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191 results on '"Savva, Christos G."'

23. The architecture of the spliceosomal U4/U6.U5 tri-snRNP

Author

Nguyen, Thi Hoang Duong, Galej, Wojciech P., Bai, Xiao-chen, Savva, Christos G., Newman, Andrew J., Scheres, Sjors H.W., and Nagai, Kiyoshi

Subjects
Brewer's yeast -- Research, Spliceosomes -- Research, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a precursor messenger RNA substrate bound to U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), and transforms into a catalytically active spliceosome after extensive compositional and conformational changes triggered by unwinding of the U4 and U6 (U4/U6) snRNAs. Here we use cryo-electron microscopy single-particle reconstruction of Saccharomyces cerevisiae tri-snRNP at 5.9 Å resolution to reveal the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3' stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the amino-terminal domain of Prp8 position U5 snRNA to insert its loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome., The protein-coding sequences of most eukaryotic genes are interrupted by non-coding segments called introns. Introns are removed from precursor mRNAs (pre-mRNAs) and the flanking coding segments (exons) are spliced together [...]

Published
2015
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24. Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex

Author

Gruszczyk, Jakub, primary, Grandvuillemin, Loic, additional, Lai-Kee-Him, Josephine, additional, Paloni, Matteo, additional, Savva, Christos G., additional, Germain, Pierre, additional, Grimaldi, Marina, additional, Boulahtouf, Abdelhay, additional, Kwong, Hok-Sau, additional, Bous, Julien, additional, Ancelin, Aurelie, additional, Bechara, Cherine, additional, Barducci, Alessandro, additional, Balaguer, Patrick, additional, and Bourguet, William, additional

Published
2022
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25. Holin triggering in real time

Author

White, Rebecca, Chiba, Shinobu, Pang, Ting, Dewey, Jill S., Savva, Christos G., Holzenburg, Andreas, Pogliano, Kit, Young, Ry, and Collier, R. John

Published
2011

44. Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2.

Author

Brotherton, Deborah H., Nijjar, Sarbjit, Savva, Christos G., Dale, Nicholas, and Cameron, Alexander David

Subjects
*CELL communication, *BLUE light, *PARTIAL pressure, *CONNEXINS, *GLUTAMIC acid
Abstract

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation. [ABSTRACT FROM AUTHOR]

Published
2024
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48. Stable micron-scale holes are a general feature of canonical holins

Author

Savva, Christos G., Dewey, Jill S., Moussa, Samir H., To, Kam H., Holzenburg, Andreas, and Young, Ry

Subjects
Viral Proteins, Cell Membrane, Cryoelectron Microscopy, Endopeptidases, Escherichia coli, Bacteriophage lambda, Models, Biological, Article
Abstract

At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S(21) 68. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.

Published
2013

49. Structural insights into Clostridium perfringens delta toxin pore formation

Author

Motta, A., Huyet, Jessica, Naylor, Claire E., Savva, Christos G., Gibert, M., Popoff, M.R., and Basak, Ajit K.

Subjects
bcs
Abstract

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.

Published
2013

50. Clostridium perfringensepsilon toxin H149A mutant as a platform for receptor binding studies

Author

Bokori-Brown, M., Kokkinidou, M.C., Savva, Christos G., Fernandes da Costa, S.P., Naylor, Claire E., Cole, Ambrose R., Moss, David S., Basak, Ajit K., and Titball, R.W.

Subjects
bcs
Abstract

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.

Published
2013

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