Synthetic cross-links arrest the C-terminal region of the relaxin-like factor in an active conformation.
All kinetic indicators suggest that the receptor-binding site of the relaxin-like factor (RLF) is located in the flexible C-terminal region of the B chain and is centered about the tryptophan in position B27. Conformational restraints of varying lengths were used to confine Trp (B27) to a narrow range of positions relative to the C terminus of the A chain. Total synthesis of variants involving residues proximate to Trp (B27) assured us that none had a role in receptor binding. Even arginine B26, next to the important tryptophan, can be replaced without deleterious effects. To fix the distance between the C-terminal end of the A chain and Trp (B27) at predetermined lengths, we synthesized RLF with covalent cross-links between a lysine, which was placed in position B26, and the alpha-carboxyl group at the C terminus of the A chain (A26). The affinity of the cross-linked ligands for the RLF receptor varied as a parabolic function of length whereby the range of 10.0-11.1 A provided the closest approach to the binding conformation. Wild-type transmembrane signaling activity, as determined by cAMP accumulation, was reached only with a glycine cross-linker.
Büllesbach
Erika E
EE
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250509, Charleston, South Carolina 29425, USA. bullesee@musc.edu
Schwabe
Christian
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007328:Insulin; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry; D014599:Uterus / Q000378:metabolism
D003432:Cross-Linking Reagents; D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; C033395:relaxin receptors; D014364:Tryptophan; D000242:Cyclic AMP; D008239:Lysine