An NMR study of the interaction of 15N-labelled bradykinin with an antibody mimic of the bradykinin B2 receptor.
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. & Muller-Esterl, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882-3892]. Bradykinin was obtained in a uniformly 15N-labelled 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-molecular-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 15N and 1H resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the 15N and 1H 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 Xaa-Pro 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.
Ottleben
H
H
Institut für Molekulare Biotechnologie, Jena, Germany.
Haasemann
M
M
Ramachandran
R
R
Görlach
M
M
Müller-Esterl
W
W
Brown
L R
LR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001920:Bradykinin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009587:Nitrogen Isotopes; D011485:Protein Binding; D011487:Protein Conformation; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D009587:Nitrogen Isotopes; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D001920:Bradykinin