Only one thousandth of a milligram of bacterial botulinum toxin is needed to kill a living organism. The toxin exerts its lethal effect by preventing the release of neurotransmitters at the stage where nerve cells attach to muscle tissue, thereby paralyzing them. As uncomplicated as it sounds, this process is actually a sophisticated, multi-step procedure. No less complex and in fact very effective is the process of poisoning complexes of toxins (Tc), virulence factors of many bacteria, including insects and human pathogens.
A bacterial syringe releases a deadly enzyme
The mechanism of action Tc toxins have only recently been more widely discovered by the work of Stefan Raunser’s team in structural biology at MPI Dortmund. “Unraveling the construction of Tc toxin subunits and their assembly using cryo-electron microscopy (cryo-EM) allowed us to understand key steps in toxin activation and membrane penetration,” says Raunser. Scientists have shown that the subunits of the Tc toxin complex work together like a syringe: once the subunits are assembled, structural changes in the complex trigger the opening of a cocoon that contains a toxic enzyme, which is then secreted in a proprietary injection mechanism by using a channel in the host cell. There, it unfolds its deadly effect by disrupting the regulation of the cell’s cytoskeleton, made up of a network of polymerized actin (F-actin) filaments involved in many essential cellular processes.
Renting the opponent by reducing the striking distance
“For a long time, we have struggled to get a full picture of the intoxication process, as we lacked the structural data of the secreted enzymes, one of which is TccC3,” Raunser explains. Until recently, it was only known that TccC3 transfers an ADP-ribose group to actin promoting its aberrant polymerization, which leads to clumping of actin filaments. “TccC3 is what we call a ‘difficult’ system for structural investigations due to its size and high flexibility,” says Hartmut Oschkinat. “Only by applying the NMR remedy could we overcome this challenge and visualize the 3D composition of the protein for the first time.” By fusing two other cryo-EM snapshots of F-actin-bound TccC3 and modified F-actin alone, scientists uncovered the enzyme’s unique mechanism of action. “TccC3 acts like a boxer who tramples his opponent to make him vulnerable to attack” explains Stefan Raunser. In the first step, the enzyme binds to a region between two consecutive actin subunits of F-actin. TccC3 then opens a gate, which brings the NAD+ molecule that contains the ADP-ribose group within striking distance of a reactive site on actin. Once the ADP-ribose bulky group is transferred to F-actin, it is no longer available for its depolymerization factors, as F-actin can no longer be broken down and therefore clump together.
In addition to this finding, the scientists’ findings helped formulate an explanation for the surprisingly high efficiency of the enzyme. When the enzyme detaches from F-actin, its gate mechanism prevents futile reconnection to already modified actin in preparation for the next attack. “It’s amazing how all of these mechanisms have evolved to increase the potency of toxins to the utmost. And the character has done a good job since botulinum toxins, ricin and other biotoxins are still considered the most toxic substances known,” concludes Raunser.
ActinEnzymeMusic BandPoisoningProteinProtein SubunitTechnetium99Toxin