An NMR Study of the Interaction of ¹⁵N‐Labelled Bradykinin with an Antibody Mimic of the Bradykinin B2 Receptor

Abstract

An isotope-edited NMR study of the peptide hormone bradykinin (RPPGFSPFR) bound to the Fab fragment of a monoclonal antibody against bradykinin (MBK3) is reported. MBK3 was previously shown to provide a binding site model of the B2 bradykinin receptor [Haasemann, M., Buschko, J., Faussner, A,, Roscher, A. A., Hoebeke, J., Burch, R. M. & Miiller-Esterl, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Irnrnunol. 147, 3882-38921. Bradykinin was obtained in a uniformly l5N-labe1led form using recombinant expression of a fusion protein consisting of the glutathione-binding domain of glutathione S-transferase fused to residues 354-375 of the high-moleculx-mass kininogen from which bradykinin was released by proteolytic digestion with its natural protease plasma kallikrein. Bradykinin forms a complex with the Fab fragment of MBK3 which exchanges slowly on the NMR time scale. The I5N and 'H resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the I5N and 'H linewidths indicate that the hydrodynamic behaviour of bound bradykinin is dominated by the high-molecular-mass Fab fragment. The NMR data indicate that essentially the entire nonapeptide is involved in binding. The kinetics of the ligand-exchange process, together with resonance assignments obtained via exchange spectroscopy, indicate that bradykinin binds to MBK3 only in the all-trans conformation at all three XaaPro amide bonds. NH-NH NOE connectivities suggest that bradykinin is bound in an extended conformation. The spectroscopic data obtained from this study are compared to recently proposed computational models of the conformation of bradykinin bound to the B2 receptor.

Cis–Trans Isomerizations of Proline Residues Are Key to Bradykinin Conformations

“…No definitive structural information about the N-terminal portion of the peptide exists. Instead, this region is described as a highly flexible 12 random coil 7,13 that is believed to be unstructured in solution. 14 Recently we found evidence for as many as 10 populations of different structural forms of free BK, that vary depending on the solution composition; 15 this plurality of states is consistent with the inability to characterize the N-terminal region of the peptide.…”

Correlations in Palmitoylation and Multiple Phosphorylation of Rat Bradykinin B2 Receptor in Chinese Hamster Ovary Cells

“…The consensus bradykinin receptor topology predicts four extracellular domains (ED 1 1-4) and intracellular domains (ID1-4), each separated by seven transmembrane helical regions (TM1-7) spanning the lipid bilayer. B 2 receptors are post-translationally modified by glycosylation (5), phosphorylation (6), and presumably by palmitoylation of the cytoplasmic surface.…”

“…Based on homology with other G protein-coupled receptors there have been indications regarding possible structural features probed by agonists, antagonists, and anti-idiotypic antibodies (5,6), functional regions (7), and sites of post-transitional modifications for the B 2 receptor (8). Site-directed mutagenesis indicated the importance of Tyr residues and of ID4 for the signaling and the uptake of the B 2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9).…”

Evidence for the Absolute Conformational Specificity of the Intestinal H+/Peptide Symporter, PEPT1

“…On the other hand, peptidyl prolyl cis/trans isomerases utilize both cis and trans isomers of peptide and protein substrates (8). For peptide receptors, a preference either for cis conformers (10) or for alltrans conformers (11,12) has been hypothesized. Whether peptide transporters are able to differentiate between cis and trans conformers of di-and tripeptides has not yet been shown.…”