Structure and dynamics of an archetypal DNA nanoarchitecture revealed via cryo-EM and molecular dynamics simulations.

DNA can be folded into rationally designed, unique, and functional materials. To fully realise the potential of these DNA materials, a fundamental understanding of their structure and dynamics is necessary, both in simple solvents as well as more complex and diverse anisotropic environments. Here we analyse an archetypal six-duplex DNA nanoarchitecture with single-particle cryo-electron microscopy and molecular dynamics simulations in solvents of tunable ionic strength and within the anisotropic environment of biological membranes. Outside lipid bilayers, the six-duplex bundle lacks the designed symmetrical barrel-type architecture. Rather, duplexes are arranged in non-hexagonal fashion and are disorted to form a wider, less elongated structure. Insertion into lipid membranes, however, restores the anticipated barrel shape due to lateral duplex compression by the bilayer. The salt concentration has a drastic impact on the stability of the inserted barrel-shaped DNA nanopore given the tunable electrostatic repulsion between the negatively charged duplexes. By synergistically combining experiments and simulations, we increase fundamental understanding into the environment-dependent structural dynamics of a widely used nanoarchitecture. This insight will pave the way for future engineering and biosensing applications.

CryoEM structure and assembly mechanism of a bacterial virus genome gatekeeper.

Numerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit ß-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage.

Suppression of amyloid-ß fibril growth by drug-engineered polymorph transformation.

Fibrillization of the protein amyloid ß is assumed to trigger Alzheimer's pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure. We employ time-resolved in situ atomic force microscopy to quantify the kinetics of growth of individual fibrils and supplement it with structure characterization by cryo-EM. We show that fibrils with structure engineered by the drug nucleate and grow substantially slower than "normal" fibrils; remarkably, growth remains stunted even in drug-free solutions. We find that the suppression of fibril growth by bexarotene is not because of the drug binding to the fibril tips or to the peptides in the solution. Kinetic analyses attribute the slow growth of drug-enforced fibril polymorph to the distinctive dynamics of peptide chain association to their tips. As an additional benefit, the bexarotene fibrils kill primary rat hippocampal neurons less efficiently than normal fibrils. In conclusion, the suggested drug-driven polymorph transformation presents a mode of action to irreversibly suppress toxic aggregates not only in Alzheimer's but also potentially in myriad diverse pathologies that originate with protein condensation.

Cryo-EM structure of a type IV secretion system.

Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.

Cryo-EM Structures of Two Bacteriophage Portal Proteins Provide Insights for Antimicrobial Phage Engineering.

Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and human infections that are especially lethal in newborns. Such pathogens are targeted by the obligate lytic phages of the Salasmaviridae and Guelinviridae families. These phages have relatively small ~20 kb linear protein-capped genomes and their compact organisation, relatively few structural elements, and broad host range, are appealing from a phage-engineering standpoint. In this study, we focus on portal proteins, which are core elements for the assembly of such tailed phages. The structures of dodecameric portal complexes from Salasmaviridae phage GA1, which targets Bacillus pumilus, and Guelinviridae phage phiCPV4 that infects Clostridium perfringens, were determined at resolutions of 3.3 Å and 2.9 Å, respectively. Both are found to closely resemble the related phi29 portal protein fold. However, the portal protein of phiCPV4 exhibits interesting differences in the clip domain. These structures provide new insights on structural diversity in Caudovirales portal proteins and will be essential for considerations in phage structural engineering.

Unwinding of a DNA replication fork by a hexameric viral helicase.

Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged ß-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.

The structural basis for Z α1-antitrypsin polymerization in the liver.

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin.

Lysis Performance of Bacteriophages with Different Plaque Sizes and Comparison of Lysis Kinetics After Simultaneous and Sequential Phage Addition.

Background: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance. Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity. Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance. Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment.

Structural transitions during the scaffolding-driven assembly of a viral capsid.

Assembly of tailed bacteriophages and herpesviruses starts with formation of procapsids (virion precursors without DNA). Scaffolding proteins (SP) drive assembly by chaperoning the major capsid protein (MCP) to build an icosahedral lattice. Here we report near-atomic resolution cryo-EM structures of the bacteriophage SPP1 procapsid, the intermediate expanded procapsid with partially released SPs, and the mature capsid with DNA. In the intermediate state, SPs are bound only to MCP pentons and to adjacent subunits from hexons. SP departure results in the expanded state associated with unfolding of the MCP N-terminus and straightening of E-loops. The newly formed extensive inter-capsomere bonding appears to compensate for release of SPs that clasp MCP capsomeres together. Subsequent DNA packaging instigates bending of MCP A domain loops outwards, closing the hexons central opening and creating the capsid auxiliary protein binding interface. These findings provide a molecular basis for the sequential structural rearrangements during viral capsid maturation.

Unravelling Ribosome Function Through Structural Studies.

Ribosomes are biological nanomachine that synthesise all proteins within a cell. It took decades to reveal the architecture of this essential cellular component. To understand the structure -function relationship of this nanomachine needed the utilisisation of different biochemical, biophysical and structural techniques. Structural studies combined with mutagenesis of the different ribosomal complexes comprising various RNAs and proteins enabled us to understand how this machine works inside a cell. Nowadays quite a number of ribosomal structures were published that confirmed biochemical studies on particular steps of protein synthesis by the ribosome . Four major steps were identified: initiation , elongation, termination and recycling. These steps lead us to the important question how the ribosome function can be regulated. Advances in technology for cryo electron microscopy: sample preparations, image recording, developments in algorithms for image analysis and processing significantly helped in revelation of structural details of the ribosome . We now have a library of ribosome structures from prokaryotes to eukaryotes that enable us to understand the complex mechanics of this nanomachine. As this structural library continues to grow, we gradually improve our understanding of this process and how it can be regulated and how the specific ribosomes can be stalled or activated, or completely disabled. This article provides a comprehensive overview of ribosomal structures that represent structural snapshots of the ribosome at its different functional states. Better understanding rises more particular questions that have to be addressed by determination structures of more complexes.Synopsis: Structural biology of the ribosome.

