Molecular Characterization of the Substance P·Neurokinin-1 Receptor Complex

DEVELOPMENT OF AN EXPERIMENTALLY BASED MODEL*

Maria PellegriniDagger , Andrew A. Bremer§, Amy L. UlfersDagger , Norman D. Boyd§, and Dale F. MierkeDagger ||

From the Dagger  Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 and the § Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, February 4, 2001, and in revised form, March 29, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. The ligand·receptor complex is based on experimental data from a series of photoaffinity labeling experiments and spectroscopic structural studies of extracellular domains of the NK-1R. Using the ligand/receptor contact points derived from incorporation of photolabile probes (p-benzoylphenylalanine (Bpa)) into SP at positions 3, 4, and 8 and molecular dynamics simulations, the topological arrangement of SP within the NK-1R is explored. The model incorporates the structural features, determined by high resolution NMR studies, of the second extracellular loop (EC2), containing contact points Met174 and Met181, providing important experimentally based conformational preferences for the simulations. Extensive molecular dynamics simulations were carried out to probe the nature of the two contact points identified for the Bpa3SP analogue (Bremer, A. A., Leeman, S. E., and Boyd, N. D. (2001) J. Biol. Chem. 276, 22857-22861), examining modes of ligand binding in which the contact points are fulfilled sequentially or simultaneously. The resulting ligand·receptor complex has the N terminus of SP projecting toward transmembrane helix (TM) 1 and TM2, exposed to the solvent. The C terminus of SP is located in proximity to TM5 and TM6, deeper into the central core of the receptor. The central portion of the ligand, adopting a helical loop conformation, is found to align with the helices of the central regions EC2 and EC3, forming important interactions with both of these extracellular domains. The model developed here allows for atomic insight into the biochemical data currently available and guides targeting of future experiments to probe specific ligand/receptor interactions and thereby furthers our understanding of the functioning of this important neuropeptide system.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The neurokinin-1 receptor (NK-1R)1 is a member of the G protein-coupled receptor family of integral membrane proteins activated by the tachykinin peptide hormones substance P (SP) and neurokinin A. Over the years, a number of different methods have been employed to characterize the NK-1R and the mode in which it interacts with these peptide ligands as well as with a number of non-peptide antagonists that target the NK-1R. Based on site-directed mutagenesis, fluorescence experiments, and engineering of zinc-binding pockets, a number of residues involved in the binding of the non-peptide antagonists has been identified (1-7). Most of these residues are located within the hydrophobic core formed by the seven transmembrane helices (TM), predominantly in the region of TM5 and TM6. Using these putative sites of ligand/receptor interaction and computational models of the receptor, insight into the mode of ligand binding of these antagonists has been developed (7-9).

The development of a similar understanding of the binding of the peptide ligands SP and neurokinin A to the NK-1R has been much more challenging. Most of the residues identified as important for SP binding are in the N terminus or extracellular loops of the NK-1R (10-12). These regions are not structurally characterized (in contrast to the bundle of transmembrane helices, homology modeling based on rhodopsin is not possible for the loops and termini); and therefore, molecular models have a much greater uncertainty associated with them.

To address these issues, we have undertaken the structural characterization of the extracellular domains of the NK-1R (13). The experimentally based conformational preferences can then be incorporated into the model of the full-length receptor. Coupling these data with the identification of ligand/receptor contact points as determined by photoaffinity labeling provides unique insight into the interactions involved in the binding of SP to the NK-1R.

