Bioenergetic coupling in P-glycoprotein: determining the relative position, topography and role of transmembrane helices six and twelve

Multidrug efflux pump P-glycoprotein (P-gp) has been the subject of significant research interest since the discovery of the protein in multi-drug resistant cancer cells 40 years ago. Overexpression of P-gp can confer resistance to a broad range of structurally and functionally distinct drugs. The mechanism of this polyspecificity is poorly understood, yet the mechanism is known to involve a complex series of binding, hydrolysis and dissociation events that are coupled to an array of conformational changes. A wealth of biochemical data implicates TM6 & 12 as mediators of bioenergetic coupling in P-gp. A major issue in attempts to determine the molecular mechanism is the lack of a high-resolution structure of human P-gp in the basal, inwardfacing state. Several crystal structures of murine P-gp in the basal state are available, and molecular dynamics have been used to simulate the structure of human P-gp. Both of these structural models are subject to controversy. Crystallisation requires solubilisation of P-gp into a detergent micelle, an environment in which the protein has been demonstrated to be inactive; the crystal structures published to date also differ significantly. MD simulations are an increasingly powerful tool for predicting protein structure; however, these must be treated with caution when not supported by experimental data. The present investigation determined the relative position of two transmembrane helices, six and twelve (TM6 & 12), by measuring the distances between residues. Site directed mutagenesis was used to generate nine double mutants, each containing one cysteine on TM6 and one on TM12. Double mutants were expressed using the baculovirus/insect cell expression system. Crude membranes containing P-gp were isolated, followed by purification by affinity chromatography. P-gp mutants were reconstituted into proteoliposomes; retention of function in the P-gp double mutants was confirmed using ATP hydrolysis assays. Inter-residue distance measurements were performed using intramolecular cross-linking with homobifunctional reagents and 5 double resonance EPR. Further ATP hydrolysis assays were used to determine the effect of intramolecular cross-linking between TM6 & 12 on the activity of P-gp. All the double mutants were functional, and most inter-residue distances were measured successfully by both techniques. The topographical map generated was compared to distance measurements in the crystal structures and MD simulations to judge whether either of these models are a good representation of human P-gp in a lipid bilayer. It was found that the 4M1M structure of murine P-gp is the most accurate representation among models derived by crystallography, while MD simulations based on this are closer still to experimental observations. The experimental results strongly suggest that the side chain orientations are accurate in the models in the membrane embedded portion of TM6 & 12. This investigation therefore demonstrated that 4M1Mbased structural models can be relied upon in the important membrane-integral region, which has been shown to be the site of drug binding. This can give greater weight to in silico methods for predicting molecular interactions with P-gp. Further details of the role of the two helices in the P-gp mechanism were added by analysis of functional changes in P-gp containing intramolecular cross-links between TM6 & 12. Specifically, the results presented here indicate that different parts of the helices require relative movement during drug-stimulated ATP hydrolysis in comparison to basal hydrolysis, suggesting that the conformational transitions of P-gp may be different in the two cases

93Zr developments at the Heavy Ion Accelerator Facility at ANU

The long-lived radionuclide 93Zr t1/2 = (1.61 +- 0.05) Ma plays an important role in nuclear astrophysics and nuclear technology. In stellar environments, it is mainly produced by neutron capture on the stable nuclide 92Zr. On Earth high amounts of radioactive 93Zr are produced in nuclear power plants directly from 235U fission, but also by neutron capture on 92Zr, as Zr-alloys are commonly used as cladding for nuclear fuel rods.This work was supported by the Australian Research Council DP140100136