Antibiotic Finds Novel Way of Sn(e)aking Across Membranes
News Jul 04, 2013
Professor Colin Kleanthous and his research group, together with colleagues in the chemistry department at the University of Oxford and at Birkbeck College, University of London, describe the snaking mechanism in their recent paper in Science (see references below).
The previously unknown process by which these protein antibiotics gain entry into bacteria could be relevant to other systems where proteins cross membranes. The work will also help studies exploring whether these antibiotics, which target closely related bacteria, have clinical potential.
The antibiotics, colicins, are part of a large family of antibacterial proteins that target the gut bacterium Escherichia coli. Similar antibiotics are produced by many other bacteria, including many pathogens, and are used to attack neighbouring bacteria competing for the same resources.
They bind to proteins on the cell surface and then assemble a complex nanomachine or 'translocon' that links the outside of the cell to the inside. Once these connections are made, the colicin is able to move into the cell where it delivers a toxic payload.
Scientists knew colicin used a protein called OmpF on the outside of the target bacteria cell as part of this process but until now, the details of how the colicin exploited OmpF were unknown.
The work from Professor Kleanthous and colleagues sheds light on the process, revealing the surprising 'snaking' mechanism.
The discovery required important technical developments in a number of areas.
One of the major advances was in mass spectrometry, in collaboration with Professor Carol Robinson and Dr Jonathan Hopper in the University of Oxford chemistry department.
"The work we've done together is really pushing the boundaries in terms of membrane protein mass spectrometry", said Professor Kleanthous. "The mass spec result was amazing - it had never been done before."
The group suspected that colicin threaded through two of OmpF's three holes, so designed a new technique sensitive enough to detect if part of the colicin molecule was occupying them.
"The mass spec approach showed that we could measure the mass of the peptide inside the holes", said Professor Kleanthous. "The peptide is only around 1% of the total mass, but we can detect this because the resolution of the technique is so good."
By engineering a mutation in colicin they were able to keep the translocon tethered in place in order to capture, purify and analyse it.
This revealed that to form the translocon colicin had indeed snaked through two of OmpF's holes.
"We found that colicin is tethered to two holes in a three-hole protein", said Professor Kleanthous. "The surprise is that the colicin not only goes into the cell by one of the holes of OmpF but also comes back out again through a second hole."
When viewed under an electron microscope the researchers saw that the threaded colicin allows another protein within the cell membrane to be held in place, making it easier to continue colicin's journey into the bacterium.
This mechanism explains how disordered proteins can burrow their way through narrow pores, as well as pass a charged signal into a cell.
Now that the group have started to piece together the molecular interactions between components of the translocon, they are keen to fill in more of the details.
"We want to find out exactly how the colicin sneaks its way in and out of OmpF and to see if this mechanism occurs in colicins that target pathogenic bacteria", said Professor Kleanthous. "We'll be focusing on all the components of the translocon, ultimately trying to assemble them in a reconstituted system in vitro."
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