Normal for NorM
An X-ray structure determined by US researchers reveals details of the only remaining class of multidrug resistance transporters that remained to be described. The work has implications for antibiotic-resistant strains of bacteria, as well as for developing hardy strains of agricultural crops.
Geoffrey Chang of the Scripps Research Institute's Department of Molecular Biology and colleagues reckon they are close to understanding one of the final pieces in the drug resistance puzzle. The researchers focused on the protein NorM, which is found in the highly infectious bacterium Vibrio cholerae, the water-born agent responsible for the lethal disease cholera. Cholera often emerges in the least developed parts of the world and during natural disasters when sanitation fails and people are living alongside foul water and have no access to fresh drinking water.
Transporting drug resistance
For its part, the NorM transporter is responsible for the widespread resistance to the primary medication used to treat cholera, ciprofloxacin, and other inexpensive, broad-spectrum antibiotics in the fluoroquinolone class. It has also led to resistance to an entirely new class of drug designed specifically to overcome antibiotic resistance, tigecycline.
NorM is a member of a family of proteins that evolved to protect organisms. They carry out an important biological function across all kingdoms of life, plants, animals, microbes by pumping out toxic chemicals before they can have any detrimental effect on the organism. Unfortunately, that means they also pump out the toxic drug compounds we try to use against those organisms in the case of the cholera bacterium. Indeed, the protein family is known as the multidrug and toxic compound extrusion (MATE) family. In addition to endowing bacteria with antibiotic resistance, MATE transporters are also associated with resistance to a commonly used diabetes drug, as well as resistance to anti-inflammatory and heart drugs. These transporters exist in human liver and kidney cells and so can reduce the effectiveness of a wide variety of drugs. In plants, MATE transporters help to neutralize the acidity of soil, directly affecting crop yields worldwide".
Crystallisation woes
Initially, the researchers struggled to produce adequate quantities of the protein in pure enough form with which to work, and then as is common with protein diffraction, obtaining X-ray quality crystals was a difficult task and even then the NorM crystals were unusually fragile under the intense gaze of the X-rays. After many attempts, the research team succeeded in producing two crystal structures of the NorM transporter as it sat on the outside surface of V. cholerae. One showed the transporter by itself and the other provided a snapshot of how the pump is powered by sodium ions. With these crystal structures to hand, scientists can for the first time figure out exactly how this transporter works, Chang explains. "This could lead to the design of drugs that evade or inhibit the transporter, or to reengineering the transporter to help some plants grow in soil they can't grow in now."
The V-shaped NorM transporter usually sits upside down on the interior of the bacterial cell membrane waiting for any toxic chemicals, antibiotics for instance, to pass through the membrane. When this happens, the protein responds by changing shape, so scooping up the chemical and then transporting it back through the cell wall to the outside of the bacterium.
The structure of this bacterial pump revealed a shape distinct from all other MDR transporter families, explain team members Xiao He and Paul Szewczyk of the University of San Diego, California, (UCSD) who worked with Chang to derive the structure. The pair also took the lead in the effort to verify the crystal structure -- a process of labelling 16 different amino acids on the protein and confirming their three-dimensional position. This alone took 18 months.
By showing how a key member of the MATE transporter family changes shape during the extrusion of toxic compounds, the Chang work might ultimately lead to new ways of blocking the transporter, with possible applications in medicine and agriculture. Bacteria have a number of different transporter systems, so it is important to design antibiotics that will not be instantly pumped out, the researchers explain.
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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