DDX3X structural analysis: Implications in the pharmacology and innate immunity.

The human DEAD-Box Helicase 3 X-Linked (DDX3X) is an ATP-dependent RNA helicase involved in virtually every step of RNA metabolism, ranging from transcription regulation in the nucleus to translation initiation and stress granule (SG) formation, and plays crucial roles in innate immunity, as well as tumorigenesis and viral infections. This review discusses latest advances in DDX3X biology and structure-function relationship, including the implications of the recent DDX3X crystal structure in complex with double stranded RNA for RNA metabolism, DDX3X involvement in the cross-talk between innate immune responses and cell stress adaptation, and the roles of DDX3X in controlling cell fate.

Assembly of complex viruses exemplified by a halophilic euryarchaeal virus.

Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.

Virus found in a boreal lake links ssDNA and dsDNA viruses.

Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1-adenovirus lineage, characterized by a major capsid protein bearing two ß-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.

Structural and functional characterization of the kindlin-1 pleckstrin homology domain.

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin ß-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) ∼100 µM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.

Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase.

Cytoplasmic terminal uridylyl transferases comprise a conserved family of enzymes that negatively regulate the stability or biological activity of a variety of eukaryotic RNAs, including mRNAs and tumor-suppressor let-7 microRNAs. Here we describe crystal structures of the Schizosaccharomyces pombe cytoplasmic terminal uridylyl transferase Cid1 in two apo conformers and bound to UTP. We demonstrate that a single histidine residue, conserved in mammalian Cid1 orthologs, is responsible for discrimination between UTP and ATP. We also describe a new high-affinity RNA substrate-binding mechanism of Cid1, which is essential for enzymatic activity and is mediated by three basic patches across the surface of the enzyme. Overall, our structures provide a basis for understanding the activity of Cid1 and a mechanism of UTP selectivity conserved in its human orthologs, suggesting potential implications for anticancer drug design.

Purification and crystallization of Kokobera virus helicase.

Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3(1)21 (or P3(2)21), with unit-cell parameters a = 88.6, c = 138.6 A, and exhibit a diffraction limit of 2.3 A.