Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release.

DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. Here, we report the biochemical characterisation of full-length human DHX8 and the catalytically active helicase core DHX8Δ547, alongside crystal structures of DHX8Δ547 bound to ADP and a structure of DHX8Δ547 bound to poly(A)6 single-strand RNA. Our results reveal that DHX8 has an in vitro binding preference for adenine-rich RNA and that RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. We demonstrate the importance of R620 and both the hook-turn and hook-loop regions for DHX8 helicase activity and propose that the hook-turn acts as a gatekeeper to regulate the directional movement of the 3' end of RNA through the RNA-binding channel. This study provides an in-depth understanding of the activity of DHX8 and contributes insights into the RNA-unwinding mechanisms of the DEAH-box helicase family.

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery.

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

Secretion systems in Gram-negative bacteria: structural and mechanistic insights.

Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I-VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone-usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.

Recent advances in the structural and molecular biology of type IV secretion systems.

Bacteria use type IV secretion (T4S) systems to deliver DNA and protein substrates to a diverse range of prokaryotic and eukaryotic target cells. T4S systems have great impact on human health, as they are a major source of antibiotic resistance spread among bacteria and are central to infection processes of many pathogens. Therefore, deciphering the structure and underlying translocation mechanism of T4S systems is crucial to facilitate development of new drugs. The last five years have witnessed considerable progress in unraveling the structure of T4S system subassemblies, notably that of the T4S system core complex, a large 1 MegaDalton (MDa) structure embedded in the double membrane of Gram-negative bacteria and made of 3 of the 12 T4S system components. However, the recent determination of the structure of -3MDa assembly of 8 of these components has revolutionized our views of T4S system architecture and opened up new avenues of research, which are discussed in this review.

Crystal structure of ATV(ORF273), a new fold for a thermo- and acido-stable protein from the Acidianus two-tailed virus.

Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV(ORF273). ATV(ORF273) forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV(ORF273) displays a new α + ß protein fold, consistent with the absence of homologues of this protein in public sequence databases.

Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar.

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded ß-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.

Chaperone role for proteins p618 and p892 in the extracellular tail development of Acidianus two-tailed virus.

The crenarchaeal Acidianus two-tailed virus (ATV) undergoes a remarkable morphological development, extracellularly and independently of host cells, by growing long tails at each end of a spindle-shaped virus particle. Initial work suggested that an intermediate filament-like protein, p800, is involved in this process. We propose that an additional chaperone system is required, consisting of a MoxR-type AAA ATPase (p618) and a von Willebrand domain A (VWA)-containing cochaperone, p892. Both proteins are absent from the other known bicaudavirus, STSV1, which develops a single tail intracellularly. p618 exhibits ATPase activity and forms a hexameric ring complex that closely resembles the oligomeric complex of the MoxR-like protein RavA (YieN). ATV proteins p387, p653, p800, and p892 interact with p618, and with the exception of p800, all bind to DNA. A model is proposed to rationalize the interactions observed between the different protein and DNA components and to explain their possible structural and functional roles in extracellular tail development.

Getting the best out of long-wavelength X-rays: de novo chlorine/sulfur SAD phasing of a structural protein from ATV.

The structure of a 14 kDa structural protein from Acidianus two-tailed virus (ATV) was solved by single-wavelength anomalous diffraction (SAD) phasing using X-ray data collected at 2.0 A wavelength. Although the anomalous signal from methionine sulfurs was expected to suffice to solve the structure, one chloride ion turned out to be essential to achieve phasing. The minimal data requirements and the relative contributions of the Cl and S atoms to phasing are discussed. This work supports the feasibility of a systematic approach for the solution of protein crystal structures by SAD based on intrinsic protein light atoms along with associated chloride ions from the solvent. In such cases, data collection at long wavelengths may be a time-efficient alternative to selenomethionine substitution and heavy-atom derivatization.

Acidianus filamentous virus 1 coat proteins display a helical fold spanning the filamentous archaeal viruses lineage.

Acidianus filamentous virus 1 (AFV1), a member of the Lipothrixviridae family, infects the hyperthermophilic, acidophilic crenarchaeaon Acidianus hospitalis. The virion, covered with a lipidic outer shell, is 9,100-A long and contains a 20.8-kb linear dsDNA genome. We have identified the two major coat proteins of the virion (MCPs; 132 and 140 amino acids). They bind DNA and form filaments when incubated with linear dsDNA. A C-terminal domain is identified in their crystal structure with a four-helix-bundle fold. In the topological model of the virion filament core, the genomic dsDNA superhelix wraps around the AFV1-132 basic protein, and the AFV1-140 basic N terminus binds genomic DNA, while its lipophilic C-terminal domain is imbedded in the lipidic outer shell. The four-helix bundle fold of the MCPs from AFV1 is identical to that of the coat protein (CP) of Sulfolobus islandicus rod-shaped virus (SIRV), a member of the Rudiviridae family. Despite low sequence identity between these proteins, their high degree of structural similarity suggests that they could have derived from a common ancestor and could thus define an yet undescribed viral lineage.