Membrane pores: from structure and assembly, to medicine and technology

09:00-09:30 Membrane pore-forming proteins in the molecular arms race between host and pathogen

Professor Helen Saibil FMedSci FRS, Birkbeck College, UK

Abstract

Pathogens have evolved weapons to invade and damage our cells, and our immune system has evolved defences against these attacks. Among the weaponry used by both sides in this continual war are proteins that punch holes in cell membranes. Membrane perforation enables pathogens to take over host cells and resources for their own replication, and also enables host immune systems to kill invading pathogens. The membrane attack complex-perforin (MACPF)/ cholesterol dependent cytolysin (CDC) superfamily of membrane pore-forming proteins is used by a wide range of pathogens as well as by host immune systems. This talk focuses on the mechanisms by which MACPF and CDC proteins convert from their soluble, monomeric forms into large arcs and rings that insert into membranes and perforate them. Studies of pore assembly on liposomes in vitro and examples of their action in vivo will be presented.

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09:30-10:00 Assembly mechanism of the pore-forming toxin Cytolysin A from Escherichia coli

Professor Rudolph Glockshuber, ETH Zurich, Switzerland

Abstract

The a-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic phenotype of several Escherichia coli and Salmonella enterica strains and is cytotoxic towards cultured mammalian cells. ClyA is a soluble, monomeric 34 kDa protein that spontaneously forms ring-shaped, homododecameric pore complexes upon contact with target membranes or detergent. The pore complex forms a hollow cylinder with an inner diameter of 70 Å that is reduced to 30 Å in the membrane-inserted part of the complex. The structural comparison between the soluble momoner and the protomer in the pore complex revealed one of the largest conformational transitions observed in a protein so far, in which more than half of the ClyA residues are reorganized and 16% of the residues are located in different secondary structure elements after protomer formation. Analysis of detergent-induced ClyA assembly in vitro showed that the rate-limiting step in pore formation at ClyA concentrations above 100 nM is the unimolecular transition of the soluble monomer to the assembly-competent protomer, which is followed by rapid protomer assembly. Single-molecule Förster resonance energy (FRET) experiments at picomolar  ClyA concentrations preventing pore assembly showed that a molten globule-like off-pathway intermediate is formed during the monomer-to-protomer transition. The global kinetic analysis of detergent-induced pore formation from picomolar to micromolar ClyA concentrations supports a model in which all sterically compatible, linear oligomeric ClyA intermediates participate in pore assembly.

10:00-10:25 Discussion

10:25-10:50 Coffee

10:50-11:20 Designing water-soluble and membrane-spanning peptide barrels

Professor Dek Woolfson, University of Bristol, UK

Abstract

This talk will describe how we have developed an understanding of natural coiled-coil sequence motifs found in proteins, which lead to specific protein oligomerization; and then how we can apply this knowledge in rational peptide and protein design.  For the latter, it is now clear that nature has almost certainly only shown us a glimpse of what is possible structurally and functionally with polypeptide chains.  To help explore past the confines of natural coiled-coil structures, and into what has been termed the dark matter of protein space, a toolkit of de novo peptides has been developed. These can be used as building blocks for the rapid construction of new protein structures and assemblies. The talk will demonstrate the utility of this approach to make water-soluble protein-like barrels and pores, which can be engineered to bind biological molecules and catalyse simple reactions.  More recently membrane-spanning variants of these α-helical barrels have been engineered, and in collaboration with the Bayley lab, it has shown that these insert into lipid bilayers and conduct ions in voltage-dependent manners.  The two systems have been explored.  In both cases, the insertion and orientation of peptides into the membranes can be directed, and the channel activities can be controlled through mutagenesis and blocking experiments.