Here we describe a model for the binding of SP to the NK-1R that incorporates all of the experimental data currently available. The model contains the structural features of the second extracellular loop (EC2) of the NK-1R as recently determined by high resolution NMR (13). The loop plays an important role in SP binding, as evidenced by previous photoaffinity studies (13-17). Both position 4 (Bpa4SP) and position 8 (Bpa8SP) of SP form covalent linkages with residues in EC2, Met174 and Met181, respectively. As described in the accompanying article (17), a residue in EC2, between residues 173 and 177, is photoaffinity labeled by Bpa3SP. Interestingly, Bpa3SP also forms a covalent bond with a residue in the proximal N terminus of the NK-1R (residues 11-21). The structural consequences of Bpa3SP interacting with two sites, either sequentially or simultaneously, are examined here by extensive molecular dynamics simulations. Incorporating all of the results from photoaffinity labeling and structural studies provides for one of the most advanced, experimentally based models of any peptide hormone G protein-coupled receptor. The results presented here may therefore provide important insight into peptide/hormone seven-TM receptors beyond those for neurokinin.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The molecular model of the human NK-1R was built using the topological arrangement of the transmembrane helices of rhodopsin (18-20) following procedures detailed previously (21, 22). The loops connecting the transmembrane helices were generated using a metric matrix distance geometry program, employing the distances between the transmembrane helices as restraints. The structural features of the second extracellular loop, NK-1R-(162-198), as determined by high resolution NMR were directly incorporated into the model. The disulfide bond between Cys105 and Cys180, in the first and second extracellular loops, respectively, was created. In an attempt to develop the conformational preferences of the other extracellular domains of the NK-1R, the corresponding sequences were submitted to a BLAST search (23). The homologous regions of each of these protein structures were analyzed for conformational features. The secondary structural features were then incorporated into the molecular model (21, 22, 24).

Preliminary simulations were run to determine the preferred docking mode of the ligand to the receptor. Using three different orientations of SP placed above the receptor, extensive simulated annealing cycles (1000 to 300 K over 100 ps) were run in conjunction with distance restraints derived from the photoaffinity labeling experiments. The ligand·receptor complexes that best fulfilled the experimental restraints were utilized as starting structures for the following simulations. Based on numerous NMR studies of SP, an alpha -helix in the center of the sequence, from Gln4 to Phe8, was assumed (25-29).

To guide the docking of the ligand to the receptor, restraints with a target value of 12 Å were applied for the following ligand/receptor pairs of atoms: CG Phe8/SG Met181, CG Gln4/SG Met174, CG Lys3/SG Met174, and CG Lys3/CG Glu21, with a force constant of 500 kJ mol-1 nm-1. The latter two distance restraints, corresponding to the results presented in the accompanying article (17), were applied individually during separate simulations as well as simultaneously. The distance of 12 Å was chosen to provide ample freedom for the receptor and ligand to adjust to the constraint, to optimize the ligand/receptor interactions, and to account for the incorporation of a Bpa-containing ligand used in the photoaffinity labeling experiments.

To refine the molecular model, molecular dynamics simulations and energy minimization were carried out with the GROMACS program (30). The membrane environment was mimicked by a 40-Å layer of decane molecules, with ~40-Å layers of water above and below (31). The simulation cell was 132 × 81 × 71 Å and consisted of 12,906 water molecules and 518 decane molecules. The seven-helix bundle of the receptor was placed in the decane layer with the extra- and intracellular regions within the water phases. All molecular dynamics (MD) simulations were carried out on an SGI Origin 2000 computer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The structure and arrangement of the transmembrane helices of our model of the NK-1R are based on the experimental data reported for rhodopsin (18-20). This model has proven to be very effective when applied to the design of zinc-binding sites in the seven-TM bundle of the NK-1R (6, 7, 32, 33), supporting the use of rhodopsin as a good template for the modeling of peptide-based G protein-coupled receptors of class I. Indeed, during the simulations, the helical bundle was stable, undergoing very minor displacements (<1.0-Å root mean square deviation); the resulting structures were found to be in accord with the results from the incorporation of zinc-binding sites.

For the modeling of the extracellular domains of the NK-1R, two different sources were used to provide structurally relevant data: experimentally based spectroscopic studies and theoretically based homology analysis. The NMR-derived structure of the receptor fragment NK-1R-(162-198), consisting of the entire second extracellular loop, was incorporated into the model (13). Homology modeling, following published procedures (21, 22, 24), was used in an attempt to obtain structural information for the remaining extracellular loops and N terminus. A number of structures with sequences homologous to these regions of the NK-1R were identified. A comparison of the secondary structure motifs of these homologous regions suggests that the central portion of both EC1 (Val94-Trp98) and EC3 (Asp276-Ile283) has a propensity to form an alpha -helix. In addition, the far N terminus, consisting of Asn3-Asp10, was found to be alpha -helical, and a turn was found for Pro13-Asn14. These secondary structural elements were introduced into the initial model of the receptor.

