An infection caused by pathogens, such as viruses, bacteria, parasites, or fungi, can be seen as a battle between the invading pathogen and our body. The role of the immune system is to protect us against disease or invading threats. But how does the body combat these intruding pathogens? To learn about one particular immune response strategy our body uses for fighting pathogens, read more.
Without even realising it, every day we come into contact with thousands of pathogens that live all around us. Normally they cause no harm, because a healthy immune system can defeat these invading disease-causing pathogens. The best way to prevent infections is to block pathogens from entering the body. But if the physical barriers, like skin and mucosa, are weakened, then pathogens can enter our body and can start multiplying causing an infection and/or killing cells and disrupting cell function.
If this happens, not all is lost. Our body is equipped to fight off invading pathogens. In response to their presence, our immune system immediately springs into action. Anything that is identified as foreign or non-self is a target for the innate immune response. The innate immune system has limited power to stop pathogens from spreading. If pathogens successfully evade the innate response, a second layer of protection (the adaptive immune system) is activated. It specifically targets each pathogen making it more accurate. To specifically target each pathogen it first needs to identify the pathogen, meaning that this type of immune response is slower than that of the innate immune system. The adaptive immune system also has an immunological memory that allows for an enhanced immune response if this pathogen is faced again.
Central to the innate and adaptive arms of the immune system is the ability to use cells, such as natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), which respectively represent the innate and adaptive immune system. Despite various fundamental biological differences, both NK cells and CTLs employ the same, single cytotoxic mechanism to initiate target cell apoptosis, namely the secretory granule-dependent death pathway, where a pore-forming protein (perforin) plays a key role.
Perforin was first characterised as a component present and stored in dense cytoplasmic granules of both cytotoxic T lymphocytes and natural killer cells. This ~70 kDa protein is synthesised as a monomer with multiple domains: the core membrane attack complex/perforin (MACPF) domain, followed by the epidermal growth factor (EGF) � like domain and ending with the membrane-docking C2 domain.
Structure of murine perforin. The MACPF domain is coloured in pink, the EGF domain in lime green and the C2 domain is shown in cyan. The C-terminal end is grey and two calcium ions are indicated as orange spheres. (PDB ID 3NSJ).
The C2 domain of perforin is important for the regulation of its activity and mediating binding to the lipid membrane of the target cell in a calcium-dependent manner. This domain comprises eight β-strands assembled into a β-sandwich fold. Currently, it is unknown how many calcium ions can be coordinated by perforin in the fully liganded state, but there is an estimate that it might be up to five. Calcium ions bind within three calcium-binding regions (CBR1-3) which contain conserved aspartic acid residues. Upon calcium binding, CBR1 and CBR3 are brought into close proximity forming a hydrophobic groove. Four hydrophobic aromatic residues - tryptophan and tyrosine - one of each in CBR1 and CBR3, have been shown to be crucial for proper interaction with the membrane of the target cell.
In the first full-length crystal structure of perforin (PDB ID 3NSJ), two calcium ions were observed. One calcium ion is coordinated between the two CBRs. The second calcium ion is coordinated by a non-conserved aspartic acid residue (Asp490) and found outside the CBR3 in an unusual binding position that appears to be unique to the perforin C2 domain. The subsequent structure of perforin C2 domain (PDB ID 4Y1T) demonstrated the binding of five calcium ions supporting previous observation in the full-length perforin structure.
Comparison of perforin C2 domain of PDB ID 3NSJ (left) and PDB ID 4Y1T (right) mediating calcium ion interaction. Orange spheres represent calcium ions. Schematic representations show residues involved in calcium ions coordination (adapted from DOI 10.1074/jbc.M115.668384).
The conformation of perforin is very different after its synthesis in the endoplasmic reticulum compared to when it resides in the granule. The low pH in the granule, along with a lack of available calcium ions, prevents premature activation of perforin. On release of the granules content, by exocytosis, higher extracellular calcium concentration and a neutral pH promote perforin binding to the target cell plasma membrane where it oligomerizes into pores.
Upon membrane insertion, two α-helical bundles known as transmembrane hairpins (TMH1, TMH2) of MACPF domain refold to form antiparallel membrane-spanning β strands. Each perforin contributes four antiparallel β strands towards assembly of transmembrane β barrel comprising 80-200 strands in a full pore. The membrane facing portion of the β barrel is lined by mostly hydrophobic residues, whereas the inner (luminal) side is mostly hydrophilic. The MACPF, EGF-like, and C2 domains of perforin stay outside the pore. The formation of pores destabilises the membrane and allows the entry of granzymes (also released from the granules alongside perforin) to the target cell cytoplasm, with the subsequent initiation of apoptosis. An alternate pathway may be in which perforin is internalised by endocytosis and then forms pores from inside the endocytic vesicle to release the granzymes.
Perforin pore formation. A. Conformational change of perforin upon insertion into the membrane. The central part of the perforin MACPF domain is shown in red and yellow. The two clusters of α helices (CH1, CH2 - yellow) unwind upon pore formation to insert into the membrane as a pair of amphipathic β strands. The transmembrane region is boxed. PDB ID 3NSJ (left), PDB ID 7PAG (right). B. Superimposition of monomeric perforin before and after pore formation. PDB ID 3NSJ (green), PDB ID 7PAG (light brown). C. Perforin pore (EMD-1769) and segment of the pore map with one perforin monomer fitted.
Human history has, in many cases, been shaped and defined by infectious disease. It is estimated that over half of all humans who ever lived on earth were killed by infectious disease. In retrospect, perhaps there is more to the phrase ‘What doesn’t kill you, makes you stronger�.
Did you know?
1. A person can come in contact with around 60 000 types of germs on a daily basis. But only 1-2% of them are potentially dangerous to a human with a healthy immune system.
2. Unlike vertebrates, plants only have an innate immune system, because they lack mobile immune cells with a highly specific recognition system, and yet they live and survive in the same harsh environment.
3. Leprosy and tuberculosis are believed to be the oldest contagious disease in the history of mankind. This makes Mycobacterium leprae and Mycobacterium tuberculosis the oldest pathogens to have infected humans.
Romana Gáborová
About the artwork
Adelya Efendieva (Year 12) from the Leys School (Cambridge, UK) learned about this protein when studying the immune system. She was intrigued at how its name sounds related to its function, verb perforate: make holes. Upon visualising the structure of the protein after searching it on the PDBe website she could see how its shape could be incorporated into art and had the idea about the membranes with pores in them. For creating her final piece, techniques like etching, printing, and painting with acrylic were used.
View the artwork in the .
Perforin structures mentioned in this article
PDB ID 3NSJ
PDB ID 4Y1T
PDB ID 7PAG
EMDB EMD-1769
Link to perforin-1 at the PDBe-KB protein pages
Sources
