Role of amino acid side chains in region 17-31 of parathyroid hormone (PTH) in binding to the PTH receptor.
The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.
Dean
Thomas
T
Endocrine Unit, Massachusetts General Hospital, and Harvard Medical School, 50 Blossom Street, Boston, MA 02114, USA.
Khatri
Ashok
A
Potetinova
Zhanna
Z
Willick
Gordon E
GE
Gardella
Thomas J
TJ
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D018929:Cell Culture Techniques; D002460:Cell Line; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism*
D000596:Amino Acids; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; C101312:parathyroid hormone (1-31); D018698:Glutamic Acid; D000409:Alanine