Photoaffinity Labeling of Mutant Neurokinin-1 Receptors Reveals Additional Structural Features of the Substance P/NK-1 Receptor Complex

Abstract

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid

“…Numerous studies have aimed at identifying regions in the NK1 receptor involved in the binding of its ligands (2,4,8,9,17,18,(35)(36)(37)(38). This receptor is of particular interest because it can bind three different endogenous peptides as well as a number of synthetic ligands, one of which is an antiemetic drug on the market (3,5,39,40).…”

“…1) is critical for NK1-SP interaction (33). Furthermore, the two loop residues Met-174(C-6) and Met-181(Cϩ1) are close to the ligand in NK1-SP complexes (15,17,19,21). Specifically, the conserved disulfide bridge positions Met-181(Cϩ1) approximately one helical turn above His-108 (colored green in Fig.…”

“…1, highlighted in green) (8 -11). A photocross-linking approach using synthetic artificial SP analogues containing an unnatural amino acid, p-benzoyl-L-phenylalanine (BzF), was also employed to investigate how SP interacts with the NK1 receptor (12)(13)(14)(15)(16)(17)(18)(19). To identify the cross-linking site of the SP analog on the NK1 receptor, this method required that receptor residues or fragments that cross-linked to the SP analog were determined using proteolytic digestion and/or mass spectrometry of the cross-linked complex.…”

“…In addition, the substitution of Phe-8 of SP, SP(BzF8), resulted in cross-linking to Met-181(Cϩ1) and possibly Met-174(C-6) (highlighted in red, Fig. 1A) (15,17,19,21). Taken together, the previous ligand binding studies indicated that the SP peptide presumably interacts with epitopes presented on the outer part of the extracellular portion of the NK1 receptor, but the overall results have not led to a satisfactory refined model of how SP binds to the NK1 receptor.…”

“…cross-linking to Met-174(C-6) and Met-181(Cϩ1) in the ECLII of NK1 had previously been suggested (15,17,19,21), we decided to further investigate the ECLII region. It has been established that Cys-180 forms an intramolecular disulfide bridge with Cys-105 that is essential for SP binding (33).…”