Praper T, Sonnen AF-P, Kladnik A, Andrighetti AO, Viero G, Morris KJ, Volpi E, Lunelli L, Serra MD, Froelich CJ, Gilbert RJC, Anderluh G et al. 2011. Perforin activity at membranes leads to invaginations and vesicle formation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 108 (52), pp. 21016-21021. | Read more

Praper T, Sonnen A, Viero G, Kladnik A, Froelich CJ, Anderluh G, Dalla Serra M, Gilbert RJC. 2011. Human Perforin Employs Different Avenues to Damage Membranes J BIOL CHEM, 286 (4), pp. 2946-2955. | Read more

Sonnen AF, Yu C, Evans EJ, Stuart DI, Davis SJ, Gilbert RJ. 2010. Domain metastability: a molecular basis for immunoglobulin deposition? J Mol Biol, 399 (2), pp. 207-213. Read abstract | Read more

We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis-trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits. Hide abstract

Flanagan JF, Namy O, Brierley I, Gilbert RJC. 2010. Direct Observation of Distinct A/P Hybrid-State tRNAs in Translocating Ribosomes STRUCTURE, 18 (2), pp. 257-264. | Read more

Gilbert RJ, Gordiyenko Y, von der Haar T, Sonnen AF, Hofmann G, Nardelli M, Stuart DI, McCarthy JE. 2007. Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex. Proc Natl Acad Sci U S A, 104 (14), pp. 5788-5793. Read abstract | Read more

In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5' end of the mRNA, from which point it scans along the 5' untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state. Hide abstract

Namy O, Moran SJ, Stuart DI, Gilbert RJ, Brierley I. 2006. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature, 441 (7090), pp. 244-247. Read abstract | Read more

The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame. Hide abstract

Gilbert RJ, Beales L, Blond D, Simon MN, Lin BY, Chisari FV, Stuart DI, Rowlands DJ. 2005. Hepatitis B small surface antigen particles are octahedral. Proc Natl Acad Sci U S A, 102 (41), pp. 14783-14788. Read abstract | Read more

The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of approximately 44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with approximately 22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at approximately 12-A resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core. Hide abstract

Gilbert RJ. 2005. Inactivation and activity of cholesterol-dependent cytolysins: what structural studies tell us. Structure, 13 (8), pp. 1097-1106. Read abstract | Read more

The homologous bacterially expressed cholesterol-dependent cytolysins (CDCs) form pores via oligomerization; this must occur preferentially once the target membrane has been engaged. Conformational changes in CDCs then drive partition from an aqueous environment to a lipidic one. This review addresses how premature oligomerization is prevented, how conformational changes are triggered, and how cooperativity between subunits brings about new functionality absent from isolated protomers. Variations are found in the answers provided by the CDCs to these issues. Some toxins use pH as a trigger of activity, but recent results have shown that dimerization in solution is an alternative way of preventing premature oligomerization, in particular for the CDC from Clostridium perfringens, perfringolysin. More controversially, there is still no resolution to the debate as to whether incomplete (arciform) oligomers form pores: recent results again suggest that they do. Hide abstract

Tilley SJ, Orlova EV, Gilbert RJC, Andrew PW, Saibil HR. 2005. Structural basis of pore formation by the bacterial toxin pneumolysin CELL, 121 (2), pp. 247-256. | Read more