was assessed using PROCHECK (Laskowski et al., 1993
). With the exception of Asp334 (VII:25) in the HH98 model, all non-glycine and non-proline residues were within the `most-favoured' (A) alpha helical region of the Ramachandran plot. Asp334 is located in the `additionally allowed' (a) region permitting the retention of a close interaction with Arg156 (III:25), equivalent to that existing between Asn310 and Arg135 in the Herzyk and Hubbard (1998) all-atom model of rhodopsin.
The reproduction of helix side chain geometry characteristic of that in native proteins was assessed by comparing rotamer probability frequency distributions in the models with those of helix data sets for transmembrane and all--helical globular proteins (see Table III
). Frequency histograms are shown in Figure 3A
for the conditional probability of finding a particular
1 side chain rotamer for a given
,
combination and residue type and in Figure 3B
, the conditional probability of a side chain possessing a particular
2 rotamer, given the
1 rotamer class. Statistically significant Spearman rank order correlation coefficient values were obtained for all pair-wise model and experimental data combinations for both rotamer probability distributions (data not presented). Despite the high correlations in this non-parametric statistical test, visual inspection of the illustrative histogram for the B97mod5 model in Figure 3A
reveals a much greater fraction of the modelled rotamer data in the highest
1 conditional probability bracket compared to experiment. This strong relative frequency peak, found for all models, persisted (>0.3) when probability data for exposed residues with relative side chain accessibilities of >45% (in vacuo) were discarded. The most likely interpretation is that the modelled bundles are not as closely packed as real structures, thus allowing the most probable
1 rotamer to be selected more often. This type of comparative analysis of side chain rotamer probability distributions may provide an increasingly useful means of assessing the quality of transmembrane bundle packing geometry as more accurate rhodopsin structures become available.
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Figure 4 shows a profile of inter-atomic C
distances obtained upon superposition of the B97 and HH98 models. Pronounced differences (of up to 7 Å) are apparent in the N-terminal sections of helix V and, to a lesser extent, helix III, that together with components from helices VI and VII are assumed to form the transmembrane ligand binding site. The extracellular sections of TM helices VI and VII of the two models superpose well and serve as a frame of reference with which to compare stereoviews of the binding pocket region, shown in Figure 5
.
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The loose packing of helices V and VI in this region of the B97 model, which we previously remarked upon in relation to the ORL1 receptor (Topham et al., 1998), is also apparent in Figure 5B
. This leaves the ligand binding site partly exposed to lipid as a result. Correlated site-directed mutagenesis studies of the
1B-adrenergic receptor reported by Hwa et al. (1995) are consistent with the spatial proximity of the helix V/VI residue pair, V:3 (Ala204)/VI:23 (Leu314). The V:3 (Lys227)VI:23 (Ile294) C
C
inter-atomic distance in the B97
-opioid receptor model is 15.4 Å, whereas the equivalent distance in the HH98 model is 8.5 Å. Two factors contribute to the better satisfaction of this inter-helical distance constraint in the HH98 model. First, as Herzyk and Hubbard (1998) note, they apply bundle-forming restraints that restrict the maximum separation of neighbouring helix ends to 11 Å. Calculations on truncated receptor models comprising 184 residues common to both frameworks show an 18% increase in the helix V/VI contact surface area in the HH98 model compared with the B97 model, although the two TM bundles appear to be equally well packed overall (Table IV
). Second, Herzyk and Hubbard (1998) employ the original TM helix definitions of Baldwin (1993) with extensions on the cytoplasmic sides of helices III and V. The N-terminus of helix V in the HH98 model is V:1 (Phe225), which allows V:3 to be sunk to a similar depth as VI:23. The revised helix definitions of Baldwin et al. (1997) place V:3 itself at the N-terminus of helix V and the (V:3/VI:23) vertical (Z) displacement is consequently greater in the B97 model.
