Elena V. Orlova

The structural basis for membrane binding and pore formation by lymphocyte perforin.

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin ‘key-shaped’ molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.

Structure of a type IV secretion system core complex

Type IV secretion systems (T4SSs) are important virulence factors used by Gram-negative bacterial pathogens to inject effectors into host cells or to spread plasmids harboring antibiotic resistance genes. We report the 15 angstrom resolution cryo–electron microscopy structure of the core complex of a T4SS. The core complex is composed of three proteins, each present in 14 copies and forming a ∼1.1-megadalton two-chambered, double membrane–spanning channel. The structure is double-walled, with each component apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and composition from the other known double membrane–spanning secretion system that has been structurally characterized.

Structure of a type IV secretion system

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3u2009megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.

Structural analysis of macromolecular assemblies by electron microscopy.

1.1. Light and Electron Microscopy and Their Impact in Biology nTo fully understand biological processes from the metabolism of a bacterium to the operation of a human brain, it is necessary to know the three-dimensional (3D) spatial arrangement and dynamics of the constituent molecules, how they assemble into complex molecular machines, and how they form functional organelles, cells, and tissues. The methods of X-ray crystallography and NMR spectroscopy can provide detailed information on molecular structure and dynamics. At the cellular level, optical microscopy reveals the spatial distribution and dynamics of molecules tagged with fluorophores. Electron microscopy (EM) overlaps with these approaches, covering a broad range from atomic to cellular structures. The development of cryogenic methods has enabled EM imaging to provide snapshots of biological molecules and cells trapped in a close to native, hydrated state.1,2 n nBecause of the importance of macromolecular assemblies in the machinery of living cells and progress in the EM and image processing methods, EM has become a major tool for structural biology over the molecular to cellular size range. There have been tremendous advances in understanding the 3D spatial organization of macromolecules and their assemblies in cells and tissues, due to developments in both optical and electron microscopy. In light microscopy, super-resolution and single molecule methods have pushed the resolution of fluorescence images to ∼50 nm, using the power of molecular biology to fuse molecules of interest with fluorescent marker proteins.(3) X-ray cryo-tomography is developing as a method for 3D reconstruction of thicker (10 μm) hydrated samples, with resolution reaching the 15 nm resolution range.(4) In EM, major developments in instrumentation and methods have advanced the study of single particles (isolated macromolecular complexes) in vitrified solution as well as in 3D reconstruction by tomography of irregular objects such as cells or subcellular structures.1,5−7 Cryo-sectioning can be used to prepare vitrified sections of cells and tissues that would otherwise be too thick to image by transmission EM (TEM).8,9 n nIn parallel, software improvements have facilitated 3D structure determination from the low contrast, low signal-to-noise ratio (SNR) images of projected densities provided by TEM of biological molecules.10−14 Alignment and classification of images in both 2D and 3D are key methods for improving SNR and detection and sorting of heterogeneity in EM data sets.(14) The resolution of single-particle reconstructions is steadily improving and has gone beyond 4 A for some icosahedral viruses and 5.5 A for asymmetric complexes such as ribosomes, giving a clear view of protein secondary structure elements and, in the best cases, resolving the protein or nucleic acid fold.15,16

Structure of bacteriophage SPP1 head-to-tail connection reveals mechanism for viral DNA gating

In many bacterial viruses and in certain animal viruses, the double-stranded DNA genome enters and exits the capsid through a portal gatekeeper. We report a pseudoatomic structure of a complete portal system. The bacteriophage SPP1 gatekeeper is composed of dodecamers of the portal protein gp6, the adaptor gp15, and the stopper gp16. The solution structures of gp15 and gp16 were determined by NMR. They were then docked together with the X-ray structure of gp6 into the electron density of the ≈1-MDa SPP1 portal complex purified from DNA-filled capsids. The resulting structure reveals that gatekeeper assembly is accompanied by a large rearrangement of the gp15 structure and by folding of a flexible loop of gp16 to form an intersubunit parallel β-sheet that closes the portal channel. This stopper system prevents release of packaged DNA. Disulfide cross-linking between β-strands of the stopper blocks the key conformational changes that control genome ejection from the virus at the beginning of host infection.

