Abstract
Anopheles gambiae mosquitoes that transmit Plasmodium falciparum malaria use a series of olfactory cues present in human sweat to locate their hosts for a blood meal. Recognition of these odor cues occurs through the interplay of odorant receptors and odorant-binding proteins (OBPs) that bind to odorant molecules and transport and present them to the receptors. Recent studies have implicated potential heterodimeric interactions between two OBPs, OBP1 and OBP4, as important for perception of indole by the mosquito (Biessmann, H., Andronopoulou, E., Biessmann, M. R., Douris, V., Dimitratos, S. D., Eliopoulos, E., Guerin, P. M., Iatrou, K., Justice, R. W., Kröber, T., Marinotti, O., Tsitoura, P., Woods, D. F., and Walter, M. F. (2010) PLoS ONE 5, e9471; Qiao, H., He, X., Schymura, D., Ban, L., Field, L., Dani, F. R., Michelucci, E., Caputo, B., della Torre, A., Iatrou, K., Zhou, J. J., Krieger, J., and Pelosi, P. (2011) Cell. Mol. Life Sci. 68, 1799-1813). Here we present the 2.0 Å crystal structure of the OBP4-indole complex, which adopts a classical odorant-binding protein fold, with indole bound at one end of a central hydrophobic cavity. Solution-based NMR studies reveal that OBP4 exists in a molten globule state and binding of indole induces a dramatic conformational shift to a well ordered structure, and this leads to the formation of the binding site for OBP1. Analysis of the OBP4-OBP1 interaction reveals a network of contacts between residues in the OBP1 binding site and the core of the protein and suggests how the interaction of the two proteins can alter the binding affinity for ligands. These studies provide evidence that conformational ordering plays a key role in regulating heteromeric interactions between OBPs.
Highlights
Combat transmission of malaria and other mosquito borne diseases, and a number of efforts are under way to discover novel reagents for this purpose
We demonstrated that a key amino acid substitution in a critical loop of the Drosophila melanogaster odorant-binding proteins (OBPs) LUSH could activate pheromone receptors in the complete absence of pheromone [23, 26, 27], providing direct evidence that the OBP is the ligand for the receptor complex
In An. gambiae it was recently demonstrated that perception of indole and 3-methyl-indole requires OBP1 [31], and this may involve the formation of interactions between OBP1 and OBP4 [32, 33]
Summary
Protein Expression and Purification—The sequences of OBP1 and -4 without the signal peptide, as predicted by SignalP [35], were PCR-amplified from a An. gambiae antennal cDNA library and subcloned into the NdeI/BamHI site of a pET13a vector [36]. OBP4 primers were (NdeI) 5ЈCTAGCATATGACCATGAAACAGCTAACC-3Ј and (BamHI) 5Ј-TACGGGATCCCTATTATGGGAACATGAAGGTGTCGG-3Ј. In both cases the restriction sites are underlined. The final proteins contain 127 and 124 amino acids (OBP1 and -4, respectively). Biophysical Analysis—Circular dichroism (CD) was performed on a Jasco-815 spectropolarimeter in the University of Colorado School of Medicine Biophysics Core using 5 M protein samples at 25 °C using a 2-mm path length cell. Backbone chemical shift assignments for OBP4 and OBP1 were made semi-automatically using the PINE server [39] followed by manual inspection from standard HNCACB, CBCA(CO)NH, HNCO, (HACA)CO(CA)NH, and heteronuclear single quantum coherence (HSQC)-NOESY-HSQC triple resonance experiments using the pulse sequences supplied by Varian. The crystal structure of OBP4 was solved by molecular replacement starting from the structure of LUSH (PDB code 1OOH) using Phaser [44] within the CCP4 suite [45] and refined using Refmac5 [46] followed by manual rebuilding in COOT [47]
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