The putative Plasmodium translocon of exported proteins (PTEX) is essential for transport of malarial effector proteins across a parasite-encasing vacuolar membrane into host erythrocytes, but the mechanism of this process remains unknown. Here we show that PTEX is a bona fide translocon by determining structures of the PTEX core complex at near-atomic resolution using cryo-electron microscopy. We isolated the endogenous PTEX core complex containing EXP2, PTEX150 and HSP101 from Plasmodium falciparum in the 'engaged' and 'resetting' states of endogenous cargo translocation using epitope tags inserted using the CRISPR-Cas9 system. In the structures, EXP2 and PTEX150 interdigitate to form a static, funnel-shaped pseudo-seven-fold-symmetric protein-conducting channel spanning the vacuolar membrane. The spiral-shaped AAA+ HSP101 hexamer is tethered above this funnel, and undergoes pronounced compaction that allows three of six tyrosine-bearing pore loops lining the HSP101 channel to dissociate from the cargo, resetting the translocon for the next threading cycle. Our work reveals the mechanism of P. falciparum effector export, and will inform structure-based design of drugs targeting this unique translocon.

INTRODUCTION DNA replication has been studied since the 1950s. It is well established that double helical DNA needs to be separated for replication by a helicase. Each strand is then copied by a DNA polymerase, continuously on the leading and discontinuously (via Okazaki fragments) on the lagging strand, where each DNA synthesis initiates from an RNA primer provided by primase. After six decades, how DNA polymerases, helicase, primase, and their accessory factors form a replisome and perform concerted leading and lagging strand synthesis at a replication fork had never been visualized in atomic detail. RATIONALE Bacteriophage T7 presents the simplest known DNA replication system, consisting of only three proteins. Helicase and primase reside in one polypeptide chain that forms a hexamer in the presence of DNA and ATP or dTTP. T7 DNA polymerase, aided by E. coli thioredoxin as its processivity factor, carries out both leading and lagging strand DNA synthesis. Based on published biochemical data, we designed a minimal DNA fork to trap these essential proteins in replication competent states. RESULTS We determined cryogenic-electron microscopy (cryo-EM) structures of the T7 replisome and showed how its essential enzymatic functions are coordinated in three dimensions. The hexameric helicase adopts a spiral “lock washer” form that encircles the coil-like lagging DNA strand, with two nucleotides (nt) bound to each protein subunit and adjacent helicase subunits linked by domain swapping. ATP hydrolysis propels each helicase domain to translocate sequentially and coaxially along DNA in a hand-over-hand fashion, advancing 2 nt per step in the 5′ to 3′ direction (Fig. A). Instead of all enzymes moving in the same direction parallel to the downstream parental DNA, a β-hairpin from the leading-strand polymerase separates the two parental DNA strands into a T-shaped fork that enables the closely coupled helicase to unspool the downstream DNA tangentially (Fig. B). By protein-protein and DNA-mediated interactions, the leading-strand DNA polymerase and helicase cooperate to determine the rate of replication. For every ATP hydrolyzed and 2 nt advanced on DNA by the helicase, the DNA polymerase incorporates two deoxyribonucelotides. T7 primase, separated from the leading-strand polymerase by the helicase domain, synthesizes the RNA primers needed to initiate lagging-strand DNA synthesis. Transfer of a short RNA primer from the primase to DNA polymerase is facilitated by a zinc-binding-domain at the N-terminus of the T7 primase-helicase protein. Two lagging strand polymerases can be attached to the hexameric primases with one actively synthesizing DNA and the other waiting for a primer (Fig. B). Such a relay system may allow the discontinuous lagging-strand synthesis to keep pace with the leading-strand synthesis. CONCLUSION We note the similarity between hexameric DNA helicases and AAA+ protein chaperones and unfoldases, which form spiral-shaped hexamers around protein substrates, bind two amino-acid residues with each su...

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryoEM structures of the yeast E complex assembled on introns, providing the first view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, which either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyzes back-splicing generating circRNA (on long exons). The model is supported by our experiments demonstrating that E complex assembled on the yeast EFM5 or HMRA1 middle exon can be chased into circRNA when the exon is sufficiently long. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Summary To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryoEM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs rotate, shift and deform extensively for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the “beyond 12/23” restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.

The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and ILS complexes revealed mechanisms of 5′ splice site (ss) recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release. Here, we report a cryoEM structure of the post-catalytic P complex at 3.3Å resolution, revealing that 3′ ss is mainly recognized through non-Watson-Crick basepairing with the 5′ ss and branch point. Furthermore, an unidentified protein becomes stably associated with the P complex, securing the 3′ exon and potentially regulating Prp22 activity. Prp22 binds nucleotides 15–21 in the 3′ exon, enabling it to pull the intron-exon or ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading or exon release, respectively.

Synthetic diblock copolypeptides were designed to incorporate oppositely charged ionic segments that form β-sheet-structured hydrogel assemblies via polyion complexation when mixed in aqueous media. The observed chain conformation directed assembly was found to be required for efficient hydrogel formation and provided distinct and useful properties to these hydrogels, including self-healing after deformation, microporous architecture, and stability against dilution in aqueous media. While many promising self-assembled materials have been prepared using disordered or liquid coacervate polyion complex (PIC) assemblies, the use of ordered chain conformations in PIC assemblies to direct formation of new supramolecular morphologies is unprecedented. The promising attributes and unique features of the β-sheet-structured PIC hydrogels described here highlight the potential of harnessing conformational order derived from PIC assembly to create new supramolecular materials.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue (“fast gate”) known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule (“slow gating”). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1–related diseases.

Suppressing cellular signal transducers of transcription 2 (STAT2) is a common strategy viruses use to establish infections, yet the detailed mechanism remains elusive due to lack of structural information of the viral-cellular complex involved. Here, we report the cryo-EM and crystal structures of human STAT2 (hSTAT2) in complex with the non-structural protein 5 (NS5) of Zika virus (ZIKV) and dengue virus (DENV), revealing two-pronged interactions between NS5 and hSTAT2. First, the NS5 methyltransferase and RNA-dependent RNA polymerase (RdRP) domains form a conserved inter-domain cleft harboring the coiled-coil domain of hSTAT2, thus preventing association of hSTAT2 with interferon regulatory factor 9. Second, the NS5 RdRP domain also binds the N-terminal domain of hSTAT2. Disruption of these ZIKV NS5–hSTAT2 interactions compromised NS5-mediated hSTAT2 degradation and interferon suppression, and viral infection under interferon-competent condition. Taken together, these results clarify the mechanism underlying the functional antagonism of STAT2 by both ZIKV and DENV.