ABSTRACT
Lipocalin 12 (Lcn12) is a recently identified epididymis-specific protein that might play a significant physiological role in male reproduction. However, the detailed structure and function of Lcn12 remain to be determined. In the present work, we cloned, expressed, and purified the rat Lcn12 (rLcn12) protein in Escherichia coli, introduced the Cys176Ala substitution to eliminate the aggregation problem associated with the wild-type protein. Homology modeling results demonstrated that rLcn12 adopted an eight-stranded, antiparallel ß-barrel conformation containing a conserved disulfide bond between Cys98 and Cys203, which was in accordance with the physicochemical properties elucidated by a combination of mass, circular dichroism, and nuclear magnetic resonance spectrometry. The purified rLcn12 protein exhibited a high binding affinity for all-trans retinoic acid in fluorescence titration experiments, implying that rLcn12 could be involved in retinoic acid transport in the epididymis.
Subject(s)
Epididymis/metabolism , Escherichia coli/metabolism , Lipocalins/chemistry , Amino Acid Sequence , Animals , Circular Dichroism , Disulfides , Lipocalins/metabolism , Magnetic Resonance Spectroscopy/methods , Male , Molecular Conformation , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Rats , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tretinoin/chemistryABSTRACT
Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to wild-type rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to wild-type rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other rat lipocalins.
Subject(s)
Cysteine/metabolism , Disulfides/metabolism , Intramolecular Oxidoreductases/metabolism , Lipocalins/metabolism , Recombinant Proteins/metabolism , Animals , Binding Sites , Cloning, Molecular , Cysteine/chemistry , Cysteine/genetics , Enzyme Stability , Hot Temperature , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/genetics , Lipocalins/chemistry , Lipocalins/genetics , Protein Folding , Rats , Recombinant Proteins/chemistryABSTRACT
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH2 to produce PGD2. Three cysteine residues, Cys65, Cys89, and Cys186 in L-PGDS, are conserved among all species, of which Cys89 and Cys186 residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.