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De novo reconstruction of transmembrane helix V
The foregoing analysis indicates that the Herzyk and Hubbard (1998) template better satisfies experimental constraints than does the Baldwin et al. (1997) template, the shortcomings of which relate to the position and orientation of the N-terminal section of TM helix V. This helix is the least clear feature in the Unger et al. (1997) rhodopsin projection map. Both a clockwise rotation of helix V, when viewed from the extracellular surface, and a repositioning closer to helix VI, appear necessary for opioid/ORL1 receptor modelling applications. However, rotation of the entire helix presents a problem, since highly conserved residue positions in the C-terminal section become exposed to the membrane. As a possible solution, we have rebuilt helix V with an alternative proline kink at position V:14 and reinserted it into the Baldwin et al. (1997) template. The transmembrane binding pocket region of the modified -opioid receptor structure (B97mod5) is shown in Figure 5C
.
The separation between the extracellular parts of helices V and VI is substantially reduced in the B97mod5 model and the ligand binding pocket is no longer exposed to the membrane. The distance between the V:3 and VI:23 C atoms is 10.1 Å, which is slightly longer than that in the HH98 model (8.5 Å) owing to the retention of the Baldwin et al. (1997) helix limits and the overall 23° tilt angle estimate of Unger et al. (1997). Construction of a two-residue extension to helix VI gives a V:3VI:27 C
C
inter-atomic distance of 7.6 Å, which accords both with the rhodopsin disulphide mutant cross-linking results (Yu et al., 1995
; Struthers et al., 1999
) and the upper distance limit of 13 Å required for the engineered zinc ion binding site in the
-opioid receptor (Thirstrup et al., 1996
). The improved packing of helices V and VI in the binding site region results in a 42% increase in the helix V/VI contact surface area with respect to the B97 model, with no overall loss in helix (IVII) packing interactions (Table IV
). There is also a 30% increase in the helix V/VI contact surface area compared with the HH98 model.
An orthogonal view of the three model helix V/VI pairs in the Baldwin et al. (1997) coordinate system is shown in Figure 6. Details of the corresponding helix axis parameters are given in Table V
. The principal difference between the B97 and B97mod5 models is a change in the helix V (x0 and y0) positional parameters, whilst a 5° difference in the
tilt angle permits the helix V axes of the B97 and HH98 models to intersect closer to the Z-axis origin. Figure 6
shows clearly how the smaller helix V axis tilt angle in the HH98 receptor model compensates for differences in the vertical displacement of equivalent residues, most apparent towards the helix N-terminus. The C-termini of the three helices occupy similar positions on the cytoplasmic side of the receptor, where they interact closely with the C-terminal section of TM helix III, which superposes well in the B97 and HH98 models (see Figure 4
).
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We have paid particular attention to the orientation of the N-terminal section of helix V with respect to the centre of the transmembrane ligand binding pocket. A profile of the amino acid residue entropy in the GPCR superfamily for helix V is shown in Figure 7. Helical periodicity is only evident in the C-terminal part of this helix. Pronounced troughs occur at highly conserved residue positions :11, :14, :18, :22 and :25. Profiles of the difference in relative side chain accessibilities for each
-opioid receptor model, calculated in the presence and absence of an artificial membrane, are also presented in Figure 7
. These profiles which highlight membrane-exposed residue positions exhibit essentially the same periodicity pattern as the entropy profile in the C-terminal section. In contrast, the absence of sequence entropy periodicity in the N-terminal section of helix V, where sequence variability is uniformly high, precludes the reconciliation of the variation in the accessibility difference profiles for the three models in the residue range V:3 through V:10.
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Discussion |
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Comparative (homology) modelling techniques are in practice largely founded on the extrapolation of positional and through-space constraints from one protein homologue to another. This is justified when the shared sequence identity of the two proteins is high as it has long since been recognized that structure is more highly conserved than is sequence. However, the diversity in class A rhodopsin-like GPCR sequences has become increasingly evident in recent years and an often neglected question regarding the modelling of the seven-helix transmembrane bundle is the extent to which the tertiary structure is conserved in the wider superfamily. This concern relates not only to the dependence on low-resolution rhodopsin structures and the associated tendency to arrive at a single `generic' model for all receptor types (Flower, 1999), but also to the application of constraints derived from experimental studies of other GPCRs.
