NMR Mapping of the Recombinant Mouse Major Urinary Protein I Binding Site Occupied by the Pheromone 2-sec-Butyl-4,5-dihydrothiazole

Abstract

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.

Detection ofα2u-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

“…This has been resolved by X-ray diffraction and NMR spectroscopy and this cavity forms the binding site for polar ligands. 6,10,[40][41][42] In pigs, the submaxillary glands express an abundant protein called pheromaxein, with a molecular mass of about 15 kDa, and this is able to bind pheromonal molecules that are involved in pheromonal communication. 20 Apart from these proteins in rodents and pigs, the human axillary region contains a gland called the apocrine gland and this releases two proteins, the ASOBPs, as a part of apocrine secretion.…”

On the saliva proteome of the Eastern European house mouse (Mus musculus musculus) focusing on sexual signalling and immunity

“…In mice, the most published studies in chemical communication focused on the major urinary proteins (MUPs)34567, which are expressed by the liver and transport volatile organic compounds (VOCs) in their beta barrel structure to the urine67891011. VOCs are slowly released from different urinary MUPs, and have been proposed to function in a variety of social signals, including identity, territorial marking, mate choice etc 3121314…”

Recombinant Major Urinary Proteins of the Mouse in Specific IgE and IgG Testing

“…The amino acid sequences of MUP1, MUP2, MUP7, MUP8 and MUP9 differ approximately by only 0.6–1.9%, while MUP4 differs approximately 25% from the others [11]. Purification of the MUPs was done as previously described [11, 22]. In short, Ni-affinity chromatography was followed by removal of the His(6) tag by treatment with factor Xa protease.…”