The contact points identified from photoaffinity labeling were used as distance restraints during the MD simulations. The different sites identified with Bpa3SP were examined individually and concurrently with separate simulations. The previously determined contact points (e.g. Bpa4SP/Met174 and Bpa8SP/Met181) (13, 15, 16) were utilized during all of the simulations. The results for Bpa3SP required special handling. The identification of two distinct regions of the NK-1R (EC2 and N terminus) that form covalent linkages with Bpa3SP presents two possible scenarios. Given the short distance required for photoaffinity covalent bond formation (values of 3.1 Å are assumed (34)), it is clear that the benzoylphenylalanine must be in close proximity to the identified contact point. The first scenario has the N terminus and EC2 of the NK-1R in close proximity to each other, and therefore, Bpa3SP can interact with both. The other possibility has the N terminus and EC2 distal from each other, and Bpa3 is in close proximity to either one at different times (e.g. during the binding process, Bpa3 is close to the N terminus and then to EC2 or vice versa); or there are two equilibrium binding modes (in one, Bpa3 is close to the N terminus; in the other, it is close to EC2). To explore these different possibilities for Bpa3SP, three different simulations were run starting with SP in the bound state using the distance restraints generated from Bpa4SP and Bpa8SP. The simulation employing a distance restraint between Bpa3SP and the N terminus alone was deemed uninformative since the distance between Bpa3 and Met174 satisfied the restraint anyway (recall that there is a restraint between SP Gln4 and NK-1R Met174 based on previous photoaffinity labeling experiments (16)). Therefore, we describe below the results from the simulations in which Lys3 of SP is restrained to Met174 alone and to both Met174 and the N terminus (residues 11-21) simultaneously.

In both of the simulations, the overall orientation of SP within the receptor is very similar. The N-terminal portion of SP, Arg1-Pro2-Lys3, projects toward TM1 and the proximal N terminus of the NK-1R. The C terminus of the ligand, Gly9-Met10-Leu11-NH2, projects toward the extracellular end of TM3 and TM6 and deeper into the core of the seven transmembrane helices. Concerning the extracellular domains, the helix centrally located in EC2 (13) and the putative helix in EC3 align in an almost parallel fashion with the helical loop in the middle of SP. The distal portion of EC2 is not involved in stabilizing the bound ligand. Many of these features are shown in Fig. 1. The C-terminal portion of EC2 lies over the ligand, forming a number of important ligand/receptor contacts.


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Fig. 1.   Model of the NK-1R with SP resulting from MD simulations using structural data from NMR and homology modeling and contact points from photoaffinity labeling experiments. The ligand SP is depicted as sticks. The N terminus (NT), TM1, and TM4-EC2-TM5 are light green, whereas TM2-EC1-TM3 and TM6-EC3-TM7 are dark green. The disulfide bridge between EC1 and EC2, Cys105-Cys180, is shown in yellow.

A superposition of the transmembrane helices and extracellular domains (backbone atoms of residues 30-56, 69-131, 144-218, and 244-306), excluding the N terminus, of the resulting structures from the two simulations produced a root mean square deviation of 1.0 Å. Including the ligand in the superposition produced a root mean square deviation of 1.2 Å. An illustration of the ligand structures from the former superposition of the receptors is shown in Fig. 2. The conformation of the ligand from the two simulations is similar; when distance restraints between both the N terminus and EC2 to SP Lys3 are applied, the ligand structure is slightly elongated, resulting from the N terminus lying over the ligand. Importantly, the orientation and displacement of the side chains of SP are almost identical, especially in the C terminus of the ligand. The variation in the N terminus of the ligand can be attributed to the close proximity of the N terminus of the NK-1R brought about by the two distance restraints applied to Lys3 in one of the simulations.


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Fig. 2.   Comparison of the orientation of SP while bound to the NK-1R from MD simulations using only Bpa3SP/Met174 as a distance restraint (blue, ball-and-sticks) and with Bpa3SP both to the N terminus and EC2 (yellow, sticks). The resulting structures from the simulations were superimposed using the heavy backbone atoms of TM1, TM2-EC1-TM3, TM4-EC2-TM5, and TM6-EC3-TM7.

