The mid-region of parathyroid hormone (1-34) serves as a functional docking domain in receptor activation.
Elucidating the bimolecular interface between parathyroid hormone (PTH) and its cognate G protein-coupled receptor (PTHR1) should yield insights into the basis of molecular recognition and the mechanism of ligand-mediated intracellular signaling for a system that is critically important in regulating calcium levels in blood. We used photoaffinity scanning (PAS) to identify key ligand-receptor interactions for residues from the unstructured mid-region domain of PTH-(1-34). Four PTH analogues, containing a single photoreactive p-benzoylphenylalanine (Bpa) residue in position 11, 15, 18, or 21, were found to photo-cross-link within receptor regions [165-176], [183-189], [190-298], and [165-176], respectively. Addition of these mid-region contacts as constraints to our previously proposed model of the PTH-PTHR1 complex and extensive molecular simulation experiments enables substantial refinement of the model. Specifically, (1) the overall receptor-bound conformation of the hormone is not extended, but bent; (2) helix [169-176] of the N-terminal extracellular domain (N-ECD) of the receptor is redirected toward the heptahelical bundle; and (3) the hormone traverses between the top of transmembrane (TM) helices 1 and 2, rather than between TM-7 and TM-1. This significantly alters the model of both the receptor-bound tertiary structure of the hormone and the topological orientation of the C-terminus of the N-ECD in the hormone-receptor bimolecular complex. We propose that the mid-region of PTH-(1-34) has a role in fixing, by extensive contacts with the receptor, the entry of the N-terminal helix of the hormone into the heptahelical bundle between TM-1 and TM-2. This anchorage would orient the amino terminus into position to activate the receptor.
Wittelsberger
Angela
A
Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA. Angela.Wittelsberger@tufts.edu
Corich
Martina
M
Thomas
Beena E
BE
Lee
Byung-Kwon
BK
Barazza
Alessandra
A
Czodrowski
Paul
P
Mierke
Dale F
DF
Chorev
Michael
M
Rosenblatt
Michael
M
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006801:Humans; D008958:Models, Molecular; D010281:Parathyroid Hormone / Q000031:analogs & derivatives / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000502:physiology*
C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone