A role for a helical connector between two receptor binding sites of a long-chain peptide hormone

The conformational freedom of single-chain peptide hormones, such as the 41 -amino acid hormone corticotropin releasing factor (CRF), is a major obstac le to the determination of their biologically relevant conformation, and th us hampers insights into the mechanism of ligand-receptor interaction, Sinc e N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for recept or activation. Here we show the existence of two segregated receptor bindin g sites at the N- and C termini of CRF, connection of which is essential fo r receptor binding and activation. Connection of the two binding sites by h ighly flexible E-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC50: 100-300 nM) independent of lin ker length. Connection of the two sites by an appropriate helical peptide l ed to a very potent analogue, which adopted, in contrast to CRF itself, a s table, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent a gonists; their significantly different biopotencies (EC50:0.6-50 nM), howev er, suggest the relative orientation between the two binding sites rather t han the maintenance of a distinct distance between them to be essential for a high potency.