W0114

Structural Perspective of Selective Binding of Mouse Pheromones by MUP4. Qin Zou¹, Tom Hurley¹ and Milos Novotny², ¹Dept. of Biochem. & Mol. Biol., Indiana Univ. Schl. of Med.and ²Dept. of Chemistry, Indiana Univ.

Mouse major urinary proteins (MUPs) help to transport various mouse pheromones. MUPs consist of various isoforms. Previous structural work has focused on MUP1. MUP4 shares about 70% sequence identity with MUP1. Much of the sequence variation is located at the pheromone binding site, which correlates with the difference in their selective binding of various pheromones. For example, MUP4 has about 23-fold higher affinity for 2-sec-butyl-4,5-dihydrothiazol (SBT) than that of MUP1 (Sharrow et. al. 2002). It is expected that the overall fold of MUP4 would be the same as that of MUP1. However, the differences between MUP1 and MUP4 with respect to the specific interaction in the binding site are likely to give rise to the binding differences.

MUP4 was crystallized in 0.2M CaCl₂ and 20%PEG3400 at 25°C and flash frozen in 25%PEG3400. The crystal diffracted to 1.5Å on our home detector system. The data was processed and refined to 1.55 Å, with 94% of completeness. The structure was solved by molecular replacement using the MUP1 crystal structure (Timm et. al. 2001) as the model.

The overall structure of MUP4 does not differ substantially from that of MUP1. It contains a β-barrel of eight antiparallel β-sheets. The rmsd of α-carbon between MUP1 and MUP4 is about 1.47Å. One significant feature in the MUP4 structure is at position 136, where the glycine residue in MUP1 is changed into glutamate in MUP4. In MUP1, two water molecules are involved in ligand binding, while in MUP4 the glutamate side-chain replaces one of the two water molecules and becomes part of the hydrogen bond network. The addition of this glutamate side chain in the binding site also changes the position of tyrosine 138 that is involved in ligand binding. These changes may explain the different binding affinities of MUP4 to pheromones.