Biophysical characterization of ligand binding and release of Anthereae polyphemus Pheromone Binding Protein 1 (ApolPBP1) and Exploring the nickel proteome

Abstract

In moths, pheromone binding proteins (PBPs) transport the hydrophobic pheromone molecules to the membrane-bound receptors across the aqueous sensillar lymph to trigger the neuronal response. We showed here that, recombinant Antheraea polyphemus PBP1 (ApolPBP1) expressed with hydrophobic molecule(s) endogenous to the Escherichia coli which keeps the protein in the ligand-bound conformation at high pH but switches to the ligand-free conformation at low pH. Two molecular switches are considered to play a role in this mechanism: (i) protonation of His70 and His95 situated at one end of the binding pocket and (ii) switch of the unstructured C-terminus at the other end of the binding pocket to a helix that enters the pocket at low pH. Here, we show the role of the histidine-driven switch in ligand release and the role of C-terminus in ligand binding or releasing for ApolPBP1 by binding of 1-aminoanthracene (AMA) by fluorescence spectroscopy and solution NMR. Mutation of His70 and His95 to alanines drives the equilibrium toward the ligand-bound conformation even at low pH thus eliminating the pH-induced histidine-dependent switch which exists in the wild type protein. The C-terminal truncated proteins exist only in the bound conformation at all pH levels and failed to undergo pH- or ligand-dependent conformational switching. Our studies revealed that these proteins can bind ligand even at low pH in contrast to the wild type protein. Although C terminal truncated proteins could bind ligands even at low pH, they had reduced affinity for the ligand at both low and high pH compared to that of wild-type ApolPBP1. This indicates that apart from helping in the ligand releasing mechanism, the C terminus might have a role in the ligand binding mechanism and/or preventing the early escape of the ligand from the binding pocket. Our results are in contrast to those reported, where C-terminal truncated proteins had similar or increased pheromone binding affinity at both high and low pH. We also examined the role of three charged residues (Asp132, Glu137 and Glu141) present inside the C-terminus tail. We discovered that, single mutations (D132N, E137Q and E141Q) and double mutations (D132NE137Q and E137QE141Q) have no effect on the conformation of the protein. They behaved as the wild type protein. Most of the bacteria and some plant species require nickel for the production of many essential enzymes inside their cells. In this current study we are trying to find out and characterize small nickel containing proteins from an Archaea, Methanothermobacter marburgensis. We also tried to use a metallobiome approach and discover any nickel-containing proteins that have not been characterized before. In the nickel-hyperaccumulating plant Streptanthus polygaloides the goal was to test if ICP-EAS was sensitive enough to detect the nickel-containing proteins during the purification steps. This detection method works really well and several nickel protein containing fractions have been isolated. More purification steps will be needed however to obtain pure proteins and to fully characterize them.