The survival of bacteria either in the environment or within a host organism relies not only on the microbe’s ability to replicate and divide rapidly, but is also contingent upon the capacity to shuttle important molecules beyond its own boundaries. Many bacteria coordinate signals to other members of their species, release toxic compounds to thwart their foes, and, most deviously, release signals to manipulate host cells they have infected. However, to accomplish these tasks, microbes must get their cargo past their own cell membranes.
Bacteria are able to accomplish these feats since they have evolved specialized structures and systems for expelling proteins. Now, researchers from The Rockefeller University have been able to resolve the atomic structure of a seemingly simple, but previously unexamined pump from the bacterium Clostridium thermocellum that escorts proteins through the organism’s cell membrane.
“This pump, called PCAT for peptidase-containing ATP-binding cassette transporter, is composed of a single protein, a sort of all-in-one machine capable of recognizing its cargo, processing it, then burning chemical fuel to pump that cargo out of the cell,” explained senior author Jue Chen, Ph.D., professor and head of the Laboratory of Membrane Biology and Biophysics at The Rockefeller University. “This new atomic-level structure explains for the first time the links between these three functions.”
The findings from this study were published today in Nature through an article entitled “Crystal structures of a polypeptide processing and secretion transporter.”
The researchers were able to observe that each PCAT contains two peptidase domains that cleave the secretion signal, a peptide sequence that tells the cell where the protein is headed, two transmembrane domains that form a translocation pathway, and two nucleotide-binding domains that use ATP for energy required to complete the entire process.
PCATs specialize in pumping proteins out of the cell, and, because they are single-molecule machines that work alone, or with two partner proteins in some bacteria, they are the most simplistic of such systems. Numerous homologs of PCAT proteins exist, spread throughout an array of bacterial species, many for which their functional role have yet to be determined.
“At this point, we have no idea how many PCATs exist, although we expect they are numerous because each specializes in a specific type of cargo. For this study, we focused on one we called PCAT1, which transports a small protein of unknown function,” stated lead author David Yin-wei Lin, Ph.D, postdoctoral researcher in Dr. Chen’s laboratory. “To get a sense of how PCAT1 changes shape when powered by energy from ATP, we examined the structure in two states, both with and without ATP.”
The Rockefeller team employed a technique called X-ray diffraction analysis, in which a pattern produced by X-rays bounced off the crystallized protein can be used to infer the structure of the molecule. What they found was a remarkable feature of a large, water-filled central channel that is an appropriate environment for hydrophilic (water-loving) proteins. Furthermore, they found that the side openings into the channel were guarded by the cargo-recognizing domain—recognizing and clipping off cargos tag before guiding the protein into the channel.
The investigators were excited by their findings and are continuing their work to determine the cargo that PCAT1 pushes out of the cell, as well as looking to see if their newly resolved crystal structure reveals any possible druggable targets for therapeutic intervention.