Bacteria use pore forming proteins to drill holes in the membranes of their target cells. Similarly, our immune system uses such proteins to kill bacteria, virus-infected and cancerous cells. In the course of their action, these proteins are first released as soluble monomers, which next bind to the target membrane and self-assemble into transmembrane pores of typically >10 nm size and consisting of up to several tens of monomeric subunits.

To understand this rather remarkable transformation and the effectiveness of the resulting membrane attack, we have carried out real-time atomic force microscopy and electron microscopy experiments, combined with mutagenesis, on proteins of the superfamily of membrane attack complex/ perforin (MACPF) and bacterial cholesterol dependent cytolysins (CDCs).

For the bacterial CDCs, we find that the self-assembly solely occurs in the so-called prepore state on the membrane. The self-assembly process results in a distribution of differently sized assemblies, which can be understood via a simple model of kinetically trapped oligomerisation. Next, these assemblies insert into the membrane, resulting in membrane lesions regardless the size of the protein assembly. For immune effector perforin, on the other hand, the assembly is greatly enhanced upon membrane insertion of short perforin oligomers, suggesting that membrane lesions help to attract perforin to thus growing transmembrane pores. In addition, we find that the addition of a non-inserting perforin mutant hampers the functionality of wild-type perforin, possibly providing a mechanism for the failure of the immune system in patients with partially deficient perforin.

Overall, our results provide a molecular basis for an important process in bacterial attack and immune defence, as well as new insights in the self-assembly of oligomeric protein machinery.

References:

[1] Leung, C., Dudkina, N. V., Lukoyanova, N., Hodel, A. W., Farabella, I., Pandurangan, A. P., . . . & Hoogenboom, B. W. (2014). Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin. eLife, 3, e04247. doi:10.7554/eLife.04247

[2] Hodel, A. W., Leung, C., Dudkina, N. V., Saibil, H. R., & Hoogenboom, B. W. (2016). Atomic force microscopy of membrane pore formation by cholesterol dependent cytolysins. Curr Opin Struct Biol, 39, 8-15. doi:10.1016/j.sbi.2016.03.005

[3] Lukoyanova, N., Hoogenboom, B. W., & Saibil, H. R. (2016). The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins. J Cell Sci, 129 (11), 2125-2133. doi:10.1242/jcs.182741

[4] Leung, C., Hodel, A. W., Brennan, A. J., Lukoyanova, N., Tran, S., House, C. M., …, & Hoogenboom, B. W. (2017). Real-time visualization of perforin nanopore assembly. Nat Nanotechnol. doi:10.1038/nnano.2016.303