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Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins 1-3 . Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators-which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements-can remodel their substrate DNA and cognate transposases to promote function., (© 2024. The Author(s).)

Prokaryotes have evolved intricate innate immune systems against phage infection 1-7 . Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB 8 . GajA functions as a DNA endonuclease that is inactive in the presence of ATP 9 . Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg 2+ . GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Structural and biochemical studies elucidate that PA N may contribute to the host protein shutdown observed during influenza A infection. Thus, inhibition of the endonuclease activity of viral RdRP is an attractive approach for novel antiviral therapy. In order to envisage structurally diverse novel compounds with better efficacy as PA N endonuclease inhibitors, a ligand-based-pharmacophore model was developed using 3D-QSAR pharmacophore generation (HypoGen algorithm) methodology in Discovery Studio. As the training set, 25 compounds were taken to generate a significant pharmacophore model. The selected pharmacophore Hypo1 was further validated by 12 compounds in the test set and was used as a query model for further screening of 1916 compounds containing 71 HIV-1 integrase inhibitors, 37 antibacterial inhibitors, 131 antiviral inhibitors and other 1677 approved drugs by the FDA. Then, six compounds ( Hit01 - Hit06 ) with estimated activity values less than 10 μM were subjected to ADMET study and toxicity assessment. Only one potential inhibitory 'hit' molecule ( Hit01 , raltegravir's derivative) was further scrutinized by molecular docking analysis on the active site of PA N endonuclease (PDB ID: 6E6W). Hit01 was utilized for designing novel potential PA N endonuclease inhibitors through lead optimization, and then compounds were screened by pharmacophore Hypo1 and docking studies. Six raltegravir's derivatives with significant estimated activity values and docking scores were obtained. Further, these results certainly do not confirm or indicate the seven compounds ( Hit01 , Hit07 , Hit08 , Hit09 , Hit10 , Hit11 and Hit12 ) have antiviral activity, and extensive wet-laboratory experimentation is needed to transmute these compounds into clinical drugs.

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease., (© 2021. The Author(s).)

Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements 1 . However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion 2-7 . Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ 7 , but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, envelope-spanning basal body and external needle with tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for these residues in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing interests, financial or personal, that would inappropriately influence the work presented here., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported here., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25-150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors., (© 2020 The Society for Applied Microbiology.)

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one β-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Magnesium transporter A (MgtA) is an active transporter responsible for importing magnesium ions into the cytoplasm of prokaryotic cells. This study focuses on the peptide corresponding to the intrinsically disordered N-terminal region of MgtA, referred to as KEIF. Primary-structure and bioinformatic analyses were performed, followed by studies of the undisturbed single chain using a combination of techniques including small-angle X-ray scattering, circular dichroism spectroscopy, and atomistic molecular-dynamics simulations. Moreover, interactions with large unilamellar vesicles were investigated by using dynamic light scattering, laser Doppler velocimetry, cryogenic transmission electron microscopy, and circular dichroism spectroscopy. KEIF was confirmed to be intrinsically disordered in aqueous solution, although extended and containing little β -structure and possibly PPII structure. An increase of helical content was observed in organic solvent, and a similar effect was also seen in aqueous solution containing anionic vesicles. Interactions of cationic KEIF with anionic vesicles led to the hypothesis that KEIF adsorbs to the vesicle surface through electrostatic and entropic driving forces. Considering this, there is a possibility that the biological role of KEIF is to anchor MgtA in the cell membrane, although further investigation is needed to confirm this hypothesis.

Structural maintenance of chromosomes (SMC) complexes organize chromosomes ubiquitously, thereby contributing to their faithful segregation. We demonstrate that under conditions of increased chromosome occupancy of the Escherichia coli SMC complex, MukBEF, the chromosome is organized as a series of loops around a thin (<130 nm) MukBEF axial core, whose length is ∼1,100 times shorter than the chromosomal DNA. The linear order of chromosomal loci is maintained in the axial cores, whose formation requires MukBEF ATP hydrolysis. Axial core structure in non-replicating chromosomes is predominantly linear (1 μm) but becomes circular (1.5 μm) in the absence of MatP because of its failure to displace MukBEF from the 800 kbp replication termination region (ter). Displacement of MukBEF from ter by MatP in wild-type cells directs MukBEF colocalization with the replication origin. We conclude that MukBEF individualizes and compacts the chromosome lengthwise, demonstrating a chromosome organization mechanism similar to condensin in mitotic chromosome formation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs) 1,2 . In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC 3 contains 16 subunits, including the ATP-dependent DNA translocase Sth1 4,5 . RSC removes nucleosomes from promoter regions 6,7 and positions the specialized +1 and -1 nucleosomes that flank NDRs 8,9 . Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements 8,10,11 that influence RSC functionality 12 . The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity 5 . The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer 13 .