Concentrating on the C terminus of the ligand, clearly the resulting mode of binding is not altered by including the distance constraint between position 3 of SP and the N terminus. During simulations utilizing only the Bpa3SP/Met174 contact point, the N terminus of the NK-1R does not approach the central TM core of the receptor, maintaining the orientation and topology of the starting structure. Importantly, the secondary structure elements postulated from homology modeling are also maintained during the simulation and therefore are energetically stable by the force field. In contrast, when the distance restraints between Lys3 of SP and the N terminus and EC2 are applied simultaneously, the N terminus of the NK-1R folds up and over the N-terminal portion of the ligand, forming a number of additional ligand/receptor contacts.

As expected, most of the ligand/receptor interactions are concentrated on the extracellular portion of the central core formed by the transmembrane helices. In Fig. 3, many of these interactions are depicted. The receptor has been cut in half, with TM2-EC1-TM3 and TM4-EC2-TM5 displayed on one panel and N terminus-TM1 and TM6-EC3-TM7 on the other. The residues of the NK-1R forming important interactions with SP are depicted.


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Fig. 3.   Illustration of many of the interactions between SP and the NK-1R. The receptor has been split in half, with N terminus-TM1 and TM6-EC3-TM7 shown in the upper panel and TM2-EC2-TM3 and TM4-EC2-TM5 shown in the lower panel. SP is depicted as sticks in light gray and denoted using the three-letter code. The residues of the receptor are shown as ball-and-sticks and denoted using the one-letter code.

Beginning with Arg1 of SP, the side chain is found to project out away from the receptor, interacting with the solvent in the simulation using only the distance restraint to EC2. In the second simulation, in which the N terminus folds over the ligand, a number of ligand/receptor interactions are formed. Favorable coulombic interactions include SP Arg1 with the side chain of receptor Asp8 and Glu21. The guanidinyl function of Arg1 is also stabilized by interactions with NK-1R Ser16.

In both simulations, SP Pro2 is directed downwards, forming a large number of interactions with the NK-1R. The side chain is found in a hydrophobic pocket formed by NK-1R Asn96, Tyr100, and Tyr104. In the simulation utilizing the distance restraint to the N terminus of the NK-1R, Val4, Met7, and Phe12 contribute to the hydrophobic pocket for SP Pro2 from the extracellular face.

The side chain of SP Lys3 is projecting away from the central core of the receptor, toward the solvent, forming only minimal contacts with the receptor. In the second conformation, interaction between the side chain amino group and NK-1R Asn18 is observed. Consistent with the application of the distance restraints derived from the photoaffinity labeling experiments, the distance between the side chains of SP Lys3 and NK-1R Leu11, Pro13, Ile15, Met174, Pro175, and Met181 are 4.0, 4.6, 5.6, 3.6, 6.5, and 5.5 Å, respectively.

The second proline of SP, Pro4, projects toward the extracellular end of TM1 and EC1, in a similar fashion as described for Pro2. Both Trp30 of the NK-1R proximal N terminus and Tyr104 of EC1 are in close proximity in both simulations. In the second simulation, Phe12 of the NK-1R far N terminus folds over, forming the pocket for SP Pro4. The distance between the CG of SP Pro4 and the CE of NK-1R Met174 is under 6.0 Å.

Continuing along SP, Gln5 and Gln6 are found to project in opposite directions, with Gln5 directed toward the solvent (with minimal interaction with the NK-1R) and Gln6 forming a number of interactions deep within the central core of the receptor. The backbone carbonyl of Gln6 forms a hydrogen bond with the side chain amino group of Lys177 of the NK-1R. The side chain amide of Gln6 is found sandwiched between a group of aromatic residues, including NK-1R Tyr99, Tyr104, Phe107, and His108.

Based on previous studies substituting the amino acids of SP, Phe7 has been shown to be vital for binding to and activation of the NK-1R. Therefore, it is not surprising to find an extremely large number of ligand/receptor interactions. In Fig. 4, all of the residues within a 6-Å radius of Phe7 are depicted. The residues are from the N terminus (Asn18, Thr19, Glu21, Gln31, and Ile32), EC1 (Phe107), EC2 (Arg177), and EC3 (Leu277, Tyr278, Leu279, Lys280, Phe282, and Gln284) of the NK-1R. During the simulations, the side chain of Phe7 undergoes transitions between the different chi 1 rotamers. Energy minimization of the three different conformations results in low energy structures, without significantly altering the ligand/receptor contacts. This flexibility within the binding pocket could indicate an important role in activation or differences between the high and low affinity states of ligand binding.


