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P-Glycoprotein Structure and Chemotherapy Resistance Print

 

A research team from the Scripps Research Institute and the Texas Tech University Health Sciences Center has obtained the first glimpse of a protein that keeps certain substances, including many drugs, out of cells. The protein, called P-glycoprotein, or P-gp for short, is one of the main reasons cancer cells are resistant to chemotherapy drugs. Understanding its structure may help scientists design more effective drugs. The structure is a nice tool for understanding how drugs are transported out of cells by P-gp and for designing drugs to evade P-gp, preventing drug resistance.

The Toll of Multidrug Resistance

The American Cancer Society reported over 12 million new cancer cases and 7.6 million cancer deaths worldwide in 2007. Many cancers fail to respond to chemotherapy by acquiring multidrug resistance (MDR), to which has been attributed the failure of treatment in over 90% of patients with metastatic cancer. Although MDR can have several causes, one major form of chemotherapy resistance has been correlated with the presence of molecular "pumps" that transport drugs out of the cell.

The most prevalent of these MDR transporters is P-gp: a particularly "promiscuous" molecule that can embrace and reject from a cell dozens of distinct types of molecules ranging from peptides to steroids as well as chemotherapy drugs. Once a cancer cell starts to produce this protein, it becomes resistant to chemotherapy drugs and it becomes much less likely that the patient will recover. By providing physical evidence in support of a direct-transport model, the structure solved by Aller et al. marks the culmination of a decades-long search for understanding of the mechanism by which this critical protein operates.

 

P-gp structure. Six transmembrane helices (TMs) and two nucleotide-binding domains (NBDs) are labeled and numbered. The two halves of the molecule (N- and C-terminal halves) are colored yellow and blue, respectively.

P-gp, a protein first identified in 1976, sits in the membrane that surrounds human cells, including those in the gut, intestine, kidney, and brain, where it functions as a gate keeper, shooing out potentially harmful agents. Problematically, P-gp not only transports substances that are harmful out of the cell, but also drugs targeted to cancer cells and HIV-infected cells, as well as some therapeutics aimed at alleviating psychiatric conditions.

The scientists succeeded in performing the x-ray crystallography on mouse protein P-gp at SSRL, APS, and ALS Beamlines 8.2.2 and 8.3.1. Once they solved the structure, they found that the mouse P-gp, which is 87 percent identical to its human counterpart, has the shape of an upside down "V" or an inverted cone with a large cavity inside. The cone's interior is lined with amino acids that bind to various substances, holding them in place. The top part of the cone resides inside the cell membrane and has two openings for substances to enter; the bottom portion protrudes into the cell, ending in two dumbbell-shaped arms.

This overall shape is strikingly similar to that of another protein, MsbA, that transports lipids out of bacteria (see previous ALS highlight, "Protein Flips Lipids Across Membranes"). This similarity suggests that P-gp works by bringing the two dumbbell-shaped arms together on the inside of the cell and opening the closed end toward the outside of the cell, essentially reversing direction of the "v" shape so any substance caught inside the protein's cavity is ejected from the cell.

Model of substrate direct transport by P-gp (gray). (a) Substrate molecules (magenta) enter a cavity lined with amino acids (cyan) that bind to a wide variety of molecules. (b) ATP molecules (yellow) bind to the NBDs, causing a large conformational change, exposing the substrate to the outside of the cell.

While the new study shows another similarity between bacterial MsbA and mammalian P-gp—both binding cavities are lined with hydrophobic (water-avoiding) amino acids—it turns out that the mammalian P-gp has many more such amino acids and a greater variety of them, including ones containing aromatic compounds (i.e., compounds with one or more benzene rings). The presence of so many hydrophobic and aromatic residues explains how, unlike the bacterial protein, the mammalian P-gp is able to accommodate a wide range of substrates.

This study also produced insights by showing structures of P-gp bound to some of its substrates. Research team members Chang and Aller collaborated with Qinghai Zhang, an assistant professor at Scripps Research who had designed several compounds that can block the function of P-gp. These compounds bind inside the P-gp cavity, preventing other substances from entering. Chang and Aller were able to obtain the structures of two of Zhang's compounds inside P-gp. They both go in the same cavity and bind to different amino acids, but with some overlap. What this tells us is that there is an extremely important core set of amino acids in P-gp that bind all substances, and there are additional amino acids for fine-tuning the binding to specific drugs.

Knowing what the P-gp binding cavity looks like and precisely where substances bind may allow researchers design better drugs, for example by using chemistry to modify portions of that drug so that it can sneak past P-gp to get inside cells. One advantage in this process is that we don't have to design brand new drugs, but rather redesign existing ones to make them work better.

 


 

Research conducted by S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, Q. Zhang, and G. Chang (The Scripps Research Institute); R. Zhuo, P.M. Harrell, Y.T. Trinh, and I.L. Urbatsch (Texas Tech University Health Sciences Center).

Research funding: U.S. Army, National Institutes of Health, Beckman Foundation, Skaggs Chemical Biology Foundation, Jasper L. and Jack Denton Wilson Foundation, Southwest Cancer and Treatment Center, and the Norton B. Gilula Fellowship. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P.M. Harrell, Y.T. Trinh, Q. Zhang, I.L. Urbatsch, and G. Chang, "Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding," Science 323, 1718 (2009).

 

 

ALSNews Vol. 298, May 27, 2009

 

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