Ribosome biogenesis is tightly regulated through stress-sensing pathways that impact genome stability, aging and senescence. In Saccharomyces cerevisiae , ribosomal RNAs are transcribed from rDNA located on the right arm of chromosome XII. Numerous studies reveal that rDNA decondenses into a puff-like structure during interphase, and condenses into a tight loop-like structure during mitosis. Intriguingly, a novel and additional mechanism of increased mitotic rDNA compaction (termed hypercondensation) was recently discovered that occurs in response to temperature stress (hyperthermic-induced) and is rapidly reversible. Here, we report that neither changes in condensin binding or release of DNA during mitosis, nor mutation of factors that regulate cohesin binding and release, appear to play a critical role in hyperthermic-induced rDNA hypercondensation. A candidate genetic approach revealed that deletion of either HSP82 or HSC82 (Hsp90 encoding heat shock paralogs) result in significantly reduced hyperthermic-induced rDNA hypercondensation. Intriguingly, Hsp inhibitors do not impact rDNA hypercondensation. In combination, these findings suggest that Hsp90 either stabilizes client proteins, which are sensitive to very transient thermic challenges, or directly promotes rDNA hypercondensation during preanaphase. Our findings further reveal that the high mobility group protein Hmo1 is a negative regulator of mitotic rDNA condensation, distinct from its role in promoting premature condensation of rDNA during interphase upon nutrient starvation., (Copyright © 2020 Shen and Skibbens.)

Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine., (Copyright © 2020 Park et al.)

The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis (Msm) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of Msm ClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.

Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems 1 . A molecular understanding of the type VII secretion mechanism has been hampered owing to a lack of three-dimensional structures of the fully assembled secretion apparatus. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/type VII secretion system from Mycobacterium smegmatis. The core of the ESX-3 secretion machine consists of four protein components-EccB3, EccC3, EccD3 and EccE3, in a 1:1:2:1 stoichiometry-which form two identical protomers. The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The domain of unknown function (DUF) adjacent to the stalk is identified as an ATPase domain that is essential for secretion. EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain. This suggests that conformational changes in the stalk domain-triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF-could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of type VII secretion systems differs markedly from that of other known secretion machines 2 , and provide a structural understanding of these systems that will be useful for the design of antimicrobial strategies that target bacterial virulence.

ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at ∼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1 Cnd3 in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1 Cnd2 The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex., (© 2019 Manalastas-Cantos et al.)

The AAA+ ATPase spastin remodels microtubule arrays through severing and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeF x that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn't show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeF x predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation., Competing Interests: JA, NG, SJ, SW, YC No competing interests declared, JL Is affiliated with 3T Biosciences and has no other competing interests to declare, GN Reviewing editor, eLife, (© 2019, Armache et al.)

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes 1-6 . After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails) 7-9 , whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails 10-12 . In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis 13 . Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.

Chromatin remodelers are diverse enzymes, and different models have been proposed to explain how these proteins work. Here we report the 3.3 Å-resolution cryogenic electron microscopy (cryo-EM) structures of Saccharomyces cerevisiae ISWI (ISW1) in complex with the nucleosome in adenosine diphosphate (ADP)-bound and ADP-BeF x -bound states. The data show that after nucleosome binding, ISW1 is activated by substantial rearrangement of the catalytic domains, with the regulatory AutoN domain packing the first RecA-like core and the NegC domain being disordered. The high-resolution structure reveals local DNA distortion and translocation induced by ISW1 in the ADP-bound state, which is essentially identical to that induced by the Snf2 chromatin remodeler, suggesting a common mechanism of DNA translocation. The histone core remains largely unperturbed, and prevention of histone distortion by crosslinking did not inhibit the activity of yeast ISW1 or its human homolog. Together, our findings suggest a general mechanism of chromatin remodeling involving local DNA distortion without notable histone deformation.

