Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach
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- 作者：Paul Monaghan ; Beena E. Thomas ; Iwona Woznica ; Angela Wittelsberger ; Dale F. Mierke ; Michael Rosenblatt
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：June 3, 2008
- 年：2008
- 卷：47
- 期：22
- 页码：5889 - 5895
- 全文大小：3241K
- 年卷期：v.47,no.22(June 3, 2008)
- ISSN：1520-4995
文摘
Efforts to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1), have relied heavily on benzoylphenylalanine- (Bpa-) based photoaffinity cross-linking. However, given the flexibility, size, and shape of Bpa, the resolution at the PTH−PTHR1 interface appears to be reaching the limit of this technique. Here we employ a disulfide-trapping approach developed by others primarily for use in screening compound libraries to identify novel ligands. In this method, cysteine substitutions are introduced into a specific site within the ligand and a region in the receptor predicted to interact with each other. Upon ligand binding, if these cysteines are in close proximity, they form a disulfide bond. Since the geometry governing disulfide bond formation is more constrained than Bpa cross-linking, this novel approach can be employed to generate a more refined molecular model of the PTH−PTHR1 complex. Using a PTH analogue containing a cysteine at position 1, we probed 24 sites and identified 4 in PTHR1 to which cross-linking occurred. Importantly, previous photoaffinity cross-linking studies using a PTH analogue with Bpa at position 1 only identified a single interaction site. The new sites identified by the disulfide-trapping procedure were used as constraints in molecular dynamics simulations to generate an updated model of the PTH−PTHR1 complex. Mapping by disulfide trapping extends and complements photoaffinity cross-linking. It is applicable to other peptide−receptor interfaces and should yield insights about yet unknown sites of ligand−receptor interactions, allowing for generation of more refined models.