AcrB is a protein that resides in the inner membrane of Escherichia coli cells. It works in unison with two other proteins to rid the bacteria of toxins. Based on earlier studies, the researchers knew that AcrB boasts a large cavity capable of binding with a vast range of antibiotics and other molecules. But precisely how this cavity accommodates so many shapes and sizes remained unclear. To witness this trickery, the team crystallized the protein in the presence of four molecules—an antibiotic, a dye, a disinfectant, and a DNA-binding molecule—and exposed the crystals to extremely bright x rays at ALS Beamline 8.2.2. The resulting 3.5- to 3.8-Å-resolution images provide the closest look yet at a phenomenon common to all living cells: the ability to expel a diverse flotilla of toxins using one pump.

Multidrug pumps such as AcrB play important, but double-edged, roles. Normally, harmless colonies of E. coli inhabit animal intestines. In this environment, scientists theorize that AcrB's chief function is to trap and expel bile salt, which is toxic to the bacteria. Unfortunately, if a mutated form of E. coli causes food poisoning, a broad regimen of antibiotics is needed to fight the infection because AcrB shields the bacteria from many more compounds in addition to bile salt. In the structures obtained at the ALS, each of the four molecules crystallized with AcrB bonded to a different location in the protein's cavity, and each bond utilized a different set of amino-acid residues. The researchers suspect the cavity possesses areas where many types of antibiotics could be captured.

Can Bacterial Resistance
Be Made Futile?

They say that what doesn't kill you makes you stronger. This also seems to apply, unfortunately, to lowly bacteria. Despite the development in the twentieth century of "miracle drugs" such as penicillin, scientists have begun to report the emergence of drug-resistant strains of bacteria that were once considered in check. Antibiotics, if used judiciously and conscientiously, can eradicate a bacterial infection; however, their inappropriate or incomplete use provides an opportunity for any drug-resistant mutations in a population to flourish. As a result, bacteria that were once considered benign or controllable, such as those responsible for food poisoning, tuberculosis, and pneumonia, for example, are reemerging in more aggressive forms that are more difficult to treat. Basic research plays a critical role in responding to this problem. The AcrB pump, as revealed through protein crystallography, is extremely versatile in trapping and expelling toxins (including antibiotics) from bacteria. Although strategies for inhibiting these pumps are still at the early stages of research, structural knowledge about such bacterial defense mechanisms can lead to more effective avenues for drug-based counterattack.

Even ciprofloxacin, an antibiotic used to treat a variety of bacterial infections including inhaled anthrax, is no match for AcrB. In this image, the green-colored drug is firmly ensnared in the protein's cavity.

These results underscore the need to pursue alternative ways of fighting drug resistance. Currently, pharmaceutical researchers combat resistance by tweaking an antibiotic's molecular structure so that it isn't compatible with a pump's binding site. This is difficult work to begin with, largely because antibiotics must adhere to strict potency and safety regulations that limit the extent to which they can be modified. Add to these restrictions a better understanding of AcrB's readiness to bind with an array of compounds, including the most carefully engineered antibiotics, and the job appears even more difficult.

Instead, the researchers believe their work supports another strategy in which the multidrug pump is disabled. In E. coli, such monkey-wrenching is possible because AcrB connects to a funnel-shaped protein embedded in the bacteria cell's outer membrane. This protein ejects the antibiotics trapped in the AcrB cavity. If a specially designed molecule could lodge inside the funnel, the pump would be rendered useless and antibiotics would be allowed to slip inside the cell unhindered.

To support these advancements in drug design, the team will next use the ALS to more fully explore how AcrB binds with a structurally diverse range of antibiotics. They believe a fuller understanding of E. coli's multidrug pump will shed light on similar pumps found in other harmful bacteria, which could lead to better treatments for a variety of infections.

Research conducted by E.W. Yu, H.I. Zgurskaya, H. Nikaido, D.E. Koshland, Jr. (University of California, Berkeley); G. McDermott (Berkeley Lab).

Research funding: Howard Hughes Medical Institute and the National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: E.W. Yu, G. McDermott, H.I. Zgurskaya, H. Nikaido, and D. Koshland, Jr., "Structural Basis of Multiple Drug-Binding Capacity of the AcrB Multidrug Efflux Pump," Science 300, 976 (2003).