Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer |
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Authors: | Peter W; Dunkel R; Stouten PFW; Vriend G; Beato M; Suske G |
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Affiliation: | Institut für Molekularbiologie und Tumorforschung, Philipps-Universit t Marburg Emil-Mannkopff-Strasse. 2, DW-3550 Marburg, Germany |
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Abstract: | The progesteronebinding 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 Cys3Cys69' and Cys69Cys3' 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 (UGl3Ser and UGl69Ser)bind progesterone with the same affinity as the wildtypesuggesting that both cysteine residues are not directly involvedin progesterone binding. In contrast to the wildtypeprotein, 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 Cys6969' and Cys33' disulfidebonds exist in UG13Ser and UG169Ser, 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 nonreducingconditions the tunnel is closed by two disulfide bridges (Cys3Cys69'(and Cys69Cys3') 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. |
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Keywords: | bacterial expression/ dimerization/ disulfide bonds/ progesterone binding/ uteroglobin |
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