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11:20-11:50 Analysis of designed and natural proton transporters

Professor William DeGrado, University of California, San Francisco, USA

Abstract

The mechanism of proton transport through membrane proteins is of general interest to multiple areas of biology.  Using a variety of spectroscopic, crystallographic, and computational methods, the mechanism by which protons are conducted through the M2 proton channel from influenza A virus was investigated, and this information was used to design new anti-influenza drugs that target highly drug-resistant forms of the virus.  A second topic of the talk will focus on the use of de novo protein design to test the mechanism by which a class of transporters uses proton gradients to drive the conduction of molecules into or out of cells. Transporters have been hypothesized to arise by physical association or gene duplication of primordial units, leading to an assembly with “frustrated symmetry” that rocks between two states with the substrate-binding site alternately accessing each side of the membrane. Rocker, a minimalist Zn2+/proton antiporter was designed to test these principles, although it bears no sequence similarity to any known natural protein. Structural, dynamic and functional studies indicate that Rocker is a primordial transporter, which recapitulates many of the properties of this class of proteins.  These studies also demonstrate the feasibility of using de novo design to design membrane proteins with complex dynamic and functional properties.

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11:50-12:15 Discussion

13:10-13:40 Structural basis for complement membrane attack complex formation

Dr Doryen Bubeck, Imperial College London, UK

Abstract

The membrane attack complex (MAC) is a fundamental component of immune defence that drills holes in bacterial membranes and kills pathogens. MAC lesions were first identified in 1964, yet half a century later details of its structure and assembly mechanism remain undiscovered. Here electron cryo-microscopy is used to visualize the human pore complex at subnanometer resolution. The protein composition of the MAC is determined and interaction interfaces that hold the assembly together are identified. Unlike closely related pore-forming proteins, the MAC’s asymmetric pore and "split-washer" shape suggest a killing mechanism that involves not only membrane rupture, but also distortion.

13:40-14:10 Pore formation assisted by lipids

Dr Jose Caaveiro, University of Tokyo, Japan

Abstract

Pore-forming toxins (PFT) constitute a fascinating group of proteins belonging to the molecular offensive and defensive machinery of virtually all kingdoms of life. This class of water-soluble proteins shares the remarkable ability to metamorphose in the presence of cell membranes, generating lytic pores and causing cell-damage. Actinoporins are a family of potent hemolytic toxins from sea anemone forming alpha-helical pores on cellular and model membranes.

In general, two requirements are sufficient to trigger pore-formation by actinoporins:

(i) the presence of the lipid sphingomyelin, and (ii) the segregation of the membrane on domains or lipid-rafts. Until recently, the molecular basis of pore-formation by actinoporins, and specially the specific requirement for sphingomyelin were unclear.

However, a number of recent studies have shed light into critical steps of their mechanism of action, such as binding of the toxins to the membrane, self-assembly via protein-protein interactions, and assembly of the transmembrane pore.

Collectively, the data suggests that sphingomyelin facilitates pore-formation at the binding and assembly stages, and reveal the first example of a hybrid lipid/protein pore by a PFT. The structural and thermodynamic basis of this novel architecture will be explained in detail during this presentation. Surprisingly, the entire process can be made reversible under mild experimental conditions by the careful selection of detergents, challenging current perceptions in the field of membrane-protein interactions.

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14:10-14:35 Discussion

14:35-14:55 Tea

14:55-15:25 New insights into Bax pore formation from advanced microscopy methods

Dr Ana-Jesus Garcia-Saez, University of Tübingen, Germany

Abstract

Bax is a key player in apoptosis that mediates of the permeabilization of the outer mitochondrial membrane. Despite intense research, the underlying process remains poorly understood. By combining biophysical approaches at different scales, new insight into the molecular mechanism of Bax is provided. Electron paramagnetic resonance data shows a key conformational change in the central hairpin of Bax that is involved in pore formation. In this configuration, Bax is present as a mixture of oligomers based on dimer units, as revealed by single molecule imaging. Moreover, the nanoscale organization of Bax at mitochondria of apoptotic cells is provided by superresolution microscopy. Based on this, a new model for the molecular mechanism of Bax is proposed.