Condensins and cohesins are highly conserved complexes that tether together DNA loci within a single DNA molecule to produce DNA loops. Condensin and cohesin structures, however, are different, and the DNA loops produced by each underlie distinct cell processes. Condensin rods compact chromosomes during mitosis, with condensin I and II complexes producing spatially defined and nested looping in metazoan cells. Structurally adaptive cohesin rings produce loops, which organize the genome during interphase. Cohesin-mediated loops, termed topologically associating domains or TADs, antagonize the formation of epigenetically defined but untethered DNA volumes, termed compartments. While condensin complexes formed through cis -interactions must maintain chromatin compaction throughout mitosis, cohesins remain highly dynamic during interphase to allow for transcription-mediated responses to external cues and the execution of developmental programs. Here, I review differences in condensin and cohesin structures, and highlight recent advances regarding the intramolecular or cis -based tetherings through which condensins compact DNA during mitosis and cohesins organize the genome during interphase., Competing Interests: Competing interestsThe author declares no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-α7 insertions within both AAA domains important for Rix7 function.

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Protein assemblies consisting of structural maintenance of chromosomes (SMC) and kleisin subunits are essential for the process of chromosome segregation across all domains of life. Prokaryotic condensin belonging to this class of protein complexes is composed of a homodimer of SMC that associates with a kleisin protein subunit called ScpA. While limited structural data exist for the proteins that comprise the (SMC)-kleisin complex, the complete structure of the entire complex remains unknown. Using an integrative approach combining both crystallographic data and coevolutionary information, we predict an atomic-scale structure of the whole condensin complex, which our results indicate being composed of a single ring. Coupling coevolutionary information with molecular-dynamics simulations, we study the interaction surfaces between the subunits and examine the plausibility of alternative stoichiometries of the complex. Our analysis also reveals several additional configurational states of the condensin hinge domain and the SMC-kleisin interaction domains, which are likely involved with the functional opening and closing of the condensin ring. This study provides the foundation for future investigations of the structure-function relationship of the various SMC-kleisin protein complexes at atomic resolution., Competing Interests: The authors declare no conflict of interest.

Periplasmic flagella are essential for the distinct morphology and motility of spirochetes. A flagella-specific type III secretion system (fT3SS) composed of a membrane-bound export apparatus and a cytosolic ATPase complex is responsible for the assembly of the periplasmic flagella. Here, we deployed cryo-electron tomography (cryo-ET) to visualize the fT3SS machine in the Lyme disease spirochete Borrelia burgdorferi. We show, for the first time, that the cytosolic ATPase complex is attached to the flagellar C-ring through multiple spokes to form the "spoke and hub" structure in B. burgdorferi. This structure not only strengthens structural rigidity of the round-shaped C-ring but also appears to rotate with the C-ring. Our studies provide structural insights into the unique mechanisms underlying assembly and rotation of the periplasmic flagella and may provide the basis for the development of novel therapeutic strategies against several pathogenic spirochetes., Competing Interests: The authors have declared that no competing interests exist.

The yeast SWR1 complex exchanges histone H2A in nucleosomes with Htz1 (H2A.Z in humans). The cryo-electron microscopy structure of the SWR1 complex bound to a nucleosome at 3.6-angstrom resolution reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at superhelical location 2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to SWR1 complex. The unwrapping, as monitored by single-molecule data, is stabilized and has its dynamics altered by adenosine triphosphate binding but does not require hydrolysis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The MinDE protein system from E. coli has recently been identified as a minimal biological oscillator, based on two proteins only: The ATPase MinD and the ATPase activating protein MinE. In E. coli, the system works as the molecular ruler to place the divisome at midcell for cell division. Despite its compositional simplicity, the molecular mechanism leading to protein patterns and oscillations is still insufficiently understood. Here we used high-speed atomic force microscopy to analyze the mechanism of MinDE membrane association/dissociation dynamics on isolated membrane patches, down to the level of individual point oscillators. This nanoscale analysis shows that MinD association to and dissociation from the membrane are both highly cooperative but mechanistically different processes. We propose that they represent the two directions of a single allosteric switch leading to MinD filament formation and depolymerization. Association/dissociation are separated by rather long apparently silent periods. The membrane-associated period is characterized by MinD filament multivalent binding, avidity, while the dissociated period is defined by seeding of individual MinD. Analyzing association/dissociation kinetics with varying MinD and MinE concentrations and dependent on membrane patch size allowed us to disentangle the essential dynamic variables of the MinDE oscillation cycle.

