Pump it up: crystal clue to bacterial resistance

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Ezine

  • Published: Mar 1, 2011
  • Author: David Bradley
  • Channels: X-ray Spectrometry
thumbnail image: Pump it up: crystal clue to bacterial resistance

Three-parts per...

Two parts of the three-part system that pumps toxins from bacteria and allows them to resist the chemical onslaught of antibiotics has been identified and described using crystallography. The structure could help in the development of new drugs that might circumvent antibiotic resistance.

Gram-negative bacteria, such as the biomedical researcher's favourite bacterium, Escherichia coli, expel toxic chemicals by recognizing and driving out heavy metals and toxins, including antibiotics, using a three-part efflux pump, a protein complex that spans the microbe's inner and outer membranes. The pump comprises a substrate-binding transporter at the inner membrane, a membrane fusion protein and an outer-membrane-anchored channel. These efflux systems are a key part of the bacterial armoury against antibiotics and evolve rapidly to allow bacteria to expel the novel toxic compounds they encounter.

Edward Yu of Iowa State University and the Ames Laboratory and colleagues Robert Jernigan, Chih-Chia Su, Feng Long and Michael Zimmermann, Iowa State, Kanagalaghatta Rajashankar of Argonne National Laboratory, report details of the co-crystal structure of two parts of the three-part efflux pump in the journal Nature.

Efflux effect

"A crystallographic model of this tripartite efflux complex has been unavailable," the researchers report, "simply because co-crystallization of different components of the system has proven to be extremely difficult." This is a common issue facing research into membrane proteins, which are, given their nature, notoriously difficult to crystallise.

The team described the first part of the pump, the inner membrane transporter known as CusA, in September and now reveal details of the inner membrane transporter and how it interacts with the pump's middle adapter, known as CusB. The two parts together are known as the CusBA complex. Yu explains that a better understanding of how all the three parts work together might one day help biomedical researchers and drug discovery chemists find ways to restore the effectiveness of antibiotics.

In the latest study, the researchers purified and co-crystallized the proteins that make up the transporter and adaptor parts of the efflux pump from E coli using the technique of sitting-drop vapour diffusion, the complete process takes about two months. Then they used an X-ray diffraction technique known as molecular replacement with single-wavelength anomalous dispersion (SAD) using the program Phaser, to determine the structure and how the two parts interact.

The study shows how the inner transporter's three molecules bind and interact with the middle adaptor's six molecules as well as revealing a funnel-like channel formed by the adaptors' six molecules, which extends from the top of the transporter. With this structure and the earlier determination in hand, the team was able to predict how the three-molecule structure of the pump's third part, the outer membrane channel known as CusC, interacts with the CusBA complex. The modelling studies of the complex show how it spans the entire bacterial cell envelope and can transport silver and copper ions out of the bacterium.

"The team is now studying how all three parts of the pump, known as CusCBA, assemble. This information will provide a platform for researchers to design inhibitors so that the pump cannot be assembled and thus cannot function," Yu told SpectroscopyNOW.

 



The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

 Two parts of the three-part system that pumps toxins from bacteria and allows them to resist the chemical onslaught of antibiotics has been identified and described using crystallography. The structure could help in the development of new drugs that might circumvent antibiotic resistance.
This ribbon diagram shows the inner membrane transporter (green) and the middle adapter (red and blue) of the CusBA pump. Credit: Edward Yu/Iowa State University, Ames Laboratory

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