Molecular Characterization of the Substance P·Neurokinin-1 Receptor Complex: DEVELOPMENT OF AN EXPERIMENTALLY BASED MODEL

Maria Pellegrini,Andrew A Bremer,Amy L Ulfers,Norman D Boyd,Dale F Mierke

doi:10.1074/jbc.m101057200

Maria Pellegrini, Andrew A Bremer + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m101057200

Copy DOI

Abstract

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 Met(174) and Met(181), 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 Bpa(3)SP 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.

Highlights

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed
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]
This model has proven to be very effective when applied to the design of zinc-binding sites in the seven-transmembrane helix (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

Summary

Introduction

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. 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 molecular models have a much greater uncertainty associated with them. The experimentally based conformational preferences can 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.

Methods

Results

Conclusion