Structural basis for complement membrane attack complex formation
Dr Doryen Bubeck, Imperial College London, UK
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.
Pore formation assisted by lipids
Dr Jose Caaveiro, University of Tokyo, Japan
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.
New insights into Bax pore formation from advanced microscopy methods
Dr Ana-Jesus Garcia-Saez, University of Tübingen, Germany
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.
Regulating Bak and Bax pore formation in the mitochondrial outer membrane
Dr Ruth Kluck, The Walter and Eliza Hall Institute of Medical Research, Australia
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.
Peptide-stabilized membrane pores: insights from simulations
Professor Themis Lazaridis, City College of New York, USA
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.