Structural and binding studies of the milk whey lipocalin, trichosurin, from the Common Brushtail Possum, Trichosurus vulpecula (*N)

Abstract

Lipocalins are small extracellular proteins which usually bind and/or transport small lipophilic molecules. In this work lipocalins from the milk whey of the brushtail possum, trichosurus vulpecula, were studied to obtain structural and binding data to establish their functional roles. The possum milk whey lipocalins late lactation protein (LLP), β-lactoglobulin (BLG), and trichosurin were recombinantly expressed in Escherichia coli and purified. Of these three, trichosurin was successfully crystallised at two different pHs, 4.6 and 8.2. The structure of trichosurin was solved by X-ray crystallography to 1.9 Å and 2.6 Å from the crystals grown at low and high pH respectively. It consists of the typical lipocalin structural fold, an eight-stranded anti-parallel β-barrel. Despite its consistency with the lipocalin fold the structure from possum trichosurin demonstrates a novel dimerisation arrangement. The N-terminal part of the polypeptide chain was found to be disordered and thus, was unable to be structurally characterised. The structure solved from crystals at low pH shows a bound Zn2+ ion tetrahedrally coordinated with two Cl- ions between two histidines of the trichosurin polypeptide. This Zn2+ is not seen in the pH 8.2 structure. The biological role of trichosurin has been postulated. It is suggested that trichosurin is involved in the transport of phenolics or polyterpenes to the pouch young to prime the liver for production of detoxifying enzymes. Possum fodder is naturally high in phenolic and terpene-based antifeedants and toxins. To investigate this, a range of potential compounds were screened for binding to trichosurin using fluorescence spectroscopy. Two compounds, 2-naphthol and 4-ethylphenol, showed binding to trichosurin, and were then structurally characterised by X-ray crystallography to determine the nature of ligand binding in detail. These experiments not only demonstrated binding of the ligand within the barrel, as typical for the lipocalin fold, but also revealed an additional secondary binding site for 2-naphthol outside the barrel. The existence of a secondary external binding site is unusual and had not been shown for a lipocalin before. Its biological significant is unclear.