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

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.

Professor Helen Saibil FMedSci FRS, Birkbeck College, UK

Professor Helen Saibil FMedSci FRS, Birkbeck College, UK

Helen Saibil was educated in biophysics at McGill University, Montreal and then at Kings College London, where she did her PhD on the structure of retinal photoreceptor membranes. In her present position at Birkbeck College, her main interests are in analyzing conformational changes in macromolecular machines and the actions of intracellular pathogens on their hosts, using cryo-electron microscopy and image processing. These studies include work on protein folding, unfolding and disaggregation by molecular chaperones, and refolding during membrane pore formation by bacterial toxins and by pore forming proteins of the immune system. She is involved in setting up the national facility for cryo EM at the Diamond synchrotron, and is a member of EMBO, a Fellow of the Royal Society and a Fellow of the Academy of Medical Sciences.

Web page: people.cryst.bbk.ac.uk/~ubcg16z/

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.

Professor Rudolph Glockshuber, ETH Zurich, Switzerland

Professor Rudolph Glockshuber, ETH Zurich, Switzerland

The research of the Rudi Glockshuber focuses on mechanisms of protein folding and assembly. Besides general topics in protein folding such as catalysis of disulfide bond formation and slow folding reactions determined by proline cis-trans isomerization, the Glockshuber group investigates the assembly of supramolecular protein complexes, including the formation of amyloid beta fibrils and the assembly of virulence factors of bacterial pathogens such as adhesive type 1 pili and the pore poring toxin ClyA from Escherichia coli.

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.

Professor Dek Woolfson, University of Bristol, UK

Professor Dek Woolfson, University of Bristol, UK

Professor Dek Woolfson took his first degree in Chemistry at the University of Oxford, UK.  He then did a PhD at the University of Cambridge followed by post-doctoral research at University College London and the University of California, Berkeley.  After 10 years as Lecturer through to Professor of Biochemistry at the University of Sussex, he moved to the University of Bristol in 2005 to take up a joint chair in Chemistry and Biochemistry. Dek’s research is at the interface between chemistry and biology, applying chemical methods and principles to understand biological phenomena.  His group is interested in the challenge of rational protein design, and how this can be developed for synthetic biology.  He places particular emphasis on making completely new protein structures and biomaterials for applications in cell biology and medicine. In 2011, Dek became the first recipient of the Medimmune Protein and Peptide Science Award of the Royal Society of Chemistry; and in 2014 he received a Royal Society Wolfson Research Merit Award.  Dek is Director of BrisSynBio, a £13.6M BBSRC/EPSRC-funded Synthetic Biology Research Centre.

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.

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

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

William (Bill) DeGrado’s work focuses on the design of small molecule drugs, peptides, proteins, and peptide mimetics. Bill is currently a professor in the Department of Pharmaceutical Chemistry at the University of California San Francisco, where he is also a member of the Cardiovascular Research Institute. Before joining UCSF in 2011, he was a member of DuPont Central Research and DuPont Merck Pharmaceutical Company from 1981 to 1996 and the Raiziss Professor in the Department of Biochemistry and Biophysics at the University of Pennsylvania (1996 – 2011). Some of Bill’s research interests include: de novo design of proteins and peptides; peptide mimetics; structure/function of membrane proteins, including integrins and viral ion channels; small molecule drug design; bioinorganic chemistry. Bill is a member of the U.S. National Academy of Science, the U.S. National Academy of Inventors, the American Academy of Arts and Sciences, and a fellow of the American Association for the Advancement of Science. Bill graduated from Kalamazoo College in 1978, received his Ph.D. in organic chemistry from the University of Chicago (1981), and joined DuPont Central Research without an intervening postdoctoral position.

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.

Dr Doryen Bubeck, Imperial College London, UK

Dr Doryen Bubeck, Imperial College London, UK

Doryen Bubeck received her PhD in Biophysics from Harvard University in 2005 where she used cryo-electron micrsocopy to investigate the cell entry mechanism of poliovirus.  As an EMBO postdoctoral fellow and Cancer Research Institute Fellow at the University of Oxford, she continued to explore the structures of membrane proteins, focusing on the complement immune pathway. A recent highlight (Hadders & Bubeck et al., Cell Rep. 2012) is the discovery that complement components associate through a sideways alignment of their central MAC-perforin domains (MACPF). These results provide a structural framework for understanding the complex protein associations underlying activation of this innate immune effector. Doryen Bubeck is a lecturer in Structural Biology  within the Department of Life Sciences and recipient of a Cancer Research UK Career Establishment Award.  Her current research adopts an integrated structural approach merging cryo-electron microscopy and Xray crystallography to investigate the role of membrane proteins in host-pathogen interactions. She aims to investigate how membrane attack complex (MAC) pore formation is controlled, a process important for fighting infections and preventing complement-mediated tissue damage.

