Interactions of Anopheles gambiae odorant-binding proteins with a human-derived repellent: implications for the mode of action of n,n-diethyl-3-methylbenzamide (DEET).

The Anopheles gambiae mosquito, which is the vector for Plasmodium falciparum malaria, uses a series of olfactory cues emanating from human sweat to select humans as their source for a blood meal. Perception of these odors within the mosquito olfactory system involves the interplay of odorant-binding proteins (OBPs) and odorant receptors and disrupting the normal responses to those odorants that guide mosquito-human interactions represents an attractive approach to prevent the transmission of malaria. Previously, it has been shown that DEET targets multiple components of the olfactory system, including OBPs and odorant receptors. Here, we present the crystal structure of A. gambiae OBP1 (OBP1) in the complex it forms with a natural repellent 6-methyl-5-heptene-2-one (6-MH). We find that 6-MH binds to OBP1 at exactly the same site as DEET. However, key interactions with a highly conserved water molecule that are proposed to be important for DEET binding are not involved in binding of 6-MH. We show that 6-MH and DEET can compete for the binding of attractive odorants and in doing so disrupt the interaction that OBP1 makes with OBP4. We further show that 6-MH and DEET can bind simultaneously to OBPs with other ligands. These results suggest that the successful discovery of novel reagents targeting OBP function requires knowledge about the specific mechanism of binding to the OBP rather than their binding affinity.

1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta.

The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα (Slater et al. J Biol Chem 272(10):6167-6173, 1997; Slater et al. Biochemistry 43(23):7601-7609, 2004), PKCε (Das et al. Biochem J 421(3):405-413, 2009) and PKCδ (Das et al. J Biol Chem 279(36):37964-37972, 2004; Das et al. Protein Sci 15(9):2107-2119, 2006) contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the (1)H, (15)N and (13)C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.