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15:25-15:55 Regulating Bak and Bax pore formation in the mitochondrial outer membrane

Dr Ruth Kluck, The Walter and Eliza Hall Institute of Medical Research, Australia

Abstract

Two members of the Bcl-2 family, Bak and Bax, drive apoptotic cell death by changing conformation and forming oligomers that permeabilise the mitochondrial outer membrane. The two proteins are activated by BH3-only family members binding to the α2-α5 hydrophobic surface groove. Newly exposed hydrophobic regions then either “collapse” onto the membrane surface to lie in-plane, or interact to generate BH3:groove symmetric dimers. We found that in each Bak dimer the N-termini are fully solvent-exposed and mobile, allowing disulphide bonding between certain residues (e.g. V61C:V61C') to specifically interrogate how dimers associate into high order complexes. These data informed mathematical simulations that support a model in which Bak dimers interact in a random manner to form compact clusters that generate lipidic pores.       

It was also found that antibodies can trigger activation of Bak and mitochondrial Bax, and do so by binding to a new activation site, the α1-α2 loop. The mechanism of antibody-mediated Bak activation involves α1 dissociation, revealed by biochemical studies and a structural model of Fab bound to Bak. Intriguingly, antibodies to the α1-α2 loop in cytosolic Bax could block its translocation to mitochondria. These data thus identify the α1-α2 loop as a new target for regulating Bak and Bax apoptotic function.

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15:55-16:20 Discussion

16:20-16:50 Peptide-stabilized membrane pores: insights from simulations

Professor Themis Lazaridis, City College of New York, USA

Abstract

The mechanism by which amphipathic peptides permeabilize biological membranes is not well understood. Do the peptides form well-defined pores or simply dissolve the membranes in a detergent-like fashion? What is the lifetime of these pores? How do the sequence and structure of these peptides determine their permeabilizing function? Both experiment and theory face formidable challenges in obtaining detailed information on such labile structures.  We have used two theoretical approaches to study peptide-induced pore formation in lipid bilayers. The first treats water and lipids implicitly and the peptide in atomistic detail. This simplified representation allows one to obtain useful insights into protein-membrane interactions. Using this approach we showed that antimicrobial peptides bind more strongly to membrane pores than to the flat membrane, consistent with the idea that they stabilize them. The second approach is fully atomistic molecular dynamics simulations, some on the 10-microsecond timescale, starting from inserted peptide aggregates. Such simulations of melittin, magainin, PGLa, alamethicin, and protegrin have revealed interesting differences between these peptides and possible explanations of the observed synergy between some of them.

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16:50-17:00 Discussion

09:00-10:30 Punching giant holes in cells: The MACPF/CDC family of pore forming toxins

Dr Michelle Dunstone, Monash University, Australia

Abstract

Membrane attack complex/perforin-like (MACPF) proteins are an excellent example of self-assembling, integral membrane proteins and comprise the largest superfamily of pore forming proteins. These pore forming proteins are an ancient family found in all kingdoms of life with diverse roles in immunity, venom toxicity, predator defense, pathogenesis and uncharacterized roles in development biology. Moreover the MACPF family share a common evolutionary ancestor with the CDC family of pore forming toxins from Gram positive bacteria.

The over-arching mechanism of this MACPF/CDC superfamily starts with soluble monomeric proteins that recognise and assemble on the target membrane into a large ring-shaped oligomer. The oligomer can then undergo a massive conformational change to become an integral membrane protein complex. These membrane complexes range from 1 – 3 Mega Daltons in size and are capable of the passive transport of folded globular proteins. Recently, the field has started to tackle the outstanding questions of the MACPF field by combining single particle cryo-electron microscopy (SP cryo-EM), combined with cutting edge computational and biophysics techniques.

