September 27, 2015

Unravelling the immune killing process

Scientists have observed for the first time how calcium ions activate the structural change required for perforin to bind to membranes.

Imaging CoE investigators along with scientists from Peter Macallum Cancer Centre, the University of Melbourne and the Monash Institute of Pharmaceutical Sciences, have revealed that calcium ions cause significant structural changes in perforin to facilitate its interactions with lipid membranes.

“These results provide us with more insight into how perforin initiates the immune killing process. In turn, this will help us design molecules able to regulate the process –upwards in the case of certain diseases such as cancer or downwards in the case of situations such as transplant rejection,” says Professor Whisstock.

Perforin is a protein secreted by immune cells; its function is to bind to the membrane of the target cell and form a pore enabling immune cells to deliver toxins to the target (virally infected or malignant) cell. A crucial step in this process is the interaction between the C2 domain of perforin and membrane of the target cell.

That calcium plays a role in this process has long been known; exactly how calcium ions mediate this event has not.

The study, recently published in the Journal of Biological Chemistry, investigated this mechanism — calcium ions and lipid binding to the C2 domain — using X-ray crystallography and NMR spectroscopy.

“Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca2+ ions bind within the calcium binding regions activating perforin with respect to membrane binding,” says Dr Conroy. “We have also determined the affinities of several of these binding sites and shown that this interaction causes a significant structural rearrangement in the first calcium binding region,” Dr Conroy further explains.

The team is now planning to use this information to better understand how perforin functions to maintain immune homeostasis and to assist in designing strategies to develop novel small molecule and biologic inhibitors to control the activity of perforin in disease.