Mapping peptide hormone-receptor interactions using a disulfide-trapping approach

Biochemistry. 2008 Jun 3;47(22):5889-95. doi: 10.1021/bi800122f. Epub 2008 May 7.


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.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Cross-Linking Reagents / chemistry
  • Disulfides / chemistry*
  • Disulfides / metabolism
  • Flow Cytometry
  • Ligands
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Parathyroid Hormone / chemistry*
  • Parathyroid Hormone / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Interaction Mapping / methods*
  • Protein Structure, Tertiary
  • Receptor, Parathyroid Hormone, Type 1 / chemistry*
  • Receptor, Parathyroid Hormone, Type 1 / metabolism
  • Transfection


  • Cross-Linking Reagents
  • Disulfides
  • Ligands
  • Parathyroid Hormone
  • Peptide Fragments
  • Receptor, Parathyroid Hormone, Type 1