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

Chromatin-remodelling factors change nucleosome positioning and facilitate DNA transcription, replication, and repair. The conserved remodelling factor chromodomain-helicase-DNA binding protein 1(Chd1) can shift nucleosomes and induce regular nucleosome spacing. Chd1 is required for the passage of RNA polymerase IIthrough nucleosomes and for cellular pluripotency. Chd1 contains the DNA-binding domains SANT and SLIDE, a bilobal motor domain that hydrolyses ATP, and a regulatory double chromodomain. Here we report the cryo-electron microscopy structure of Chd1 from the yeast Saccharomyces cerevisiae bound to a nucleosome at a resolution of 4.8 Å. Chd1 detaches two turns of DNA from the histone octamer and binds between the two DNA gyres in a state poised for catalysis. The SANT and SLIDE domains contact detached DNA around superhelical location (SHL) -7 of the first DNA gyre. The ATPase motor binds the second DNA gyre at SHL +2 and is anchored to the N-terminal tail of histone H4, as seen in a recent nucleosome-Snf2 ATPase structure. Comparisons with published results reveal that the double chromodomain swings towards nucleosomal DNA at SHL +1, resulting in ATPase closure. The ATPase can then promote translocation of DNA towards the nucleosome dyad, thereby loosening the first DNA gyre and remodelling the nucleosome. Translocation may involve ratcheting of the two lobes of the ATPase, which is trapped in a pre- or post-translocation state in the absence or presence, respectively, of transition state-mimicking compounds.

Human transcription factor IIH (TFIIH) is part of the general transcriptional machinery required by RNA polymerase II for the initiation of eukaryotic gene transcription. Composed of ten subunits that add up to a molecular mass of about 500 kDa, TFIIH is also essential for nucleotide excision repair. The seven-subunit TFIIH core complex formed by XPB, XPD, p62, p52, p44, p34, and p8 is competent for DNA repair, while the CDK-activating kinase subcomplex, which includes the kinase activity of CDK7 as well as the cyclin H and MAT1 subunits, is additionally required for transcription initiation. Mutations in the TFIIH subunits XPB, XPD, and p8 lead to severe premature ageing and cancer propensity in the genetic diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, highlighting the importance of TFIIH for cellular physiology. Here we present the cryo-electron microscopy structure of human TFIIH at 4.4 Å resolution. The structure reveals the molecular architecture of the TFIIH core complex, the detailed structures of its constituent XPB and XPD ATPases, and how the core and kinase subcomplexes of TFIIH are connected. Additionally, our structure provides insight into the conformational dynamics of TFIIH and the regulation of its activity.

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4 E233Q hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4 E233Q subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward. The motor ATPase PilB powers pilus assembly. Here we report the structures of the core ATPase domains of Geobacter metallireducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMP-PNP, at 3.4 and 2.3 Å resolution, respectively. These structures reveal important differences in nucleotide binding between chains. Analysis of these differences reveals the sequential turnover of nucleotide, and the corresponding domain movements. Our data suggest a clockwise rotation of the central sub-pores of PilB, which through interactions with PilC, would support the assembly of a right-handed helical pilus. Our analysis also suggests a counterclockwise rotation of the C2 symmetric PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction.

Chromatin remodellers are helicase-like, ATP-dependent enzymes that alter chromatin structure and nucleosome positions to allow regulatory proteins access to DNA. Here we report the cryo-electron microscopy structure of chromatin remodeller Switch/sucrose non-fermentable (SWI2/SNF2) from Saccharomyces cerevisiae bound to the nucleosome. The structure shows that the two core domains of Snf2 are realigned upon nucleosome binding, suggesting activation of the enzyme. The core domains contact each other through two induced Brace helices, which are crucial for coupling ATP hydrolysis to chromatin remodelling. Snf2 binds to the phosphate backbones of one DNA gyre of the nucleosome mainly through its helicase motifs within the major domain cleft, suggesting a conserved mechanism of substrate engagement across different remodellers. Snf2 contacts the second DNA gyre via a positively charged surface, providing a mechanism to anchor the remodeller at a fixed position of the nucleosome. Snf2 locally deforms nucleosomal DNA at the site of binding, priming the substrate for the remodelling reaction. Together, these findings provide mechanistic insights into chromatin remodelling.

