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

Abstract

Lipocalins 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, Trichosurus vulpecula, has been recombinantly expressed in Escherichia coli, 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 A (1 A=0.1 nm) and 2.6 A resolution, from crystals grown at low and high pH values respectively. Trichosurin has the typical lipocalin fold, an eight-stranded anti-parallel beta-barrel but dimerizes in an orientation that has not been seen previously. The putative binding pocket in the centre of the beta-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 beta-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.

The identification of immune genes in the milk transcriptome of the Tasmanian devil (Sarcophilus harrisii)

“…The expression of trichosurin, a marsupial-specific protein with unknown function, was much lower in the devil (0.04%) than in the wallaby (1.80%) ( Lefevre et al, 2007 ) at mid-lactation. This finding supports the previous suggestion that the primary role of trichosurin is in enabling digestion of plant-derived phenolic compounds when marsupial young move onto a solid diet ( Watson et al, 2007 ). As devils are omnivores, they have a much lower percentage of plant-based materials in their diet than herbivorous wallabies.…”

Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum, Trichosurus vulpecula

“…Vulpeculin was modelled against the RCSB Protein Data Bank (PDB) structure of trichosurin (2R73), that had been experimentally determined by X-ray crystallography [ 62 ] using Modeller 9.16 [ 70 ] and model quality was assessed using Ramachandran assessment in MolProbity [ 71 ] and QMEAN score [ 72 ]. The model of vulpeculin was viewed and annotated in PyMOL [ 73 ].…”

“…Lipocalin-like proteins are excreted in milk during lactation of the Tamar wallaby [ 56 – 58 ], red kangaroo [ 59 ], grey kangaroo [ 59 ], quokka [ 60 ] and the brushtail possum [ 61 ], some of which are homologous to marsupial β-lactoglobulins [ 59 , 61 ], and another more closely resembles rodent MUPs [ 61 ]. Trichosurin (a MUP-like lipocalin) was the first solved 3D structure of a lipocalin from a metatherian [ 62 ] and has been conjectured to bind small phenolic compounds that might prime the neonate liver to produce enzymes required to metabolize toxic plant phenols, reducing dietary limitations [ 62 ]. In this study, we have characterized the protein content of the urine of brushtail possum to explore the potential role of proteins in possum urine-mediated chemosignalling.…”

Characterisation of the immune compounds in koala milk using a combined transcriptomic and proteomic approach

“…Trichosurin, a protein unique to marsupials, was highly abundant in both early and late lactation. Its function is unknown, but a role in priming the neonate liver to produce detoxifying enzymes has been suggested 65 . Such a role might explain its particularly high abundance in koala milk, as their diet of eucalypt leaves is rich in phytotoxins.…”