Detection and separation of heterogeneity in molecular complexes by statistical analysis of their two-dimensional projections

Progress in molecular structure determination by cryo electron microscopy and single particle analysis has led to improvements in the resolution achievable. However, in many cases the limiting factor is structural heterogeneity of the sample. To address this problem, we have developed a method based on statistical analysis of the two-dimensional images to detect and sort localised structural variations caused, for example, by variable occupancy of a ligand. Images are sorted by two consecutive stages of multivariate statistical analysis (MSA) to dissect out the two main sources of variation, namely out of plane orientation and local structural changes. Heterogeneity caused by local changes is detected by MSA that reveals significant peaks in the higher order eigenimages. The eigenimages revealing local peaks are used for automated classification. Evaluation of differences between classes allows discrimination of molecular images with and without ligand. This method is very rapid, independent of any initial three-dimensional model, and can detect even minor subpopulations in an image ensemble. A strategy for using this technique was developed on model data sets. Here, we demonstrate the successful application of this method to both model and real EM data on chaperonin-substrate and ribosome-ligand complexes.

Structural biology of the p53 tumour suppressor.

The p53 tumour suppressor protein has presented a challenge for structural biology for more than two decades. The complete p53 molecule has eluded numerous attempts to determine its structure, presumably owing to the intrinsic conformational flexibility that is essential to the proteins function. Recent data obtained by X-ray crystallography, NMR spectroscopy and electron microscopy provide new insight into the quaternary architecture of the whole molecule and new strategies for examining how these structures correlate with the cell and molecular biology of the Guardian of the Genome.

Capsid Structure and Its Stability at the Late Stages of Bacteriophage SPP1 Assembly

ABSTRACT The structure of the bacteriophage SPP1 capsid was determined at subnanometer resolution by cryo-electron microscopy and single-particle analysis. The icosahedral capsid is composed of the major capsid protein gp13 and the auxiliary protein gp12, which are organized in a T=7 lattice. DNA is arranged in layers with a distance of ∼24.5 Å. gp12 forms spikes that are anchored at the center of gp13 hexamers. In a gp12-deficient mutant, the centers of hexamers are closed by loops of gp13 coming together to protect the SPP1 genome from the outside environment. The HK97-like fold was used to build a pseudoatomic model of gp13. Its structural organization remains unchanged upon tail binding and following DNA release. gp13 exhibits enhanced thermostability in the DNA-filled capsid. A remarkable convergence between the thermostability of the capsid and those of the other virion components was found, revealing that the overall architecture of the SPP1 infectious particle coevolved toward high robustness.

Genome Gating in Tailed Bacteriophage Capsids

Tailed bacteriophages use a portal system for genome entry and exit from viral capsids. Here, we review the mechanisms how these movements are controlled by the genome gatekeeper that assembles at the portal structure. Phage DNA is packaged at high pressure inside the viral capsid by a powerful motor. The viral genome is translocated through the central channel of the portal protein found at a single vertex of the capsid. Packaging is normally terminated by endonucleolytic cleavage of the substrate DNA followed by disassembly of the packaging motor and closure of the portal system, preventing leakage of the viral genome. This can be achieved either by conformational changes in the portal protein or by sequential addition of proteins that extend the portal channel (adaptors) and physically close it preventing DNA exit (stoppers). The resulting connector structure provides the interface for assembly of short tails (podoviruses) or for attachment of preformed long tails (siphoviruses and myoviruses). The connector maintains the viral DNA correctly positioned for ejection that is triggered by interaction of the phage particle with bacterial receptors. Recent exciting advances are providing new molecular insights on the mechanisms that ensure precise coordination of these critical steps required both for stable viral genome packaging and for its efficient release to initiate infection.

Methods for three-dimensional reconstruction of heterogeneous assemblies.

Electron microscopy (EM) has developed into an important method for determining the three-dimensional (3D) structures of biological complexes, in particular of isolated macromolecular complexes in vitrified solution (cryo-EM of single particles). One of the consequences of studying complexes in solution rather than in a crystal lattice is that they are less constrained to adopt a single conformation. It is a common problem in single-particle analysis that samples of purified macromolecules can be structurally heterogeneous, with molecules adopting different conformations, corresponding to different functional states. In the case of multisubunit assemblies, there may also be heterogeneity of assembly or ligand binding. Heterogeneity limits the accuracy and resolution of 3D structures, since different conformations will contribute to a single 3D map and variable parts of the structure will be smeared out. Therefore, a new group of image processing methods has been developed to deal with the problems of detecting and sorting structural heterogeneity. The basic problem is to discriminate the source of image variations, and then to separate the images into homogeneous subsets for separate reconstruction. Variations in image features can arise from different particle orientations, variations in conformation and/or ligand binding, and noise fluctuations in the low signal-to-noise ratio images typical of cryo-EM. Here, we present a review of approaches developed to deal with these problems, along with examples of the application of a method based on multivariate statistical analysis to both model and real data. The methods have been used to discriminate small differences in size, conformation and ligand binding, and to obtain high quality, reliable reconstructions of multiple structures from mixed data sets.