Progesterone binding to uteroglobin: two alternative orientations of the ligand.
Progesterone binding to a homodimer of uteroglobin takes place in a hydrophobic cavity formed by the two subunits. Previous mutational analyses have shown that the tyrosine (21 and 21') and threonine (60 and 60') residues of the uteroglobin dimer are directly involved in progesterone binding. To analyze the contribution of each of the two tyrosines and threonines in the dimer, we have constructed a covalently linked uteroglobin dimer (UGcl) by fusing two uteroglobin cDNAs via a synthetic linker sequence. Escherichia coli expressed UGcl bound progesterone with the same affinity as the native dimeric protein. Replacement of both tyrosines by phenylalanines abolished progesterone binding. Replacement of either the C-terminal tyrosine 21 or the N-terminal tyrosine 21' separately, reduced the affinity for progesterone 3- to 4-fold, suggesting that both tyrosines participate in progesterone binding. In contrast, substitutions of the threonine residues of the C- or N-terminal moities had no effect, whereas the replacement of both threonines reduced the affinity for progesterone 2- to 3-fold. These data, together with computer models, suggest that progesterone docks in the internal binding pocket of uteroglobin in two different orientations.
Dunkel
R
R
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
Vriend
G
G
Beato
M
M
Suske
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014598:Uteroglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D013912:Threonine; D014443:Tyrosine; D011374:Progesterone; D014598:Uteroglobin; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase