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The increasing interest of the biopharmaceutical industry to exploit plants as a commercially viable production system is demanding the development of new strategies to maximize product recovery. Aqueous two-phase systems (ATPSs) are a primary recovery technique that has shown great potential for the efficient extraction and purification of biological products, from organelles to proteins and low-molecular-weight compounds. The evaluation of different system parameters upon the partitioning behavior can provide the conditions that favor the concentration of contaminants and the desired target protein in opposite phases. The protocols described here provide the basic strategy to explore the use of ATPSs for the isolation and partial purification of native and recombinant proteins from plants and plant-derived extracts.

The formation of the chromosome axis is key to meiotic recombination and hence the correct distribution of chromosomes to meiotic products. A key component of the axis in Arabidopsis is the HORMA domain protein (HORMAD) ASY1, the homolog of Hop1 in yeast and HORMAD1/2 in mammals. The chromosomal association of ASY1 is dynamic, i.e., ASY1 is recruited to the axis at early prophase and later largely removed when homologous chromosomes synapse. PCH2/TRIP13 proteins are well-known regulators of meiotic HORMADs and required for their depletion from synapsed chromosomes. However, no direct interaction has been found between PCH2/TRIP13 and the presumptive HORMAD substrates in any organism other than in budding yeast. Thus, it remains largely elusive how the dynamics of ASY1 and other meiotic HORMADs are controlled. Here, we have identified COMET, the Arabidopsis homolog of human p31 comet , which is known for its function in the spindle assembly checkpoint (SAC), as a central regulator of ASY1 dynamics in meiosis. We provide evidence that COMET controls ASY1 localization by serving as an adaptor for PCH2. Because ASY1 accumulates in the cytoplasm in early prophase and is persistently present on chromosomes in comet, we conclude that COMET is required for both the recruitment of ASY1 to the nucleus and the subsequent removal from the axis. The here-revealed function of COMET as an adaptor for PCH2 remarkably resembles the regulation of another HORMAD, Mad2, in the SAC in yeast and animals, revealing a conserved regulatory module of HORMA-domain-containing protein complexes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V-ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer-associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF-κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl-xL and Bcl-2A1) and decreased expression of proapoptotic proteins (Bax, Apaf-1, caspase-3, caspase-6, and caspase-7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF-α and IL-8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer-associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Type VII secretion systems (T7SSs) are found in many disease related bacteria including Mycobacterium tuberculosis (Mtb). ESX-1 [early secreted antigen 6 kilodaltons (ESAT-6) system 1] is one of the five subtypes (ESX-1~5) of T7SSs in Mtb, where it delivers virulence factors into host macrophages during infection. However, little is known about the molecular details as to how this occurs. Here, we provide high-resolution crystal structures of the C-terminal ATPase 3 domains of EccC subunits from four different Mtb T7SS subtypes. These structures adopt a classic RecA-like ɑ/β fold with a conserved Mg-ATP binding site. The structure of EccCb1 in complex with the C-terminal peptide of EsxB identifies the location of substrate recognition site and shows how the specific signaling module "LxxxMxF" for Mtb ESX-1 binds to this site resulting in a translation of the bulge loop. A comparison of all the ATPase 3 structures shows there are significant differences in the shape and composition of the signal recognition pockets across the family, suggesting that distinct signaling sequences of substrates are required to be specifically recognized by different T7SSs. A hexameric model of the EccC-ATPase 3 is proposed and shows the recognition pocket is located near the central substrate translocation channel. The diameter of the channel is ~25-Å, with a size that would allow helix-bundle shaped substrate proteins to bind and pass through. Thus, our work provides new molecular insights into substrate recognition for Mtb T7SS subtypes and also a possible transportation mechanism for substrate and/or virulence factor secretion.

When purifying a membrane protein, finding a detergent for solubilization is one of the first steps to master. Ideally, only little time is invested to identify the best-suited detergent, which on the one hand would solubilize large amounts of the target protein but on the other hand would sustain the protein's activity. Here we describe the solubilization screen and subsequent activity assay we have optimized for the bacterial P-type ATPase KdpFABC. In just 2 days, more than 70 detergents were tested for their solubilization potential. Afterwards, a smaller selection of the successful detergents was assayed for their ability to retain the activity of the membrane protein complex.