Cluster analyses of multiple sequence alignments as a probe of structure and function
Our cluster analysis of a GPCR sequence alignment database of seven-helix folds (Figure 2A) suggests that the transmembrane regions of non-rhodopsins bear a closer structural resemblance to each other than to those of the vertebrate rhodopsins. However, 88% of the non-rhodopsin receptors were found to share a sequence identity of >20% with human rhodopsin over the 198 residue positions in the fold. This implies that their core main chain atoms should superimpose on those of rhodopsin with a maximum r.m.s.d. value of 1.8 Å, according to the Chothia and Lesk (1986) empirical equation. Indeed, this limit may be somewhat lower, dependent upon the magnitude of structural constraints imposed by the membrane. These statistics are particularly encouraging in the light of recent progress in the determination of rhodopsin structures by electron cryomicroscopy (Krebs et al., 1998
; Schertler 1998
).
Clustering methods may also be used to assess how well local inter-helical geometry is conserved within and between receptor families. Bench-marking against global distance relationships pertaining to the overall fold should enable regions of locally high sequence conservation to be identified in family sub-sets. Functionally important regions within a receptor family or functionally common to two or more families are expected to be particularly well conserved structurally, in much the same way as the conserved structural architecture of (homologous) enzyme active sites extends beyond the spatial disposition of conserved residues intimately involved in the catalytic act. Levels of local sequence identity are therefore likely to be higher in functionally important regions of GPCRs than in other regions that are better able to tolerate sequence, if not structural, variation.
The transmembrane cavity delimited by the extracellular parts of helices III, V, VI and VII is believed to be used by a number of different amine and peptide GPCRs (see Bikker et al., 1998). In information terms, a functional link can be said to be established between these receptor families. By combining sequence information with a rudimentary knowledge of the GPCR seven-helix fold, our objective is to identify independently receptor families sharing this functional link with the opioid/ORL1 receptor family. The demonstration of high levels of local sequence similarity, above that existing globally, would provide strong evidence that the helix sections forming this cavity are spatially similarly disposed in these families. This is of particular importance since much of the experimental evidence concerning the spatial disposition of the extracellular sections of helices V and VI stems from biochemical studies of amine receptors.
We specify the transmembrane ligand binding site by what we refer to as a three-dimensional sequence fingerprint of residue positions that are close in three-dimensional space, but more widely dispersed in one-dimensional sequence space. The migration of the opioid/ORL1 binding site sequence fingerprints towards the amine receptor cluster observed in Figure 2B is consistent with a locally higher degree of structural conservation compared with that over the entire seven-helix fold. The functional link and implied local structural similarity existing between these receptor families may extend to similarities in ligandreceptor binding modes, such as the deployment of the conserved aspartate at position III:7 as a counterion, receptor-mediated stacking interactions stabilizing heterocycle and F1/Y1 phenyl ring binding, fine tuned by interactions of hydroxyl and other polar groups to TM helices V, VI and VII. Indeed there is evidence that dopamine receptor ligands (Boublik and Funder, 1984
; Fortin et al., 1991
) and substituted benzamides (Chivers et al., 1988
) bind to opioid receptors.
Locally high levels of sequence conservation are expected in functionally important receptor regions. The converse is also likely to hold true within the constraints of structurally permissible sequence variation, as is demonstrated by the scatter in the chemokine receptor family cluster for the opioid/ORL1 binding pocket fingerprint (Figure 2B). Approaches that seek to identify changes in distance relationships between and within receptor families thus appear to provide a useful means of probing functional architecture. Potential applications in the GPCR field include the identification of transmembrane binding sites used by orphan receptors, that cannot be otherwise inferred from global sequence comparisons alone. Systematic searching of sequence variation patterns in different combinations of helixhelix contact interface fingerprints may also offer clues as to which helices undergo structural rearrangement during receptor activation. By combining sequence alignment data with a structural knowledge of one or more homologous proteins in this way, three-dimensional sequence fingerprint cluster analysis may find more general use in functional genomics as a means of validating biochemical annotations in families of homologous proteins whose functions have diversified throughout evolution.
We go on to consider below how well the available low-resolution rhodopsin templates satisfy spatial constraints deduced from the extensively studied amine receptor family, when extrapolated to the transmembrane ligand binding pocket of opioid/ORL1 receptors.
