C^α methylation in molecular recognition

Abstract

Two binding sites NK-1M (major, more abundant) and are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the C a methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven C a methylated analogues of the undecapeptide SP (from position 5±11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of C a methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven C a methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [aMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with C a methylation of SP are sequence dependent. Moreover, a single C a methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an a-aminoisobutyric acid. Furthermore, C a methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4±8 domain of other potent SP analogues.Keywords: binding sites; C a methylation; conformational analysis; molecular recognition; tachykinins.The structure determination of peptide/receptor complexes is still indirect and mainly based on the comparison of binding affinities and biological potencies of modified peptides and/or mutated receptors. These structure±activity relationships are nowadays enriched by the evolution of peptide chemistry, with new strategies for the design of constrained peptides, by spectroscopic analyses combined with calculations and modelization [1±4] and also by molecular biology leading to mutated receptors [5]. Over the past years, the tools to restrict the conformational space of a peptide have been clearly identified and their conformational behaviours have been analysed by spectroscopy, mainly NMR and CD, but also by X-ray diffraction associated with potential energy calculations. However, all these chemical modifications designed to stabilize a`b ioactive' conformation can also create repulsive interactions in the binding site, and/or induce conformational change of the peptide and (or) the receptor. Therefore, a combination of various nonredundant constraints must be introduced in the endogenous peptide to ascertain its postulated bioactive conformation.In this study, the C a methylation constraint [6] was used to probe the bioactive conformation(s) of substance ...

Involvement of the Second Extracellular Loop (E2) of the Neurokinin-1 Receptor in the Binding of Substance P

“…This analog also activates with potencies similar to those of SP the phospholipase C and adenylate cyclase second messenger pathways (Table I 11 ]SP-(7-11) (for NK-1m binding site), and potencies to activate phospholipase C and adenylate cyclase were determined as described under "Experimental Procedures." Data presented are the mean Ϯ S.E.…”

“…Because the mass of the photoreactive SP analog, [BAPA 0 ,(p-Bz)Phe 8 ]SP, is (MH ϩ ) ϭ 1808.89, the two peptides identified by MS analysis corresponded to peptide fragments from the receptor with a mass of 1171.5 and 1008.5 atomic mass units. Taking into account the uncertainties on the mass measurements (Ͻ0.1 atomic mass unit) and considering that the ligand-receptor complex was digested both by trypsin and also, as described previously, by trypsin-derived chymotrypsin enzymatic activities (14) 11 ]SP-(7-11) on the rat or human NK-1 receptor gave by MS analysis two peptides with quasi-molecular ions at m/z 2446.1 and 2283.0 (Fig. 2a), respectively.…”

“…Binding experiments with SP analogs in tissues and in cells transfected with the NK-1 receptor have shown that two types of non-stoichiometric binding sites with distinct pharmacological profiles are associated with the NK-1 receptor (5)(6)(7)(8)(9)(10)(11)(12). In CHO cells, B max values for the two binding sites were found to be 6000 fmol/mg proteins and 800 fmol/mg proteins for the major binding site ( 11 ]SP-(7-11), respectively (7). With the same clone it has been shown that in the presence of cholera toxin, the tachykinin NK-1 receptor is uncoupled to G-proteins (7).…”

“…In the CHO clone used herein, the more and less abundant binding sites represent 85 and 15% of the total population of receptors (6 pmol/mg of proteins), respectively (7). Substance P binds the two binding sites with high affinity, whereas some C-terminal fragment analogs of substance P and some substance P-(1-11) analogs, as well as the endogenous tachykinin NK-2 ligand neurokinin A, bind only the less abundant one (7)(8)(9)(10)(11)(12). Although several structure-activity relationship studies have been carried out, little is known about the differences in the molecular recognition of these two binding sites (8 -12).…”

“…Both photoactivatable SP analogs, [BAPA-Lys 6 ,(p-Bz)Phe 8 ,Pro 9 ,-Met (O 2 ) 11 ]SP- (7)(8)(9)(10)(11) and [BAPA 0 ,(p-Bz)Phe 8 ]SP, contain a biotinyl sulfone moiety at the N-terminal end, which is separated from the first amino acid by an aminopentanoic acid flexible spacer (BAPA). After photolabeling and enzymatic digestion(s), this biotinyl sulfone moiety is used to purify the fragment of interest via streptavidin-coated magnetic beads (14,15).…”