Three-dimensional structure and ligand binding properties of trichosurin, a metatherian lipocalin from the milk whey of the common brushtail possum Trichosurus vulpecula

摘要

pLipocalins are extracellular proteins (17–25 kDa) that bind and transport small lipophilic molecules. The three-dimensional structure of the first lipocalin from a metatherian has been determined at different values of pH both with and without bound ligands. Trichosurin, a protein from the milk whey of the common brushtail possum, iTrichosurus vulpecula/i, has been recombinantly expressed in iEscherichia coli/i, refolded from inclusion bodies, purified and crystallized at two different pH values. The three-dimensional structure of trichosurin was solved by X-ray crystallography in two different crystal forms to 1.9 ? (1 ?=0.1 nm) and 2.6 ? resolution, from crystals grown at low and high pH values respectively. Trichosurin has the typical lipocalin fold, an eight-stranded anti-parallel β-barrel but dimerizes in an orientation that has not been seen previously. The putative binding pocket in the centre of the β-barrel is well-defined in both high and low pH structures and is occupied by water molecules along with isopropanol molecules from the crystallization medium. Trichosurin was also co-crystallized with a number of small molecule ligands and structures were determined with 2-naphthol and 4-ethylphenol bound in the centre of the β-barrel. The binding of phenolic compounds by trichosurin provides clues to the function of this important marsupial milk protein, which is highly conserved across metatherians./p