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Fig. 4.   Residues of the NK-1R creating the binding pocket for Phe7 of SP in the ligand·receptor complex. The backbone of the receptor is shown as dark-green ribbons. The residues of the receptor contributing to the binding pocket for Phe7 are illustrated. The ligand is displayed as lines in gray (carbon), red (oxygen), and blue (nitrogen); Phe7 is depicted as Corey-Pauling-Koltun spheres. NT, N terminus.

Position 8 of SP has been utilized for the incorporation of residues containing a photolabile moiety during the course of the photoaffinity labeling experiments with only a modest decrease in affinity for the NK-1R. Our results illustrate a number of interactions between Phe8 and hydrophobic residues of EC2 (Met181, Ile182, and Trp184), close to the disulfide bond that covalently connects EC2 to EC1 (i.e. Cys105-Cys180).

The hydrophobic side chains of the C-terminal residues of SP, Leu10-Met11, are both projecting into the central core formed by the transmembrane helices, occupying hydrophobic pockets formed from a large number of residues mainly from TM3, TM6, and TM7. Phe107 and His108 from TM3; Trp184 from EC2; and Ile283, Gln284, and Trp297 from TM7 contribute to the binding pocket for SP Leu10. The binding pocket for SP Met11 is formed by Ile113 (TM3); Trp196 (TM5); and Pro268, Pro271, and Trp272 (TM6). The C-terminal amide forms a number of important contacts with the receptor, including the backbone of Pro271 and side chains of Lys106, Trp184, Tyr272, and Lys280. The binding pocket for SP Met11-NH2 is illustrated in Fig. 5.


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Fig. 5.   Representation of the binding pocket for the C terminus of SP, Met11-NH2, in the NK-1R. The portions of the receptor contributing to the pocket are shown as dark-green ribbons, with the residues labeled using the one-letter code. The residues of SP are labeled using the three-letter code.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A number of studies aiming to identify the regions of the NK-1R involved in the binding of SP have appeared. These investigations fall into two general classes: mutational studies of the receptor and photoaffinity labeling studies. In mutational studies, the receptor is altered, and the effects on ligand binding are monitored. This classification includes engineering of zinc-binding sites and introducing chemically reactive cysteine residues for labeling (e.g. fluorescent tag) or formation of disulfide bonds. Photoaffinity labeling studies provide evidence for direct contacts between ligand and receptor by formation of covalent bonds. Results from the former were used to establish the starting structure (particularly the zinc-binding pockets), whereas findings from the latter were used during the simulations as experimentally based restraints for the interaction of SP with the NK-1R.

One of the major topological features of the extracellular region of the NK-1R is the presence of a disulfide bond connecting the extracellular end of TM3 and EC2. This bond is observed in many seven-TM receptors and, in some cases, is the most conserved feature between distantly related receptors. In the NK-1R, the disulfide bond between Cys105 (on the extracellular end of TM3) and Cys180 (located centrally in EC2) has been shown to be essential for high affinity binding of the SP ligand (14). Interestingly, engineering of additional cysteines into the NK-1R does not disrupt the wild-type disulfide bond (35). The presence of the disulfide linkage leads to two pseudo-loops out of EC2, connecting TM3 to TM4 and TM3 to TM5. Importantly, many of the residues that have been implicated in SP binding by mutational or photoaffinity labeling studies are in close proximity to this disulfide bond (14). Thus, the convergence of three extracellular domains of the receptor at the top of TM3, centrally located in the core of the seven-TM receptor, seems to be intimately involved in the binding of SP. Indeed, the resulting binding mode shows a parallel arrangement of the helix-like structure of SP with the helix in the center of EC2, with a large number of ligand/receptor interactions. The C terminus of SP is in close proximity to the extracellular ends of TM3, TM6, and TM7. The interactions with TM3 are particularly interesting because of the unusual orientation within the seven-TM bundle, traversing the membrane diagonally with respect to the other six transmembrane helices (20). The crystal structure of rhodopsin has TM3 in the center of the TM bundle, involved in several key interhelical interactions.

