Abstract
In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.
Highlights
Chemoreception in insects is mediated via sensing of a variety of small, volatile organic compounds
The current view on insect pheromone-binding proteins (PBPs) is that the unliganded protein exists in two conformations as follows: form A (PBPA), the acidic or “ligand releasing” conformation that exists at low pH, and form B (PBPB), the basic or “ligand binding” conformation that exists at high pH (7)
We report that recombinant ApolPBP1 is bound to the hydrophobic ligand endogenous to the E. coli host regardless of whether the protein is expressed in soluble form or in inclusion bodies
Summary
Mutagenesis, Overexpression, and Purification— The ApolPBP1 gene, previously cloned into pHN1ϩ vector (4), was amplified using the following primers: forward, 5Ј-GGAATTCCATATGTCGCCAGAGATCATGAAG-3Ј, and reverse, 5Ј-GCGGATCCCTAAACTTCAGCTAAGACCTC-3Ј (restriction sites underlined) and cloned into the NdeI and BamHI sites of pET-21a vector. Uniformly 15N-labeled delipidated ApolPBP1wt (310 l of 220 M in 50 mM phosphate buffer, pH 6.5, containing 5% D2O, 1 mM EDTA, and 0.01% (w/v) NaN3) was titrated with increasing concentrations of various ligands (0 –2.2 mM), and the corresponding two-dimensional {1H,15N} HSQC spectra were recorded. The data were fitted to a nonlinear regression of the plot of (FC Ϫ Fmin)/(F0 Ϫ Fmin) against [C] with the equation corresponding to a single binding site From these graphs, the IC50 values were determined at the ligand concentrations where the AMA fluorescence was quenched to half of its maximal intensity. Samples, AMA displacement assays were carried out for the undelipidated and delipidated forms of ApolPBP1wt and ApolPBP1H70A/H95A at pH 6.5 and 4.5, using a single ligand, the acetate pheromone
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