Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin
[Originally posted on 31 December 2014]
Comment on Leung et al., Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin; http://dx.doi.org/10.7554/eLife.04247
Robert JC Gilbert [1], Andreas F-P Sonnen [2], Mauro Dalla Serra [3], Gregor Anderluh [4,5]
1 Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK
2 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
3 National Research Council of Italy - Institute of Biophysics and Bruno Kessler Foundation, Via alla Cascata 56/C, 38123 Trento, Italy
4 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
5 Department of Biology, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
eLife has just published a very interesting study of the pore structure and pore formation mechanism of the bacterial toxin suilysin by Leung et al, http://dx.doi.org/10.7554/eLife.04247. This paper should be seen in a broader context and we would like to highlight ways in which it complements pre-existing data.
The AFM data in the paper by Leung et al indicate that both complete rings and arcs of suilysin subunits can form pores, and from their work the authors conclude that a kinetic mechanism governs the point at which pore formation occurs. Leung et al are, of course, correct in this but the model has been proposed before (Gilbert, 2005). In fact, it was this model which led to the work described in a recent paper by some of us (Sonnen et al., 2014) which Leung et al reference several times.
Firstly, it is important to note that arc pore structures for cholesterol-dependent cytolysins (CDCs) were originally proposed by Sucharit Bhakdi in 1985 (Bhakdi et al., 1985) (see Figure 8 of Bhakdi et al for a model with a toroidal lipid edge which Leung et al echo in their Figure 6 and we reproduce in Figure 1a below). Professor Bhakdi’s lab then worked hard to provide data in support of their model, in the face of much opposition claiming it was impossible. The best data they obtained were described in an important paper in 1998 which showed the correlation of capped oligomerisation and smaller pore size (Palmer et al., 1998) (also in Figure 1a below). A remaining question however was whether pore formation was an “all-or-nothing” event or was gradual, as in fact argued by Bhakdi and colleagues (Palmer, 2001).
So, secondly, Rodney Tweten and colleagues showed around the year 2000 that in fact pore formation by CDCs is “all-or-nothing”, and there is a definite transition between the prepore state and the pore. In other words, oligomerisation ends before pore formation occurs – as pointed out by Leung and colleagues on the basis of their own data. Leung et al cite a relevant paper (Hotze et al., 2001) which, like their work, made use of disulphide trapping of oligomers in the prepore state.
In 2004 Tweten and colleagues published an important paper which also used AFM and which the work of Leung et al echoes (Czajkowsky et al., 2004). Leung et al do cite Czajkowsky et al, but only to say that a vertical reduction in oligomeric height accompanies pore formation. Much more importantly, Czajkowsky et al also showed data which like those of Leung et al reveal arcs of subunits with the tell-tale reduced pore height above the targeted membrane (Figure 1a below). The AFM images of Tweten and colleagues also made use of a disulphide-locked mutant which cannot undergo the prepore-to-pore transition; this was followed by an article by one of us (Gilbert, 2005) which presents a model for pore formation by kinetically trapped arcs as well as completed rings the same as that proposed by Leung and colleagues (see again Figure 1a below). The strength of Leung et al’s paper is that they have confirmed this model and quantified it, modelling the distribution of arcs and rings and showing that a minimal pore-forming arc has 5 subunits.
There are a great deal of electrophysiology and other data on CDCs and proteins related to the CDCs which support the model of pore formation by arcs with a toroidal lipidic component, alongside pore formation by rings of subunits (Figures 1a and 1b below). Leung et al do cite one review on this (Marchioretto et al., 2013), but they could have been more generous in their recognition of the contributions of others, over many years. Among the most important papers on CDCs are those by Gianfranco Menestrina (e.g. (Menestrina et al., 1990)) on perfringolysin (Figure 1a) and another CDC on which complementary data were obtained recently is listeriolysin (Bavdek et al., 2012).
