PARATHYROID HORMONE

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Parathyroid hormone (PTH) has been the object of numerous studies for many years now, owing to its crucial role in the regulation of extracellular calcium homeostasis. More recently, a growing interest in PTH and PTH-derived agonists as anabolic agents for bone remodeling and as possible drug candidates for the treatment of osteoporosis has been witnessed. From previous structural investigations, alpha helices in the N-and C-terminal domains have been identified. From sequence truncation studies, the C-terminus has been shown to be important for binding to the membrane embedded G-protein coupled receptor. Given the amphipathic nature of this helix, we proposed that the role of this helix is to interact with the membrane surface, to act as an anchor while the N-terminal helix of the PTH peptide activates the receptor. To examine this hypothesis, we began the investigation of an analog in which the amino acids 22-31 have been substituted by the sequence E(22)-L-L-E-K-L-L-E-KL(3 1), designed to model a perfect amphiphilic alpha-helix, with little sequence homology in this region (other than the amphiphilic nature) to the native PTH hormone. If an amphiphilic alpha-helix in the binding domain is the motif recognized by the receptor, then the modification should produce an effective agonist. Of course, this conclusion presupposes that the modification will be helical within the peptide sequence. To address this point, an NMR investigation of this peptide was initiated. To mimic the environment of the membrane the peptide was investigated in the presence of DPC micelles. Under these conditions the line widths of the proton signals are significantly broadened resulting in even worse spectral resolution. Because of this, it was impossible to even assign the proton signals with the instrumentation in house. Proton-proton spectra collected on the 750 MHz spectrometer allowed for the unambiguous assignment of all of the proton signals and identification of a large number of NOEs, previously unresolved. The chemical shift index and NOEs clearly indicated the presence of a helix in the C-terminus and allowed the development of a high resolution structure of this analog.