Predicted structure of a Minus-C OBP from Batocera horsfieldi (Hope) suggests an intermediate structure in evolution of OBPs.

Odorant binding proteins (OBPs) transport hydrophobic odorants from the environment to odorant receptors and play an important role in specific recognition of volatiles. Here, we expressed and purified a minus-C OBP, BhorOBPm2, from Batocera horsfieldi, a major pest of Popolus, to determine its binding characteristics with 58 candidate volatiles using a fluorescence competition-binding assay. We showed that BhorOBPm2 exhibited high binding affinity with chain volatiles and that ligands were selected based on chain length. In order to elucidate the binding mechanism, homology modeling and molecular-docking experiments were performed to investigate interactions between BhorOBPm2 and volatiles. The predicted structure with only two disulfide bonds showed one continuous channel for ligand binding, similar to classic OBPs AgamOBP1 and CquiOBP1. Unexpectedly, we observed a larger binding pocket for BhorOBPm2 and broader specificity for ligands than classic OBPs due to the expansive flexibility of BhorOBPm2 resulting from a lack of disulfide bonds. These findings suggested that BhorOBPm2 might present an intermediate structure in the evolution of OBPs. Furthermore, we designed two mutant proteins to simulate and verify functions of the C-terminal region. The changes in binding affinity observed here indicated a novel action differing from that of the "lid" described in previous studies.