Journal:IUCrJ:S2052252520005709

Structural Definition of Polyspecific Compensatory Ligand Recognition by P-glycoprotein

Christina A. Le, Daniel S. Harvey and Stephen G. Aller ^[1]

Molecular Tour
Most cell types including healthy- and cancerous- cells deal with threats from chemicals and toxins by a number of different mechanisms that allow them to develop resistance to toxic side-effects. A major mechanism includes the actual pumping of the threatening molecules out of the cell before they can pass all the way into the cell to cause damage. In the case of cancer chemotherapies, FDA-approved drugs act as the toxins to cancer cells, but the cancer cells can also adapt by increasing the number of molecular pumps at the membranous cell surface to deal with the threat and acquire resistance. One of the most dominant molecular pumps in the human body is called P-glycoprotein (MDR1/ABCB1) or Pgp for short. Also referred to as the multidrug resistance transporter, Pgp is capable of recognizing and expelling perhaps thousands of small molecule drugs and toxins to protect cells. An early idea involved developing a second drug that might 'inhibit' Pgp and allow the original chemotherapeutic drug to penetrate the cell and become more effective again. The most potent Pgp inhibitors developed, however, appeared to inhibit Pgp systemically, i.e. at important locations such as the liver, where the continual function of Pgp in detoxification is critical. Such inhibitors resulted in worse toxic side effects and even fatality. A more recent idea is to carefully design/modify the chemotherapeutics themselves such that they might not be efficiently recognized by Pgp and might evade transport. The latter idea is certainly very daunting given the polyspecific nature of Pgp - in other words, the fact that the Pgp transporter seems to have evolved to be able to recognize and transport a very large number of chemically different molecules suggests that the design of evaders will be very challenging.

Of high scientific value would be a better understanding of the molecular mechanisms of Pgp that confer its polyspecific abilities. A major part of this quest by several groups has been the determination of Pgp structure to near-atomic precision, including in the presence of ligands (drugs and drug-like molecules that are recognized and bound to the Pgp protein). The Pgp pump is now known to rock back-and-forth from an inverted "V"-shape to a "V"-shape conformation during its pumping activity. Pgp uses (hydrolyzes) ATP as an energy source to power the pumping process as it changes protein conformations. The binding location of several ligands is known to shift depending on even slight changes in protein conformation, hinting at a mechanism of polyspecificity. Furthermore, distinct ligand binding sites have been described for certain ligands whereas some ligands share overlapping sites with other ligands. What was not known until now is that, if the protein conformation of Pgp was fixed in place, but the normal ligand binding site was slightly perturbed (i.e. by mutagenesis of a single ligand-interacting amino acid residue), would the ligand stay in the same site or would it shift to new sites?

In our work here, we used an environmental toxin, called BDE-100, that is known to bind to Pgp in previous work by a highly precise and accurate technique called x-ray crystallography (XRC). We reproduced the exact conformation of Pgp including the precise location of BDE-100 binding. Next, we mutated each of five single amino acids that were in direct contact with, or very close to, the BDE-100 ligand. Of particular importance, most of the amino acid residues that form ligand binding sites are of a specific class called aromatic residues such as tyrosine (Y) and phenylalanine (F). We mutated five aromatic residues, one at a time, that interacted with BDE-100, to a much smaller amino acid (alanine, A) that is not aromatic in character (F979A, Y303A, Y306A, F724A and F728A), and determined if BDE-100 stayed in the same place or moved to another location. BDE-100 also has strong advantages because it contains bromine atoms which are easy to localize by XRC using a process called anomalous diffraction. Two mutants, Y303A and Y306A showed strong BDE-100 occupancy at the original ligand binding site (we call site 1), but also revealed a novel site 2 located on the opposing pseudo-symmetric half of the drug binding pocket (DBP). Surprisingly, the F724A structure of Pgp had no detectable binding in site 1 but exhibited a novel site shifted 11 Å from site 1. ATPase studies revealed shifts in ATPase kinetics for the five mutants but otherwise indicated catalytically active transporter that was inhibited by BDE-100 similar to unmutated (wildtype) Pgp. Our results emphasize a high degree of compensatory drug recognition in Pgp made possible by aromatic amino acid sidechains concentrated in the DBP. Compensatory recognition forms the underpinning of polyspecific drug transport but also highlights the challenges associated with the design of therapeutics that evade efflux altogether.

Six Pgp mutants and localization of BDE-100 by X-ray crystallography:

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BDE-100 in each structure is shown in stick representation with Br atoms colored red. Phe974, which undergoes a disorder-to-order transition upon binding in site 2, is labeled in all six panels.

References

1. ↑ Le CA, Harvey DS, Aller SG. Structural definition of polyspecific compensatory ligand recognition by P-glycoprotein. IUCrJ. 2020 Jun 6;7(Pt 4):663-672. doi: 10.1107/S2052252520005709. eCollection, 2020 Jul 1. PMID:32695413 doi:http://dx.doi.org/10.1107/S2052252520005709