NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in so lution. N-15-labeled and N-15,C-13-doubly-labeled recombinant MUP-I were pr oduced in a bacterial expression system and purified to homogeneity. Racemi c 2-sec-butyl-4,5-dihydrothiazole was produced synthetically. An equilibriu m diffusion assay and NMR titration revealed that both enantiomers of the p heromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constan t and slow-exchange regime dissociation kinetics were determined for the ph eromone-protein complex. H-1, N-15, and C-13 chemical shifts of MUP-I were assigned using triple resonance and N-15-correlated 3D NMR experiments. Cha nges in protein H-1(N) and N-15(H) chemical shifts upon addition of pheromo ne were used to identify the ligand binding site. Several amide signals, co rresponding to residues on one side of the binding site, were split into tw o peaks in the saturated protein-ligand complex. Similarly, two overlapping Ligand spin systems were present in isotope-filtered NMR spectra of labele d protein bound to unlabeled pheromone. The two sets of peaks were attribut ed to the two possible chiralities of the pheromone. Intermolecular NOEs in dicated that the orientation of the pheromone in the MUP-I binding cavity i s opposite to that modeled in a previous X-ray structure.