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeF x , and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N 1 N o -type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c 17 ring is H + specific, demonstrating that the ATPase is proton-coupled. The c 17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

A fundamental requirement in nature is for a cell to correctly package and divide its replicated genome. Condensin is a mechanical multisubunit complex critical to this process. Condensin uses ATP to power conformational changes in DNA to enable to correct DNA compaction, organization, and segregation of DNA from the simplest bacteria to humans. The highly conserved nature of the condensin complex and the structural similarities it shares with the related cohesin complex have provided important clues as to how it functions in cells. The fundamental requirement for condensin in mitosis and meiosis is well established, yet the precise mechanism of action is still an open question. Mutation or removal of condensin subunits across a range of species disrupts orderly chromosome condensation leading to errors in chromosome segregation and likely death of the cell. There are divergences in function across species for condensin. Once considered to function solely in mitosis and meiosis, an accumulating body of evidence suggests that condensin has key roles in also regulating the interphase genome. This review will examine how condensin organizes our genomes, explain where and how it binds the genome at a mechanical level, and highlight controversies and future directions as the complex continues to fascinate and baffle biologists.

Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for determining the structures of biological macromolecules. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resolution frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calculations on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer. Furthermore, SGD with Bayesian marginalization allows ab initio 3D classification, enabling automated analysis and discovery of unexpected structures without bias from a reference map. These algorithms are combined in a user-friendly computer program named cryoSPARC (http://www.cryosparc.com).

The spliceosome excises introns from pre-mRNAs in two sequential transesterifications-branching and exon ligation-catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA). Recently reported structures of the spliceosomal C complex with the cleaved 5' exon and lariat-3'-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5' splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site. Here we present, at 3.8 Å resolution, the cryo-electron microscopy structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but before exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75° compared to the C complex and is stabilized in a new position by Prp17, Cef1 and the reoriented Prp8 RNase H-like domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3' exon docking, and restructures the pairing of the 5' splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNase H-like domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3' exon, suggesting a possible basis for mRNA release after exon ligation. Together with the structure of the C complex, our structure of the C* complex reveals the two major conformations of the spliceosome during the catalytic stages of splicing.

ATP-binding cassette (ABC) exporters mediate vital transport of a variety of molecules across the lipid bilayer in all organisms. To explore the allosteric effect of ATP binding at the asymmetric ATPase sites, molecular dynamics simulations were performed on the nucleotide-binding domains (NBDs) of a heterodimeric exporter TM287/288 in 4 different ATP-bound states. The results showed that ATP bound at the degenerate site can maintain a semi-open conformation of NBD1-NBD2, which may be defective in ATP hydrolysis. By contrast, when bound at the consensus site, ATP can induce an intra-domain rotation of the α-helical subdomain towards the RecA-like subdomain of NBD2 at the degenerate site. The rotation of the α-helical subdomain rearranged the hydrogen bond networks at the NBD1-NBD2 interface, induced a significant conformational change in the D-loop at the degenerate site and inter- and intra-domain communications at both sites, and eventually elicited dimerization of NBD1-NBD2. These findings indicate that the asymmetric ATPase sites of the heterodimeric exporter are structurally and functionally distinct.

SMC (structural maintenance of chromosomes) complexes - which include condensin, cohesin and the SMC5-SMC6 complex - are major components of chromosomes in all living organisms, from bacteria to humans. These ring-shaped protein machines, which are powered by ATP hydrolysis, topologically encircle DNA. With their ability to hold more than one strand of DNA together, SMC complexes control a plethora of chromosomal activities. Notable among these are chromosome condensation and sister chromatid cohesion. Moreover, SMC complexes have an important role in DNA repair. Recent mechanistic insight into the function and regulation of these universal chromosomal machines enables us to propose molecular models of chromosome structure, dynamics and function, illuminating one of the fundamental entities in biology.

P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring., (Copyright © 2015 Elsevier Inc. All rights reserved.)