AN NMR-STUDY OF THE INTERACTION OF N-15-LABELED BRADYKININ WITH AN ANTIBODY MIMIC OF THE BRADYKININ B2 RECEPTOR

An isotope-edited NMR study of the peptide hormone bradykinin (RPPGFSP FR) bound to the Fab fragment of a monoclonal antibody against bradyki nin (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. & Muller-Ester l, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882-3892]. Bradykinin was obtai ned in a uniformly N-15-labelled form using recombinant expression of a fusion protein consisting of the glutathione-binding domain of gluta thione S-transferase fused to residues 354-375 of the high-molecular-m ass kininogen from which bradykinin was released by proteolytic digest ion with its natural protease plasma kallikrein. Bradykinin forms a co mplex with the Fab fragment of MBK3 which exchanges slowly on the NMR time scale. The N-15 and H-1 resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the N-15 and H-1 linewidths indicate that the hydrodynamic behaviour of bound bradykinin is dominated by the high-mo lecular-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 exc hange spectroscopy, indicate that bradykinin binds to MBK3 only in the all-trans conformation at all three Xaa-Pro amide bonds. NH-NH NOE co nnectivities suggest that bradykinin is bound in an extended conformat ion. The spectroscopic data obtained from this study are compared to r ecently proposed computational models of the conformation of bradykini n bound to the B2 receptor.