Mutations in the B2 Bradykinin Receptor Reveal a Different Pattern of Contacts for Peptidic Agonists and Peptidic Antagonists

• Published in: The Journal of biological chemistry 1996-11, Vol.271 (45), p.28277-28286
• Main Authors: Jarnagin, Kurt, Bhakta, Sunil, Zuppan, Patty, Yee, Calvin, Ho, Teresa, Phan, Thu, Tahilramani, Ram, Pease, JoeH.B., Miller, Aaron, Freedman, Richard
• Format: Article
• Language: English
• Subjects: Adrenergic beta-Antagonists - pharmacology; Amino Acid Sequence; Animals; Binding Sites - genetics; Binding, Competitive; Bradykinin - analogs & derivatives; Bradykinin - metabolism; Bradykinin - pharmacology; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Rats; Receptor, Bradykinin B2; Receptors, Bradykinin - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.45.28277

The B2 bradykinin receptor, a seven-helix transmembrane receptor, binds the inflammatory mediator bradykinin (BK) and the structurally related peptide antagonist HOE-140. The binding of HOE-140 and the binding of bradykinin are mutually exclusive and competitive. Fifty-four site-specific receptor mutations were made. BK's affinity is reduced 2200-fold by F261A, 490-fold by T265A, 60-fold by D286A, and 3-10-fold by N200A, D268A, and Q290A. In contrast, HOE-140 affinity is reduced less than 7-fold by F254A, F261A, Y297A, and Q262A. The almost complete discordance of mutations that affect BK binding versus HOE-140 binding is surprising, but it was paralleled by the effect of single changes in BK and HOE-140. [Ala9]BK and [Ala6]BK are reduced in receptor binding affinity 27,000- and 150-fold, respectively, while [Ala9]HOE-140 affinity is reduced 7-fold and [Ala6]HOE-140 affinity is unchanged. NMR spectroscopy of all of the peptidic analogs of BK or HOE-140 revealed a β-turn at the C terminus. Models of the receptor-ligand complex suggested that bradykinin is bound partially inside the helical bundle of the receptor with the amino terminus emerging from the extracellular side of helical bundle. In these models a salt bridge occurs between Arg9 and Asp286; the models also place Phe8 in a hydrophobic pocket midway through the transmembrane region. Models of HOE-140 binding to the receptor place its β-turn one α-helical turn deeper and closer to helix 7 and helix 1 as compared with bradykinin-receptor complex models.