Yorgo Modis | University of Cambridge (original) (raw)

Papers by Yorgo Modis

Journal of molecular biology, Jan 27, 2015

mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. A... more mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g. RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. To define how these factors control Dbp5 and mRNA export, an analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT ~ 4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD ~ 0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RN...

Reviews in medical virology, Jan 17, 2015

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two proph... more Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational ...

F1000posters, Feb 17, 2012

Recognition of viral genomic RNA in the cytoplasm of vertebrate cells is mediated by the DExD/H-b... more Recognition of viral genomic RNA in the cytoplasm of vertebrate cells is mediated by the DExD/H-box RNA helicases RIG-I and MDA5. RIG-I recognizes short dsRNA oligonucleotides bearing 5'-triphosphates, while MDA5 signaling is stimulated by dsRNA > 1-2 kb. The basis for length discrimination by MDA5 is poorly understood. We show MDA5 cooperatively binds short dsRNA ligands as a dimer with a 16-18-bp footprint. A crystal structure of the MDA5 helicase-insert domain reveals structural divergence that suggests MDA5 signaling is regulated differently than Rig-I. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signaling model in which the CARDs on MDA5-RNA filaments of a certain length and/or lifetime, nucleate the assembly of MAVS filaments with the same polymeric geometry.

Hepatology (Baltimore, Md.), Jan 21, 2015

Citation: Dessau, M.A., Modis, Y. Protein Crystallization for X-ray Crystallography.

Enveloped viruses rely on fusion proteins in their envelope to fuse the viral membrane to the hos... more Enveloped viruses rely on fusion proteins in their envelope to fuse the viral membrane to the host-cell membrane. This key step in viral entry delivers the viral genome into the cytoplasm for replication. Although class II fusion proteins are genetically and structurally unrelated to class I fusion proteins, they use the same physical principles and topology as other fusion proteins to drive membrane fusion. Exposure of a fusion loop first allows it to insert into the host-cell membrane. Conserved hydrophobic residues in the fusion loop act as an anchor, which penetrates only partway into the outer bilayer leaflet of the host-cell membrane. Subsequent folding back of the fusion protein on itself directs the C-terminal viral transmembrane anchor towards the fusion loop. This fold-back forces the host-cell membrane (held by the fusion loop) and the viral membrane (held by the C-terminal transmembrane anchor) against each other, resulting in membrane fusion. In class II fusion proteins, the fold-back is triggered by the reduced pH of an endosome, and is accompanied by the assembly of fusion protein monomers into trimers. The fold-back occurs by domain rearrangement rather than by an extensive refolding of secondary structure, but this domain rearrangement and the assembly of monomers into trimers together bury a large surface area. The energy that is thus released exerts a bending force on the apposed viral and cellular membranes, causing them to bend towards each other and, eventually, to fuse.

Enveloped viruses must fuse their lipid membrane to a cellular membrane to deliver the viral geno... more Enveloped viruses must fuse their lipid membrane to a cellular membrane to deliver the viral genome into the cytoplasm for replication. Viral envelope proteins catalyze this critical membrane fusion event. They fall into at least three distinct structural classes. Class II fusion proteins have a conserved three-domain architecture and are found in many important viral pathogens. Until 2013, class II proteins had only been found in flaviviruses and alphaviruses. However, in 2013 a class II fusion protein was discovered in the unrelated phlebovirus genus, and two unexpectedly divergent envelope proteins were identified in families that also contain prototypical class II proteins. The structural relationships of newly identified class II proteins, reviewed herein, shift the paradigm for how these proteins evolved.

Front. Immunol., 2014

Most organisms rely on innate immune receptors to recognize conserved molecular structures from i... more Most organisms rely on innate immune receptors to recognize conserved molecular structures from invading microbes. Two essential innate immune receptors, RIG-I and MDA5, detect viral double-stranded RNA in the cytoplasm. The inflammatory response triggered by these RIG-I-like receptors (RLRs) is one of the first and most important lines of defense against infection. RIG-I recognizes short RNA ligands with 5 -triphosphate caps. MDA5 recognizes long kilobase-scale genomic RNA and replication intermediates. Ligand binding induces conformational changes and oligomerization of RLRs that activate the signaling partner MAVS on the mitochondrial and peroxisomal membranes. This signaling process is under tight regulation, dependent on post-translational modifications of RIG-I and MDA5, and on regulatory proteins including unanchored ubiquitin chains and a third RLR, LGP2. Here, we review recent advances that have shifted the paradigm of RLR signaling away from the conventional linear signaling cascade. In the emerging RLR signaling model, large multimeric signaling platforms generate a highly cooperative, self-propagating, and context-dependent signal, which varies with the subcellular localization of the signaling platform.

Trends in Parasitology, 2012

Trends in Cell Biology, 2014

The endocytic pathway is the principal cell entry pathway for large cargos and pathogens. Among t... more The endocytic pathway is the principal cell entry pathway for large cargos and pathogens. Among the wide variety of specialized lipid structures within endosomes, the intraluminal vesicles (ILVs) formed in early endosomes (EEs) and transferred to late endosomal compartments are emerging as critical effectors of viral infection and immune recognition. Various viruses deliver their genomes into these ILVs, which serve as vehicles to transport the genome to the nuclear periphery for replication. When secreted as exosomes, ILVs containing viral genomes can infect permissive cells or activate immune responses in myeloid cells. We therefore propose that endosomal ILVs and exosomes are key effectors of viral pathogenesis.

The EMBO Journal, 2012

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytopl... more Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16-18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARDdeleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5-RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry.

Proceedings of the National Academy of Sciences, 2008

Journal of Virology, 2006

The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement t... more The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-Å crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel ␣-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure.

Journal of Structural Biology, 2013

LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homol... more LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homolog of Drosophila flightless I, through a highly conserved 87-amino acid domain. Upon binding nucleic acid ligands, LRRFIP1 recruits and activates β-catenin, leading to the IRF3-dependent production of type I interferon. However, the molecular mechanism of LRRFIP1 signaling is not well understood. Here we show that the FLI-interacting domain of LRRFIP1 forms a classic parallel, homodimeric coiled coil with 10 heptad repeats and 22 helical turns. The coiled coil domain is also a dimer in solution. However, a longer LRRFIP1 construct spanning the coiled coil and DNA binding domains assembles into higher order oligomers in solution. The structure of LRRFIP1-CC constitutes a valuable tool for probing the mechanism of LRRFIP1 signaling and for structural studies of larger LRRFIP1 constructs.

Journal of Molecular Biology, 2011