Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer

The progesterone–binding protein uteroglobin has beenexpressed in Escherichia coli in an unfused, soluble form. likemature uteroglobin from rabbit endometrium (UG), the E.coliproduceduteroglobin (UG1) dimerizes in vitro, forms an antiparalleldimer with Cys3–Cys69' and Cys69–Cys3' disulfidebonds and binds progesterone under reducing conditions. In orderto analyze the dimerization and the reduction dependence ofprogesterone binding in more detail, we separately replacedcysteine 3 and cysteine 69 by serines. Under reducing conditions,both uteroglobin variants (UGl–3Ser and UGl–69Ser)bind progesterone with the same affinity as the wild–typesuggesting that both cysteine residues are not directly involvedin progesterone binding. In contrast to the wild–typeprotein, both cysteine variants also bind progesterone withhigh affinity in the absence of reducing agents. In addition,UGl-3Ser and UGl-69Ser both form covalently linked homodimers.Thus, unnatural Cys69–69' and Cys3–3' disulfidebonds exist in UG1–3Ser and UG1–69Ser, respectively.These data together with computer models based on X-ray diffractiondata strongly support the idea that progesterone reaches itsbinding site located in an internal hydrophobic cavity via ahydrophobic tunnel along helices 1 and 4. Under non–reducingconditions the tunnel is closed by two disulfide bridges (Cys3–Cys69'(and Cys69–Cys3') that lie in the most flexible regionof the dimer. Reduction or replacement of a cysteine residueenables conformational changes that open the channel allowingprogesterone to enter.