Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy.
Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.
Fralish
Gregory B
GB
Department of Biochemistry and Molecular Biology, Life Sciences Building, University of Georgia, Athens 30602-7229, USA.
Dattilo
Brian
B
Puett
David
D
D000818:Animals; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D015951:Factor Xa / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011974:Receptors, LH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D015951:Factor Xa