Modelling of opioid and ORL1 receptors from low-resolution rhodopsin structures
Several recently published molecular models of the opioid (Strahs and Weinstein, 1997; Paterlini et al., 1997
; Pogozheva et al., 1998
) and ORL1 (Topham et al., 1998
) receptors have relied, albeit to varying extents, on structural data from the 9 Å resolution Schertler et al. (1993) electron density projection map of the bovine rhodopsin TM domain. Whereas helices IV, V, VI and VII are seen as strong peaks normal to the membrane plane in this map, helices IIII are tilted and overlap in an arc-shaped patch of unresolved 2-D electron density. However, this arc-shaped feature extends to helix V in the Schertler and Hargrave (1995) projection map of frog rhodopsin at 6 Å resolution and only helices IV, VI and VII are clearly defined. Unger et al. (1997) have now estimated the position and tilt angles of all seven helices from a 7.5 Å resolution projection map of frog rhodopsin. Contrary to earlier indications from the Schertler et al. (1993) map that helix V is untilted, Unger et al. (1997) report a 23° tilt angle. Helices IIII are also heavily tilted (up to 29°) and helix III is more deeply buried inside the receptor than had been previously anticipated. Baldwin et al. (1997) have constructed a rhodopsin C
template, by combining the Unger et al. (1997) helix axis parameter estimates with a comprehensive sequence analysis of the GPCR superfamily. The most recent (bovine) rhodopsin 2-D projection map, obtained at 5 Å resolution by Krebs et al. (1998), is consistent with the Baldwin et al. (1997) model and confirms helix V to be highly tilted. Herzyk and Hubbard (1998) have also built a rhodopsin model, using information from the Schertler and Hargrave (1995) projection map and a conformational search technique directed by structural restraints obtained from other experimental data. Herzyk and Hubbard (1998) point to the overall similarity of their model and in particular, the positions of the helix axes, with respect to the Baldwin et al. (1997) template.
Structural examination of the transmembrane binding site in two models of the -opioid receptor TM helix bundle, based on the most recent rhodopsin templates of Baldwin et al. (1997) and Herzyk and Hubbard (1998), reveals significant differences both in the physical separation of the extracellular parts of TM helices V and VI and in the orientation of helix V in the transmembrane crevice. The lack of periodicity in GPCR sequence variability in the N-terminal (:3 to :10) section of TM helix V (Figure 7
) gives little indication as to which residues make up the interior and exterior faces of the helix. However, measurements of relative cysteine alkylation rates in wild-type and engineered
2A-adrenergic receptor mutants by Marjamäki et al. (1998, 1999) appear to have resolved this question. Their findings that residue positions V:3, V:6, V:7 and V:10 are the most reactive and therefore the most accessible to ligands are in full agreement with a survey of `loss of function' site-directed mutagenesis studies of amine receptors from 10 families (Table VI
). Side chain accessibility profiling of the B97 and HH98 membrane-embedded
-opioid receptor models (Figure 8
) shows that the Herzyk and Hubbard (1998) template-derived model provides a closer fit to the expected residue exposure pattern. Distance constraints involving residue clusters at the extremities of TM helices V and VI, deduced from protein engineering studies of the
1B-adrenergic (Hwa et al., 1995
),
-opioid (Thirstrup et al., 1996
) and rhodopsin (Yu et al., 1995
; Struthers et al., 1999
) receptors, are also better met by this template.
We have remodelled TM helix V in the Baldwin et al. (1997) template in order to satisfy amine and other receptor experimental data. This was achieved by a displacement in the plane of the membrane bringing it closer to TM helix VI, thereby also improving the structural integrity of the N-terminal Y1/F1GGF tetrapeptide opioid/ORL1 receptor binding pocket and a reconstruction of the proline kink, facilitating reorientation of the extracellular section.
Does rotation of transmembrane helix V play a role in receptor activation?
Marjamäki et al. (1999) have proposed that a rotation of TM helix V in the same sense as that described here is an integral part of the activation mechanism of the 2A-adrenergic receptor. Their conclusions are based on an analysis of the reaction kinetics of engineered receptors with the sulphydryl chemical modification reagents, CEC and MTSEA, coupled with a modelling study of the covalent complexes constructed using the Baldwin et al. (1997) rhodopsin template. Rotation of TM helix V, observed during unconstrained minimization, permits the simultaneous hydrogen bonding of the protonated nitrogen of the CEC imidazole ring with Asp113 (III:7) and the formation of covalent bond with the most reactive cysteine at position V:6. On the basis that MTSEA is both too short to interact simultaneously with the III:7 aspartate and the V:6 cysteine and reacts five times more slowly with this mutant receptor than does CEC, Marjamäki et al. (1999) reason that CEC recognizes the active state of the receptor, whereas MTSEA binds and reacts with an inactive form.
