Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer.
The progesterone-binding protein uteroglobin has been expressed in Escherichia coli in an unfused, soluble form. Like mature uteroglobin from rabbit endometrium (UG), the E.coli produced uteroglobin (UG1) dimerizes in vitro, forms an antiparallel dimer with Cys3-Cys69' and Cys69-Cys3' disulfide bonds and binds progesterone under reducing conditions. In order to analyze the dimerization and the reduction dependence of progesterone binding in more detail, we separately replaced cysteine 3 and cysteine 69 by serines. Under reducing conditions, both uteroglobin variants (UG1-3Ser and UG1-69Ser) bind progesterone with the same affinity as the wild-type suggesting that both cysteine residues are not directly involved in progesterone binding. In contrast to the wild-type protein, both cysteine variants also bind progesterone with high affinity in the absence of reducing agents. In addition, UG1-3Ser and UG1-69Ser both form covalently linked homodimers. Thus, unnatural Cys69-69' and Cys3-3' disulfide bonds exist in UG1-3Ser and UG1-69Ser, respectively. These data together with computer models based on X-ray diffraction data strongly support the idea that progesterone reaches its binding site located in an internal hydrophobic cavity via a hydrophobic tunnel along helices 1 and 4. Under non-reducing conditions the tunnel is closed by two disulfide bridges (Cys3-Cys69' and Cys69-Cys3') that lie in the most flexible region of the dimer. Reduction or replacement of a cysteine residue enables conformational changes that open the channel allowing progesterone to enter.
Peter
W
W
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
Dunkel
R
R
Stouten
P F
PF
Vriend
G
G
Beato
M
M
Suske
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D005822:Genetic Vectors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D014598:Uteroglobin / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; D011374:Progesterone; D014598:Uteroglobin; D003545:Cysteine