MONOCYCLIC AND BICYCLIC ANALOGS OF PARATHYROID HORMONE-RELATED PROTEIN .2. CONFORMATIONAL-ANALYSIS OF ANTAGONISTS BY CD, NMR, AND DISTANCE GEOMETRY CALCULATIONS

The conformation of the three cyclic antagonist analogs of parathyroid hormone-related protein (PTHrP)-(7-34) {[Lys(13), Asp(17)]PTHrP-(7-34 )NH2, [Lys(26), ASp(30)]PTHrP-(7-34)NH2, [Lys(13), Asp(17), Lys(26), A sp(30)]PTHrP-(7-34)NH2} is investigated by CD, NMR, and extensive comp uter simulations in aqueous solution and a TFE:water mixture. The stru ctural analysis of these peptides, designed to stabilize different reg ions of the sequence in a-helical conformations, is an important step in addressing the correlation between helical content and binding affi nity and bioactivity in this hormone-receptor system. Results from CD and NMR spectroscopy of all three analogues in aqueous solution indica te the presence of or-helix only in regions containing a 20-membered l actam ring. Upon addition of TFE, the three analogues display differen ces in the anticipated increase in helical content. The high-resolutio n structures produced at 50:50 TFE:water indicate specific differences in the extent and location of the helical regions. These conformation s provide insight into the biological profiles of these analogues, rep orted in the previous manuscript [Bisello et al. (1997) Biochemistry 3 6, 3293-3299]. Since all three analogues are a-helical in the C-termin al region (residues 25-34 have been previously identified as containin g the binding domain) and display similar binding affinities, we concl ude that this conformational feature is important for the interaction between the peptide and the receptor. The extent of the helix (toward the N-terminus) and the presence of a hinge in the central region of t he peptide play roles in the observed efficacy as measured by antagoni sm of PTH-stimulated adenylyl cyclase activity. The most active analog ue consists of helical segments from residues 13-18 and 20-34, separat ed by a kink centered at Arg(19).