Single molecule compression reveals intra-protein forces drive cytotoxin pore formation

Abstract

Perfringolysin O (PFO) is a prototypical member of a large family of pore-forming proteins that undergo a significant reduction in height during the transition from the membrane-assembled prepore to the membrane-inserted pore. Here, we show that targeted application of compressive forces can catalyze this conformational change in individual PFO complexes trapped at the prepore stage, recapitulating this critical step of the spontaneous process. The free energy landscape determined from these measurements is in good agreement with that obtained from molecular dynamics simulations showing that an equivalent internal force is generated by the interaction of the exposed hydrophobic residues with the membrane. This hydrophobic force is transmitted across the entire structure to produce a compressive stress across a distant, otherwise stable domain, catalyzing its transition from an extended to compact conformation. Single molecule compression is likely to become an important tool to investigate conformational transitions in membrane proteins.Proteins are made up of chains of amino acids that need to fold into intricate three-dimensional shapes to work correctly. But some proteins also have to change their shape drastically when they work. Mechanical forces that change the shape of a protein can therefore be used to determine how a protein folds and how it changes its structure when working.Although researchers have developed techniques to analyze the effect of force on single proteins, most studies carried out so far have investigated the effect of stretching (or tensile forces) to understand structural changes that naturally involve an extension within the protein. However, many proteins undergo structural changes that involve a compaction in their shape. How these changes occur remains poorly understood because, for these, methods to apply compressive forces to single proteins are required.Perfringolysin O (PFO for short) is a protein that is made by a bacterium that causes food poisoning in humans. PFO makes pores in the membrane that surrounds cells. This causes the cell’s contents to leak out, killing the cell. When inserting into the membrane, PFO changes from an elongated “prepore” state to a compact pore-forming state.Czajkowsky et al. now use a combination of single molecule techniques and computer simulations to investigate how PFO undergoes this compaction. Previous work had identified a mutant PFO protein that arrests at the prepore state. Applying a compressive force to the top of this prepore-trapped PFO as it sits on the membrane transmitted forces across the entire PFO protein. This ultimately produced a compressive force across a distant part of the protein that caused the protein to change from the elongated prepore state to the compact, pore-like shape. If a compressive force was not applied, the PFO protein remained in the prepore state. Czajkowsky et al. further found that this compressive force is naturally produced by distant water-repellent parts of the naturally occurring protein interacting with the cell membrane. Therefore, internal forces can transmit across proteins to drive shape changes in distant regions.In the future, the methods developed in this study could be applied to analyze other naturally occurring changes in proteins where shape compaction happens when working.