The combined structural data of key distinct clades of the MACPF/CDC family now shed light on how the MACPF/CDC family evolved and help us understand the common mechanism of MACPF/CDC proteins. More importantly, by studying a diverse range of MACPF/CDC proteins, it is now possible to see how the common MACPF/CDC fold has evolved for specific activities within the organism and how the ancillary domains modulate the activity of the common MACPF/CDC fold.

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09:30-10:00 Mechanisms of cell-to-cell spread by Listeria monocytogenes

Professor John Brumell, The Hospital for Sick Children, Canada

Abstract

Listeria monocytogenes has a remarkable ability to disseminate within its host during infection. My laboratory is examining how these bacteria can spread from cell-to-cell using the actin-based motility mechanism powered by ActA. In a recent study we showed that ActA-mediated Arp2/3 recruitment promotes actin polymerization in the cytosol (comet tail formation) but is primarily not responsible for the formation of cell surface protrusions. Instead, we find that protrusion formation requires recruitment and activation of mDia formins. Thus, Listeria exploits two types of actin polymerization to enable cell-to-cell spread. We also showed that efferocytosis, the process by which dying/dead cells are removed by phagocytosis, promotes cell-to-cell spread by Listeria during infection. We showed that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 contributes to efficient cell-to-cell spread by Listeria in macrophages in vitro and growth of these bacteria is impaired in TIM-4-/- mice. Thus, Listeria promotes its dissemination in a host by exploiting efferocytosis.

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10:00-10:25 Discussion

10:25-10:50 Coffee

10:50-11:20 Pilus biogenesis at the outer membrane of bacterial pathogens

Professor Gabriel Waksman FMedSci FRS, UCL and Birkbank College, UK

Abstract

Gram-negative pathogens commonly exhibit adhesive pili on their surface that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher (CU) pathway. Type 1 and P pili have served as model systems for the elucidation of the CU biosynthetic pathway. Pilus assembly requires a periplasmic chaperone (FimC and PapD for type 1 and P pili, respectively) and an outer-membrane assembly platform termed “usher” (FiimD and PapC for type 1 and P pili, respectively). CU pilus subunits are produced in the cytoplasm, translocated to the periplasm by the Sec translocation machinery, and then taken up by a chaperone to cross the periplasmic space to reach the outer-membrane. At the outer-membrane, chaperone-subunit complexes are recruited to an outer-membrane assembly platform, the usher, which orchestrates recruitment and polymerization of subunits. Previous work has elucidated the molecular basis of chaperone function. Recent progress has shed light into the mechanism of pilus subunit assembly at the usher, leading to the elucidation of the entire cycle of pilus subunit incorporation.

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11:20-11:50 Structural insight into the biogenesis of beta barrel membrane proteins

Dr Susan Buchanan, National Institute of Health, USA

Abstract

β-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β -barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Structures of BamA, the central and essential component of the BAM complex, were determined from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features speak to the mechanism by which BamA catalyzes β-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, evocatively suggesting a route from the interior cavity in BamA into the outer membrane. A new structure of the BamACDE complex illustrates how the BamC, BamD, and BamE lipoproteins assemble on the BamA periplasmic domain and provides further evidence for lateral opening of the β -barrel.

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11:50-12:15 Discussion

13:10-14:40 From DNA-based pores toward membrane motors

Professor Hendrik Dietz, Technische Universität München, Germany

Abstract

It is notoriously difficult to observe, let alone control, the position and orientation of molecules because of their small size and the constant thermal fluctuations that they experience in solution. Molecular self-assembly with DNA provides a route for placing molecules and constraining their fluctuations in user-defined ways and with up to Angstroem-scale precision. These positioning options open attractive and unprecedented avenues for scientific and technological exploration, in particular with respect to the creation of artificial membrane pores membrane-embedded mechanisms. This talk will introduce some of the key concepts and methods, and highlight a number of recent developments.

