Dr Doryen Bubeck
| Research Area: | Protein Science and Structural Biology |
|---|---|
| Technology Exchange: | Crystallography, Microscopy (EM) and Protein interaction |
| Keywords: | structural biology, Complement Terminal Pathway, membrane proteins and Electron Microscopy |
Figure 3. Five-fold symmetric three-dimensional reconstruction of native poliovirus bound to ...
Ribonuclease H2 (RNaseH2) is the major nuclear enzyme involved in the degradation of RNA/DNA ...
Throughout my career, my research has taken a structural approach to investigating the molecular mechanism of disease-related macromolecules. I have investigated a wide range of biological processes including virus entry, immunity, and development. During my Ph.D., I sought to uncover how an RNA translocation pore is formed during poliovirus cell entry. Through the development of a novel liposome system for cryo-electron microscopy, I identified the symmetry break that initiates membrane attachment and solved the structure of the first virus-receptor-membrane complex. At Oxford, I broadened my expertise in structural biology to include X-ray crystallography and defined a role in genome replication and repair for RNase H2, a protein associated with the autoimmune disorder Aicardi-Goutières Syndrome. Meanwhile, I continued to pursue my research interests in electron microscopy applied to a range of projects including the Wnt signaling pathway and innate immunity. I used both negative stain and cryo-EM to provide insight into the assembly and regulation of the membrane attack complex, an innate immune effector. These results provide a structural framework for understanding the complex protein associations underlying pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.
| Name | Department | Institution | Country |
|---|---|---|---|
| Prof Susan Lea | University of Oxford | United Kingdom | |
| B.Paul Morgan | Cardiff University | United Kingdom | |
| Dr Andrew Andrew Jackson | MRC Human Genetics Unit | UK | |
| Prof Antione van Oijen | University of Groningen | Netherlands | |
| Prof Oscar Llorca | Centro de Investigaciones Biológicas/Centre for Biological Research | Spain |
2008. Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry. J Struct Biol, 162 (3), pp. 480-490. Read abstract | Read more
During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement. Here we analyze how errors in the subjective assignment of this position affect the determination of symmetry, and the accuracy of calculating Euler angles for each raw image. In this study we have optimized the method and applied it to study the membrane-attachment complex of Semliki Forest virus (SFV), a model system for enveloped virus fusion. The resulting reconstruction of the SFV-membrane complex with a fiducial provides the first experimental evidence that this pre-fusion cell entry intermediate approaches the membrane along the viral 5-fold axis. The analysis reported here, and its subsequent application to enveloped virus fusion, indicate that this is a robust tool for solving the structures of mixed-symmetry complexes. Hide abstract
2007. Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus-receptor-membrane complex at 30 A resolution. J Struct Biol, 160 (2), pp. 200-210. Read abstract | Read more
As a long-term goal we want to use cryoelectron tomography to understand how non-enveloped viruses, such as picornaviruses, enter cells and translocate their genomes across membranes. To this end, we developed new image-processing tools using an in vitro system to model viral interactions with membranes. The complex of poliovirus with its membrane-bound receptors was reconstructed by averaging multiple sub-tomograms, thereby producing three-dimensional maps of surprisingly high-resolution (30 A). Recognizable images of the complex could be produced by averaging as few as 20 copies. Additionally, model-free reconstructions of free poliovirus particles, clearly showing the major surface features, could be calculated from 60 virions. All calculations were designed to avoid artifacts caused by missing information typical for tomographic data ("missing wedge"). To investigate structural and conformational variability we applied a principal component analysis classification to specific regions. We show that the missing wedge causes a bias in classification, and that this bias can be minimized by supplementation with data from the Fourier transform of the averaged structure. After classifying images of the receptor into groups with high similarity, we were able to see differences in receptor density consistent with the known variability in receptor glycosylation. Hide abstract
2006. Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging. Structure, 14 (6), pp. 1039-1048. Read abstract | Read more
Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments. Hide abstract
2006. Characterization of early steps in the poliovirus infection process: receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles. J Virol, 80 (1), pp. 172-180. Read abstract | Read more
The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry. Hide abstract
2005. Cryo-electron microscopy reconstruction of a poliovirus-receptor-membrane complex. Nat Struct Mol Biol, 12 (7), pp. 615-618. Read abstract | Read more
To study non-enveloped virus cell entry, a versatile in vitro model system was developed in which liposomes containing nickel-chelating lipids were decorated with His-tagged poliovirus receptors and bound to virus. This system provides an exciting opportunity for structural characterization of the early steps in cell entry in the context of a membrane. Here we report the three-dimensional structure of a poliovirus-receptor-membrane complex solved by cryo-electron microscopy (cryo-EM) at a resolution of 32 A. Methods were developed to establish the symmetry of the complex objectively. This reconstruction demonstrates that receptor binding brings a viral five-fold axis close to the membrane. Density is clearly defined for the icosahedral virus, for receptors (including known glycosylation sites) and for the membrane bilayer. Apparent perturbations of the bilayer close to the viral five-fold axis may function in subsequent steps of cell entry. Hide abstract
2012. Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9. Cell Rep, 1 pp. 1-8. Read abstract
Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage. Hide abstract