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.

Dr Jose Caaveiro, University of Tokyo, Japan

Dr Jose Caaveiro, University of Tokyo, Japan

Dr Caaveiro graduated in the University of the Basque Country (Spain), where he studied the modulation of peptides and proteins by lipids. After a postdoctoral period in USA (MIT and Brandeis University) he then joined the University of Tokyo (since 2008). Dr. Caaveiro’s research program combines structural and thermodynamic data to address a broad range of biologically inspired phenomena. His ultimate goal is to understand the physicochemical basis underpinning biomolecular recognition at the atomic scale. Recently, Dr. Caaveiro and co-workers has revealed an unprecedented role for lipids in the pore-forming mechanism of the potent hemolytic class of actinoporins.

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.

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

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

Ana-Jesus Garcia-Saez is academic background includes Professor (W3) at the Interfaculty Institute for Biochemistry (IFIB), Universität Tübingen, Germany (October 2013), Max Planck Research Group Leader and Deutsches Krebsforschungzentrum (DKFZ) Junior Group Leader. Bioquant, Germany (2010 - current), Post-doc at the BioTec, TU Dresden, Germany (2005-2009) and PhD at the Department of Biochemistry and Molecular Biology, University of Valencia, Spain (2000-2005)

Fellowships and awards include:

• European Research Council (ERC) Starting Grant (project acronym: APOQUANT), 2012-2017.
• Max-Planck Gesellschaft Postdoctoral scholarship, Jan 2009- Dec 2009.
• Marie Curie Intra European fellowship. 6th Framework Programm, Marie Curie Actions. BIOTEC, TU Dresden (Germany). Jan 2007-Dec 2008.
• Short-term Fellowship. Federation of European Biochemical Societies. Biotechnologisches Zentrum, Technische Universität, Dresden (Germany). May-July 2005.
• Predoctoral Fellowship. Formación de Profesorado Universitario, Ministerio Español de Educación y Cultura. Faculty of Biology, University of Valencia. 1st April 2001- 31st March 2005
• Predoctoral Fellowship. Formació de Personal Investigador, Conselleria de Cultura i Educació de la Generalitat Valenciana. 1st March – 1st April 2001.
• Predoctoral Fellowship. Formación de Personal Investigador, Ministerio Español de Ciencia y Tecnología. 13th July 2001- 13th July 2001.
• Undergraduate Student Fellowship. Beca de Colaboración, Ministerio Español de Educacion y Cultura. Faculty of Biology, University of Valencia. Sept. 1999-June 2000

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.

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

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

Dr Kluck is a cell biologist who has studied apoptotic cell death since her PhD at the Queensland Institute for Medical Research, in collaboration with John Kerr who discovered apoptosis. As a postdoc at the La Jolla Institute for Allergy and Immunology (1995-2002), she made the seminal discovery that Bcl-2 proteins inhibit apoptosis by blocking mitochondrial release of cytochrome c. In 2002, she moved to the Walter and Eliza Hall Institute for Medical Research in Australia where she has focused on the regulation and function of the Bak and Bax pore-forming proteins.

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.

Professor Themis Lazaridis, City College of New York, USA

Professor Themis Lazaridis, City College of New York, USA

Themis Lazaridis received a Diploma in Chemical Engineering from Aristotle University in Thessaloniki, Greece in 1987.  He went on to earn a Ph.D. in Chemical Engineering from the University of Delaware, USA.  His Ph.D. research involved, among other topics, the development of a practical approach to calculate the entropy of hydration in water, elucidating the molecular origin of the unfavorable entropy of hydrophobic hydration.  He was a postdoctoral fellow at Harvard University from 1992 to 1998, continuing to work on the statistical thermodynamics of solvation.  He also worked on the energetics of protein folding and developed an implicit solvation model for proteins in solution.  Since 1998 he has been a faculty member at the City College of New York.  His current research interests focus on modeling protein-membrane interactions and especially the process of pore formation in membranes by antimicrobial peptides or protein toxins using implicit solvent models and all-atom simulations.