Since 2007 it has been known that CDCs and membrane attack complex-perforin (MACPF) proteins are structural homologues (Anderluh and Gilbert, 2014; Hadders et al., 2007; Rosado et al., 2007). The discovery that pores formed by MACPFs can be made from combinations of protein arcs and lipid is even older than the equivalent discovery with CDCs. Membrane attack complex arc pores were first noted in 1964 (Borsos et al., 1964) (Figure 1b) with a toroidal lipid edge to the pore later proposed (Bhakdi and Tranum-Jensen, 1991; Tschopp, 1984), and perforin arc pores in 1983 (Podack and Dennert, 1983) (Figure 1b). More recent biophysical data have strongly supported the idea that functional pores are generated by perforin arcs (including double arcs, as seen with suilysin by Leung et al) (Praper et al., 2011) (Figure 1b) and these data were complemented this year by a paper describing studies in cells and using AFM (Metkar et al., 2014) (Figure 1b), where the ongoing oligomerisation process was blocked at a premature, but fully functional, stage with the mAB pf-80 and evidence for a minimal functional arc assembly size was also reported (Metkar et al., 2014).
Finally, there have been fascinating insights gained into the actual biological relevance of smaller pores formed by arcs of MACPF/CDC subunits in a number of studies with which the beautiful work of Leung and colleagues should be compared – such as with the CDC listeriolysin (Birmingham et al., 2008; Shaughnessy et al., 2006), perforin within target cells (Thiery et al., 2011) and structural work on membrane attack complex proteins which argues for the pore-forming role of arcs and their gradual increase in size without in that case a prepore state (Aleshin et al., 2012) (i.e. membrane attack complex pores, unlike those of CDCs, are not “all-or-nothing”).
The paper by Leung et al is an important and helpful contribution to a mature field of research in which discussion concerning the structure and biological significance of pores formed by arcs of protein subunits has been ongoing for 30 years and more (Gilbert et al., 2014). Its impact can only be increased if it is seen in that context.
References
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Figure 1. Examples of evidence for and models of pores formed by arcs of MACPF/CDC protein subunits.
(a) The original proposal made by Bhakdi and colleagues in 1985 on the basis of their work on streptolysin (Bhakdi et al., 1985) is shown alongside supporting electrophysiology from Menestrina and colleagues demonstrating variable pore size and dynamic pore opening and closing (Menestrina et al., 1990), capped oligomers of streptolysin which form smaller functional pores (Palmer et al., 1998), ring and arc pores observed by AFM in 2004 (Czajkowsky et al., 2004) and the original figure from 2005 proposing a mechanism involving kinetically-trapped arc prepores transitioning to pores (Gilbert, 2005).
These images are reprinted with permission from Figure 8, Bhakdi et al. (1985), Infection and Immunity(© copyright American Society for Microbiology, 1985, All Rights Reserved); Figure 4A, Menestrina et al. (1990), Toxicon (© copyright Pergamon Press plc, All Rights Reserved); Figures 8 and 9, Palmer et al. (1988), EMBO Journal (© copyright Oxford University Press, 1988, All Rights Reserved); Figure 3A, Czajkowsky et al. (2004), EMBO Journal (© copyright European Molecular Biology Organisation, 2004, All Rights Reserved); and Figure 1B, Gilbert (2005), Structure (© copyright Elsevier Ltd, 2005, All Rights Reserved).
(b) Evidence for membrane attack complex and perforin forming arc pores with a protein-lipid interface. We show in the top row the original arc pore observation for the membrane attack complex from 1964 (Borsos et al., 1964) alongside models from papers in 1984 and 1985 arguing for the same kind of pore structure Leung et al propose (Amiguet et al., 1985; Tschopp, 1984). In the middle row we show similar data for perforin from 1983 (Podack and Dennert, 1983) and biophysical data in support of arc and double-arc pores from 2011 (Praper et al., 2011) and on cells and using AFM and further electrophysiology this year (Metkar et al., 2014).
These images are reprinted with permission from Figure 1, Borsos et al. (1964), Nature (© copyright Nature Publishing Group, 1964, All Rights Reserved); Figure 8, Tschopp et al., (1984), The Journal of Biological Chemistry (© copyright The American Society of Biological Chemists, Inc., 1984, All Rights Reserved); Figure 7, Amiguet et al., (1985), Biochemistry (© copyright The American Chemical Society, 1985, All Rights Reserved); Figure 1, Podack and Dennert, (1983), Nature (© copyright Nature Publishing Group, 1983, All Rights Reserved); Figure 5, Praper et al., (2011), The Journal of Biological Chemistry (© copyright The American Society of Biological Chemists, Inc., 2011, All Rights Reserved); and Figures 4, 5 and 6, Metkar et al., (2014), Cell Death and Differentiation (© copyright Macmillan Publishers Limited, 2014, All Rights Reserved).
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