Dynamics and pathways of assembly in membrane pore formation

Pore-forming proteins are crucial armaments in the continuous battle between living organisms and the pathogens that threaten their fitness and survival. These proteins act on cells, which are the micrometre-scaled, basic units of all forms of life. Cells are separated and protected from their environment by a thin membrane. Pathogens such as bacteria can release pore-forming proteins ("toxins") that drill holes in the membranes of healthy cells in the host organism, to release nutrients for the bacteria, to invade these cells and/or kill them. Patients affected by bacterial pneumonia, for example, suffer from the devastating effects of such a toxin, pneumolysin, on lung tissue. The immune system, however, uses a similar mechanism to kill germs and infected or cancerous cells, thus preventing them from doing further damage to the organism. It secretes related, but somewhat different pore-forming proteins to perforate the membranes of such unwanted invaders.

To perform these tasks, pore-forming proteins have developed sophisticated drilling mechanism. These proteins can convert from a soluble form in the aqueous, cellular environment into a very different form, in which 20-50 protein molecules assemble into a ring-shaped pore bound to the membrane. We can look at these forms with X-rays or electrons to deduce their three-dimensional structures. Thanks to such experiments, we now have a reasonably clear picture of the soluble proteins and their pore structure in the membrane. For some pore-forming proteins, scientists have even identified the changes inside the proteins which make this transition possible.

However, if we wish to design drugs that prevent such pores from being formed, as in the example of bacterial pneumonia indicated above, it would be useful to know more about the steps in their formation. It is exactly this pore assembly that is still largely enigmatic. In this project, we will try to answer some specific questions about membrane pore formation. We would like to know how the proteins assemble on the membrane. Do they assemble one by one, or do they first form larger units that subsequently assemble in a pore? Do the proteins first need to assemble on the membrane, or can they dock in the membrane and assemble in pores afterwards? And at what point in this process will the membrane that is surrounded by the assembled protein be extruded to create a hole?

To investigate the dynamics of this process, we rely on a technique called atomic force microscopy. Atomic force microscopy is the small-scale equivalent of reading Braille: With a tiny artificial finger, we feel the pore-forming proteins while they assemble on the membrane. Whereas X-ray crystallography and electron microscopy are limited to static samples, atomic force microscopy can probe active proteins while they are at work. We will thus apply atomic force microscopy to the membranes that are being exposed to attack by pore-forming proteins. Meanwhile, we will benefit from the more detailed views provided by electron microscopy to identify intermediate assemblies of pore-forming proteins, that are trapped by chemical bonds or by lowering the temperature. Electron microscopy will thus provide highly detailed pictures of pore forming proteins in different states of assembly and atomic force microscopy will enable us to see how the proteins transit between these different states.

We will use real-time atomic force microscopy (AFM), single particle electron microscopy (EM) and electron tomography to track the dynamics and the pathways of assembly of pore-forming proteins on and in membranes. We will focus on the superfamily of cholesterol dependent cytolysins (CDCs) and membrane attack complex/perforin (MACPF) pore-forming proteins. Though atomic structures of CDC and MACPF proteins have been determined for the soluble monomers, along with low resolution maps of the membrane-inserted pores, the dynamics and pathways of assembly are much harder to address experimentally. To find assembly pathways, we will trap intermediate assemblies with disulphide locked variants and reduced temperatures, and determine their structures by EM. Moreover, benefitting from recent developments on fast-scanning AFM, we will follow the assembly and pore formation by real-time imaging of the functional proteins on planar-supported model membranes. Combining the two methods, we will determine the sequential steps in the assembly of the prepore and pore state of bacterial CDCs and of the MACPF protein perforin. By probing inside the rings with AFM, we will also investigate the fate of the lipid bilayer in the pores upon assembly of bacterial CDCs. We will elucidate biophysical mechanisms that are fundamental to pathogen attack and immune defence. This information is anticipated to create opportunities for new drug design, either by stabilising pore-forming proteins to prevent pore formation, or in stabilising or enhancing their membrane-bound forms, in case of deficient pore-forming activity.

This proposal aims at elucidating the biological and biophysical mechanisms of membrane pore formation by bacterial toxins and immune mediators. The superfamily of membrane attack complex/perforin (MACPF) and cholesterol-dependent cytolysin (CDC) proteins that it focuses on are of significant medical importance. The CDC perfringolysin O rapidly induces irreversible cellular injury in a deadly form of gangrene that is caused by the bacterium Clostridium perfringens. The CDC pneumolysin is a major virulence factor of Streptococcus pneumoniae, at the root of bacterial pneumonia, still a major cause of death and illness throughout the world despite the widespread use of antibiotics. When released in the lungs, pneumolysin damages the lung tissue and its blood vessels. Antibiotics may exacerbate lung damage because they are designed to kill the bacteria by breaking them open, which leads to the further release of pneumolysin. Perforin is the main weapon of natural killer cells. It punches holes in virally infected or cancerous cells that have been detected by the immune system, and delivers lethal granzymes through these holes. Babies born with defective perforin succumb to viral infections or tumours early in life. On the other hand, if perforin is too active, normal cells can be incorrectly killed.

By elucidating the mechanism of membrane pore formation by pore-forming proteins, we will create new opportunities for drug design: e.g., the prevention of pore formation by pneumolysin would be a significant advance in the treatment of bacterial pneumonia; the ability to control the activity of MACPF proteins in the human immune system could be an important means of regulating the immune response during and after tissue and organ transplantation or could alleviate the perforin-dependent cytotoxicity in autoimmune diabetes.

In addition, the proposed methodology for studying membrane pore formation is not restricted to pore-forming proteins alone. It can be applied to a variety of other medically relevant interactions between membranes and macromolecules. Examples of this are antimicrobial peptides that are currently investigated as new therapies against bacterial infections, as well as pH-sensitive polymers that are used for intracellular drug (e.g., gene and RNA therapies) delivery across the barrier formed by endosomal membranes after cellular drug in-take via endocytosis. We anticipate that the development of such novel therapeutic approaches will be enhanced by molecular-scale understanding as can be achieved with the methods outlined in this proposal.

On the shorter term (several years), this research will thus benefit pharmaceutical industry and biomedical SMEs. On the longer term (10-20 years, given the lead times for drug development), it will have an impact on healthcare practitioners and patients.

Further impact can be achieved on the AFM technology. With this project, we propose a new application of fast-scanning AFM techniques that have only recently been developed. For such a young technology, feedback based on relevant applications is crucial. It helps to adjust the technology such that it can make a larger impact over a wider range of applications, which will benefit nanotechnological SMEs.

Given the power of high-quality images and movies such as can be produced by in particular AFM, we also anticipate an educational impact to the lay audience by demonstrating a powerful and biomedically relevant application of basic sciences and nanotechnology.