Research Article: The Structure, Stability and Pheromone Binding of the Male Mouse Protein Sex Pheromone Darcin

Date Published: October 3, 2014

Publisher: Public Library of Science

Author(s): Marie M. Phelan, Lynn McLean, Stuart D. Armstrong, Jane L. Hurst, Robert J. Beynon, Lu-Yun Lian, Paulo Lee Ho.

http://doi.org/10.1371/journal.pone.0108415

Abstract

Mouse urine contains highly polymorphic major urinary proteins that have multiple functions in scent communication through their abilities to bind, transport and release hydrophobic volatile pheromones. The mouse genome encodes for about 20 of these proteins and are classified, based on amino acid sequence similarity and tissue expression patterns, as either central or peripheral major urinary proteins. Darcin is a male specific peripheral major urinary protein and is distinctive in its role in inherent female attraction. A comparison of the structure and biophysical properties of darcin with MUP11, which belongs to the central class, highlights similarity in the overall structure between the two proteins. The thermodynamic stability, however, differs between the two proteins, with darcin being much more stable. Furthermore, the affinity of a small pheromone mimetic is higher for darcin, although darcin is more discriminatory, being unable to bind bulkier ligands. These attributes are due to the hydrophobic ligand binding cavity of darcin being smaller, caused by the presence of larger amino acid side chains. Thus, the physical and chemical characteristics of the binding cavity, together with its extreme stability, are consistent with darcin being able to exert its function after release into the environment.

Partial Text

While urine provides a means for eliminating waste liquid from the body, many species also utilize urine as a vehicle to deposit species-specific scent signals in the environment. Whilst these scent signals are often low molecular weight biochemicals, mouse urine contains a polymorphic mixture of major urinary proteins (MUPs) [1] at very high concentration [2]. These are synthesised in the liver, secreted into serum and filtered efficiently into urine, with typical concentrations of 10–30 mg/ml in adult male house mice under laboratory conditions [3]–[5] and even higher levels under naturalistic conditions [6]. Both MUP production and scent marking are particularly elevated in male mice, with males excreting approximately twice as much MUP as females kept under similar conditions [5]–[7].

Darcin exhibits physicochemical properties that set it apart from other MUPs. Both the native protein from mouse urine and the E. coli expressed recombinant protein migrate at a higher mobility on SDS-PAGE, travelling further on the gel, than other MUPs (Fig. 1A, B). This enhanced mobility of darcin (in reduced or oxidised forms) is consistent with it retaining a more compact, partially collapsed structure that can penetrate the gel matrix more easily. When darcin and other central MUPs (with the same number of protonatable sites) are subjected to electrospray ionization mass spectrometry, the charge state distribution of darcin is skewed towards a lower degree of protonation than other MUPs (Fig. 1C), consistent with a compact structure that is not completely unfolded during the conditions of electrospray ionisation that were used.

We present here the structure, physico-chemical and binding characteristics of the peripheral MUP darcin, which is a male-specific pheromone that plays a key role in mouse sexual attraction. We compare darcin with a central MUP, MUP11, which was analysed using the same methodologies. Darcin and MUP11 are both eight-stranded beta barrel proteins. Four short hairpin loops are found at the N-terminal, top end of the barrel whereas larger loops are found at the other C-terminal, bottom end (Fig. 2). The closest distance is between L1 and L5 at the C-terminal end, as in other MUP structures, with L1 occluding the bottom, C terminal end of the internal hydrophobic cavity. The enclosed hydrophobic cavity is similar to other MUPs and, more generically, to lipocalins.

 

Source:

http://doi.org/10.1371/journal.pone.0108415