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.

Alternative splicing of telomerase catalytic subunit hTERT generated by apoptotic endonuclease EndoG induces human CD4+ T cell death.

Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.

Structural Analysis of Protein Complexes by Cryo Electron Microscopy.

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.

The ribosome and its role in protein folding: looking through a magnifying glass.

Protein folding, a process that underpins cellular activity, begins co-translationally on the ribosome. During translation, a newly synthesized polypeptide chain enters the ribosomal exit tunnel and actively interacts with the ribosome elements - the r-proteins and rRNA that line the tunnel - prior to emerging into the cellular milieu. While understanding of the structure and function of the ribosome has advanced significantly, little is known about the process of folding of the emerging nascent chain (NC). Advances in cryo-electron microscopy are enabling visualization of NCs within the exit tunnel, allowing early glimpses of the interplay between the NC and the ribosome. Once it has emerged from the exit tunnel into the cytosol, the NC (still attached to its parent ribosome) can acquire a range of conformations, which can be characterized by NMR spectroscopy. Using experimental restraints within molecular-dynamics simulations, the ensemble of NC structures can be described. In order to delineate the process of co-translational protein folding, a hybrid structural biology approach is foreseeable, potentially offering a complete atomic description of protein folding as it occurs on the ribosome.

Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation.

Recent studies have shown that conjugation systems of Gram-negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we characterized the OMCC by focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMCpKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. The findings establish that OMCCs are highly structurally and functionally conserved - but also intrinsically conformationally flexible - scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function.

Structures of biomolecular complexes by combination of NMR and cryoEM methods.

CryoEM is presently providing structures of biocomplexes considered intractable to analysis by other structural techniques. NMR is playing an important role in delivering structural information on dynamics events and conformational heterogeneity. Impressive results were obtained by combining cryoEM and either liquid- or solid-state NMR, revealing the structures of cellular machines, filaments and amyloid fibrils. NMR solution structures of proteins and nucleic acids were fitted, together with crystallographic structures, into cryoEM maps of large complexes, to decipher their assembly mechanisms and describe their functional dynamics. Modelling based on solid-state NMR and cryoEM data provided 3D structure of filaments and fibrils. These NMR approaches validated, but also corrected, atomic models built de novo in cryoEM maps, and provided new structural data on flexible or structurally heterogeneous systems. Combination of cryoEM and NMR became an established hybrid approach in structural biology that significantly contributes to our understanding of functional mechanisms in supramolecular assemblies.

Brain protection during cephalosomatic anastomosis.

Cephalosomatic anastomosis requires neuroprotective techniques, such as deep hypothermia, to preserve brain activity. Despite the failure of pharmacologic neuroprotection, new strategies, including ischemic pre- and postconitioning and the use of Perftoran, have to be explored to complement hypothermia. This article summarizes the field of brain protection during CSA and these promising strategies.

Two p53 tetramers bind one consensus DNA response element.

p53 tumor suppressor is a transcription factor that controls cell cycle and genetic integrity. In response to genotoxic stress p53 activates DNA repair, cell cycle arrest, apoptosis or senescence, which are initiated via p53 binding to its specific DNA response elements (RE). The consensus p53 DNA RE consists of two decameric palindromic half-site sequences. Crystallographic studies have demonstrated that two isolated p53 DNA-binding core domains interact with one half-site of the p53 DNA REs suggesting that one p53 tetramer is bound to one RE. However, our recent 3D cryo-EM studies showed that the full-length p53 tetramer is bound to only one half-site of RE.Here, we have used biochemical and electron microscopy (EM) methods to analyze DNA-binding of human and murine p53 tetramers to various p53 DNA REs. Our new results demonstrate that two p53 tetramers can interact sequence-specifically with one DNA RE at the same time. In particular, the EM structural analysis revealed that two p53 tetramers bind one DNA RE simultaneously with DNA positioned between them. These results demonstrate a mode different from that assumed previously for the p53-DNA interaction and suggest important biological implications on p53 activity as a transcriptional regulator of cellular response to stress.

Novel Inter-Subunit Contacts in Barley Stripe Mosaic Virus Revealed by Cryo-Electron Microscopy.

Barley stripe mosaic virus (BSMV, genus Hordeivirus) is a rod-shaped single-stranded RNA virus similar to viruses of the structurally characterized and well-studied genus Tobamovirus. Here we report the first high-resolution structure of BSMV at 4.1 Å obtained by cryo-electron microscopy. We discovered that BSMV forms two types of virion that differ in the number of coat protein (CP) subunits per turn and interactions between the CP subunits. While BSMV and tobacco mosaic virus CP subunits have a similar fold and interact with RNA using conserved residues, the axial contacts between the CP of these two viral groups are considerably different. BSMV CP subunits lack substantial axial contacts and are held together by a previously unobserved lateral contact formed at the virion surface via an interacting loop, which protrudes from the CP hydrophobic core to the adjacent CP subunit. These data provide an insight into diversity in structural organization of helical viruses.

Structural basis for DNA strand separation by a hexameric replicative helicase.

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.