The binding mode of SP described here is in perfect accord with the results from a series of fluorescence-based experiments (4). Placing fluorescent probes at the N and C termini of SP and using aqueous soluble quenching agents, it was clearly demonstrated that both of the termini of SP are solvent-accessible when bound to the NK-1R. Importantly, positions 1 and 3 of SP were deemed to be much more solvent-accessible than the C terminus (4).

According to the results of the MD simulations carried out here, the covalent linkage between Bpa3SP and the N terminus of the NK-1R requires the N terminus to fold over on top of the seven-TM bundle, forming a number of contacts with the ligand. In the model, Lys3 of SP is found in close proximity to a number of hydrophobic residues of the NK-1R N terminus (Leu11, Pro13, and Ile15), suggesting that the substitution with Bpa may enhance this interaction and indeed result in the covalent linkage to the N-terminal fragment of residues 11-21. These residues are substantially closer together than the upper bound of 12 Å imposed during the simulations as distance restraints.

The conformation of the N terminus of the NK-1R, determined from the MD simulations carried out here, must be regarded as only one possible result consistent with all of the experimental data in hand. Current computational methodologies do not allow for a full exploration of all possibilities. Nonetheless, the conformation resulting from the simulations not only fulfills the experimental restraints applied during the simulation, but can also account for previous results from site-directed mutagenesis and chimera studies (10, 11). These studies illustrate that the N terminus of the NK-1R, including residues 14-29, is required for high affinity peptide binding (10, 11), with Asn23, Gln24, and Phe25 being particularly important (36, 37). Asn23 and Gln24 are conserved between the NK-1R and NK-3R, and Phe25 is conserved among all three tachykinin receptor types; it is therefore intriguing that the covalent attachment site of Bpa3SP is just upstream from these residues. The conservation of these residues has led to the speculation that the Asn23-Phe25 region of the NK-1R may play an important structural role in positioning or stabilizing the tertiary structure of the N terminus. Indeed, in the resulting model, these residues are at the point where the N terminus bends; the residues preceding Asn23 are projecting over the seven-TM core, whereas the residues after Phe25 are largely associated with the membrane (e.g. the hydrophobic residues Trp30, Ile32, Val33, and Leu34) before the beginning of TM1 at Trp35.

The N-linked glycosylation of the N terminus of the NK-1R (Asn14 and Asn18) would certainly play an important role in the proposed folding of the N terminus over the ligand in the central core of the seven transmembrane helices. The presence of the carbohydrate side chain residues does not appear to be necessary for ligand binding to the NK-1R as determined independently by photoaffinity labeling experiments (38) and receptor mutational analysis (10). It has been suggested that differential N-linked glycosylation may function to maintain membrane receptors in different agonist binding states in vivo (39), a hypothesis that may receive more attention now that both SP and neurokinin A are reported to be high affinity NK-1R functional agonists (40-42). The N-linked carbohydrate residues may also function to kinetically modulate the conformational conversion from a low affinity agonist binding state on the NK-1R into a high affinity agonist binding state. One possibility is that the N terminus folds over the central core of the receptor, inhibiting the dissociation of SP, thereby producing the high affinity state. In the binding mode proposed here, both Asn14 and Asn18 are projecting out away from the ligand, therefore consistent with glycosylation. Currently, the role of the N terminus of the receptor in ligand binding is still unresolved, and additional experimental data are required. Experiments toward this end are currently underway in our laboratories.

    FOOTNOTES

* This work was supported in part by Grants NS-31346 (to N. D. B.) and GM-54082 (to D. F. M.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology and Experimental Therapeutics, L-611, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4387; Fax: 617-638-4329; E-mail: nboyd@bu.edu.

|| To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Div. of Biology and Medicine, P. O. Box G-B491, Brown University, Providence, RI 02912. Tel.: 401-863-2139; Fax: 401-863-1595; E-mail: dale_mierke@brown.edu.

Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M101057200

    ABBREVIATIONS

The abbreviations used are: NK-1R, neurokinin-1 receptor; SP, substance P; TM, transmembrane helix; EC, extracellular loop; Bpa, p-benzoylphenylalanine; MD, molecular dynamics.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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