Mapping peptide hormone-receptor interactions using a disulfide-trapping approach.
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.
Monaghan
Paul
P
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Thomas
Beena E
BE
Woznica
Iwona
I
Wittelsberger
Angela
A
Mierke
Dale F
DF
Rosenblatt
Michael
M
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1