These findings further underline the inconsistency of the orientation of the N-terminal segment of helix V in the Baldwin et al. (1997) rhodopsin template with the cysteine reactivity patterns at positions V:3 and V:6V:10 towards both CEC and MTSEA. However, the evidence in favour of a rotation of TM helix V in receptor activation is less convincing. First, Marjamäki et al. (1999) make the assumption that the two binding interactions can only be made by CEC with the active receptor form. Second, the observed rate constants under conditions of quasi-equilibrium binding (Kitz and Wilson, 1962; Cornish-Bowden, 1979
) and non-saturating modifier concentrations should be considered as specificity constants (kX/KX), where KX is the dissociation constant of the complex (RX) formed between the modifier (X) and the receptor (R) and kX is the first-order rate constant for the modification reaction within the RX adsorptive complex. Receptor efficiencies (the ratio of active to inactive receptor forms in the presence of the agonist) are likely to be of the order of 102103 (Colquhoun, 1998
) and this will be reflected in the denominator (KX) of any experimentally determined specificity constant for modification. The 5-fold increase in the observed second-order rate constant for the reaction of CEC compared with MTSEA is thus somewhat less than might be expected for the selective interaction with the active conformation of the receptor. It can also be explained by differences in kX for the two modification reagents. Indeed, whereas CEC is known to react with water (Marjamäki et al., 1998
), thiosulphonates are found to be stable up to pH 11 (K.Brocklehurst, personal communication), which suggests that CEC is intrinsically the more reactive. Moreover, Struthers et al. (1999) conclude that large movements on the extracellular side of helix V relative to helix VI are not required for the activation of rhodopsin since helix V/VI cross-linked disulphide mutants retain wild-type spectral and light-dependent transducin activation properties.
Conclusions
Analysis of a multiple sequence alignment of G protein-coupled receptor seven-helix folds suggests that rhodopsin is a reasonable template from which to model the majority of non-rhodopsin GPCRs. We have compared models of the -opioid receptor seven-helix bundle constructed from the most recent rhodopsin templates of Baldwin et al. (1997) and Herzyk and Hubbard (1998). The Herzyk and Hubbard (1998) template is found to be in better accord with experimental studies of amine, opioid and rhodopsin receptors. This is partly due to the reduced physical separation of the extracellular sections of TM helices V and VI, presumed to form the rear wall of the transmembrane ligand binding site in the opioid/ORL1 receptors, but also to differences in the rotational orientation of the N-terminus of helix V. Cluster analysis of the GPCR alignment data using a three-dimensional sequence fingerprint to define the opioid/ORL1 receptor transmembrane binding site suggests that it is structurally similar to the amine receptor binding site. Our approach is based on the expectation that functionally conserved regions in homologous proteins will exhibit locally high sequence conservation, manifest by (increased) cluster compactness and relative shifts in the projections of sequence sub-sets (fingerprints) representing functional sites in principal component space. Appropriately automated, the method may be of value in functional genomics to annotate sequence data.
TM helix V in the Baldwin et al. (1997) template has been remodelled to satisfy experimental constraints better. Displacement in the membrane plane permits the closer approach of helix V to helix VI. A modified proline kink in TM helix V permits reorientation of the extracellular section such that relative side chain accessibilities in the membrane-embedded receptor model correlate with relative alkylation rates of engineered cysteine residues in the 2A-adrenergic receptor (Marjamäki et al., 1998
, 1999
). Marjamäki et al. (1999) have proposed that rotation of helix V is associated with receptor activation. We caution, however, that their conclusions are based on an incomplete kinetic analysis of the reaction of cysteine mutant receptors with sulphydryl chemical modification reagents.
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Deposition of model coordinates |
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Notes |
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Acknowledgments |
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References |
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Received December 24, 1999; revised April 10, 2000; accepted April 14, 2000.