A variety of candidate ligands showed a preference for elongated cylindrical molecules

Despite these differences, the binding pockets of PBPs and OBPs are structurally similar and formed by six a-helices, stabilized by disulfide bridges between six cysteines. We used the published AgamOBP1 structure to model the binding of indole into its binding pocket. Molecular mechanics calculations show possible orientations of indole in the binding pocket as well as the feasibility of 3-methyl-indole binding. Further in silico studies of AgamOBP1 binding to a variety of candidate ligands showed a preference for elongated cylindrical molecules with no side chains, but small flat ring structures can be accommodated in the binding site as well. Additionally, polar groups may also be accommodated in some regions of the binding cavity. Modeling of the AgamOBP1 dimers suggests that binding of two ligand molecules may occur readily. The steep response curves we Ginsenoside-Rc observed in the FlashPlate assays are indicative of a cooperative ligand binding and therefore may represent the binding of indole to AgamOBP1 dimers and/or trimers. The essential role of AgamOBP1 in the perception of indole was ultimately demonstrated in this study by the EAG responses of A. gambiae females subjected to AgamOBP1-dsRNA injections, which caused a drastic reduction in AgamOBP1 accumulation. Most of these mosquitoes showed complete loss of the EAG response to indole. As predicted from the ligand-binding and modeling studies, the EAG responses to 3-methyl indole were also affected in the same mosquitoes. The specificity of gene expression inhibition induced by the injection of females with AgamOBP1-dsRNA was demonstrated by the unaltered EAG responses of the mosquitoes to the terpene, geranylacetone. No loss of the EAG responses to indole and 3-methyl indole was recorded in control females injected with AgamOBP7-dsRNA. These females were shown to contain levels of AgamOBP1 mRNA and protein comparable to those of controls. These results constitute the first record of blocking Pneumocandin B0 olfactory perception of critical ligands in a mosquito and support the claim that OBPs are valuable targets for interference with the olfactory response in mosquitoes.

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