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13:40-14:00 Carbon Nanotube Porins: A biomimetic membrane pore for nanofluidics studies

Dr Alex Noy, Lawrence Livermore National Laboratory, USA

Abstract

Living systems control transport of ions or small molecules across biological membranes using ion channels that form pores in lipid bilayers.  Although bottom-up synthesis and top-down fabrication could produce pores of comparable size, an unresolved challenge remains to build a simplified membrane nanopore scaffold.  Carbon nanotube porins (CNTPs) formed by ultra-short carbon nanotubes (CNTs) modified with lipid surfactants show a set of behaviors that come remarkably close to imitating biological nanopores.

The defining features of these nanostructures are their inner pores that have atomically smooth hydrophobic walls, which confine water on a molecular level, and, in some cases, down to a single-file configuration. We present experimental results that use bulk-scale and individual nanopore measurements to explore the physical origins of efficient transport in CNT porins, and focus on the role that molecular confinement plays in determining the transport characteristics and selectivity of the pore for water, protons, and ions.  Overall, CNT porins represent a simplified biomimetic system that is ideal for studying fundamentals of nanofluidic transport and transport in biological channels, and for building complex engineered mesoscale structures.

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14:00-14:25 Discussion

14:25-14:55 Tea

14:55-15:25 Toroidal pore formation in lipid membranes

Professor Mark Wallace, King's College London, UK

Abstract

A wide range of important biological processes involve the formation of toroidal pores in lipid membranes. Here we employ a combination of single-molecule fluorescence imaging and optical Single Channel Recording in Droplet Interface Bilayers to help understand the biophysics of this process. We image the formation of individual punctate mobile defects both in pure lipid bilayers, and in bilayers where this process if mediated by the presence of a range of peptides and proteins.

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15:25-15:55 Single-protein analysis with a nanopore test-tube

Dr Giovanni Maglia, University of Groningen, Netherlands

Abstract

The specific modulation of the ionic current through biological nanopores reconstituted in artificial membranes has been used to identify and analyze individual molecules. Here we show that ClyA, a biological nanopores with a 6.5 nm diameter and a 13 nm length, can be used as a nanoscale test-tube to study proteins. We found that the electrophoretic forces inside the nanopore can be used to trap and orient individual proteins for tens of minutes. The binding of analytes to the internalized protein is then reported by specific ionic current blockades. Remarkably, despite the diffusion of charged analytes inside the nanopore is strongly affected by the electric field, the binding constants for proteins inside the nanopore are identical to the values measured in bulk, indicating that the confinement and the electrophoretic forces inside the nanopore do not alter the folding and the binding properties of proteins. This technique will allow the label-free investigation of enzymatic reaction at the single molecule level, while nanopores with internal protein sensors might be incorporated into low-cost and portable analytical devices.

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15:55-16:20 Discussion

16:20-16:50 Mass spectrometry of membrane pores - the lipid connection

Dame Carol Robinson FMedSci FRS, University of Oxford, UK

Abstract

The realization that the lipid environment is crucial for maintaining the structure and function of membrane proteins prompts new methods to understand lipid interactions. One such method, mass spectrometry, is emerging with the potential to monitor different modes of lipid binding to membrane protein complexes. Initial studies monitored the addition of lipids and deduced the kinetic and thermodynamic effects of lipid binding to proteins. Recent studies have focused on identifying lipids already present, explicitly in plugs, annular rings or cavities. Lipids that bind within these orifices to membrane proteins will have higher residence times than those in the bulk lipid bilayer and consequently can be quantified and characterized by mass spectrometry. In special cases, lipids identified within cavities have been proposed as substrates following activity assays. Alternatively, a gas phase unfolding protocol can be used to distinguish lipids that are important for stability. This lecture will provide an overview of recent advances in mass spectrometry, with a particular focus on the distinction of the various modes of lipid binding and their implications for structure and function of membrane pores.

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16:50-17:00 Discussion