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.

Since spermatheca is able to transport spermatozoa and maintain a specific microenvironment for the storage of viable sperm cells for long periods of time, specific morphofunctional features must be involved in this capacity, and an efficient nutritional and oxygen supply must be required. In this study, we investigated the histological features of spermathecae and fat bodies in six species of three genera of epidemiological importance for Chagas' disease. The association of the reproductive system with the fat bodies and tracheal system was also focused in these species. The reproductive system, tracheae and fat bodies were fixed in 4% formaldehyde, and embedded in glycol methacrylate. The sections were stained with H.E., picrosirius red and Periodic-Acid Schiff methods for morphological analyses. Paraffin-embedded spermatheca sections were submitted to immunofluorescence for detection of V-ATPase. In P. lignarius, R. montenegrensis and R. prolixus, the spermatheca contains a slightly dilated tubular distal portion. In P. megistus and T. tibiamaculata, the spermatheca shows a large bulbous distal portion, and in T. infestans, a large oval-shaped distal portion. In all species, this portion was surrounded by a thin muscular layer, and the epithelial height varied according to the shape of this terminal portion. All spermathecal proximal portions showed simple columnar epithelium surrounded by a thick muscular layer. The epithelial cells of spermathecae showed PAS-positive cytoplasm and V-ATPase immunofluorescence in the apical surface. Tracheoles and polysaccharide-rich fat body cells were found next or in close contact to the oviduct or spermathecal tissues. The results indicate that the spermatheca proximal portion is related to contraction and sperm transport, whose oxygen and energy supply is guaranteed by the associated tracheal branches and fat bodies. In the storage portion, fat bodies and tracheae seem to be crucial for the maintenance of an optimal spermathecal microenvironment and storage of viable sperm cells. The participation of V-ATPase in the spermathecae epithelial cells may contribute for the maintenance of an optimal luminal milieu to spermatozoa, by alkalinization and/or acidification of lumen, similarly to the other epithelial cell types in insects. Further studies are necessary to clarify the role of this proton pump in the spermathecal epithelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS 9 ). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS 9 and TerL are functional, and that they also function with gp74. Purified TerS 9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW., Competing Interests: The authors declare no conflict of interest.

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors. Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1-26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, 1 H-, and 13 C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200-0.350 μM) of NTPDase-1, twelve (Ki = 0.071-1.060 μM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700-4.03 μM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ± 0.007, 6.39 ± 0.89 and 1.180 ± 0.002 μM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.

Chaperone proteins are required to maintain the overall fold and function of proteins in the cell. As part of the Hsp70 family, Ssa1 acts to maintain cellular proteostasis through a variety of diverse pathways aimed to preserve the native conformation of target proteins, thereby preventing aggregation and future states of cellular toxicity. Studying the structural dynamics of Ssa1 in vitro is essential to determining their precise mechanisms and requires the development of purification methods that result in highly pure chaperones. Current methods of expressing and purifying Ssa1 utilize affinity tagged constructs expressed in Escherichia coli, however, expression in an exogenous source produces proteins that lack post-translational modifications leading to undesired structural and functional effects. Current protocols to purify Ssa1 from Saccharomyces cerevisiae require large amounts of starting material, multiple steps of chromatography, and result in low yield. Our objective was to establish a small-scale purification of Ssa1 expressed from its endogenous source, Saccharomyces cerevisiae, with significant yield and purity. We utilized a protein A affinity tag that was previously used to purify large protein complexes from yeast, combined with magnetic Dynabeads that are conjugated with rabbit immunoglobulin G (IgG). Our results show that we can produce native, highly pure, active Ssa1 via this one-step purification with minimal amounts of starting material, and this Ssa1-protein A fusion does not alter cellular phenotypes. This methodology is a significant improvement in Ssa1 purification and will facilitate future experiments that will elucidate the biochemical and biophysical properties of Hsp70 chaperones., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na^(+)-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca^(2+)-ATPases of SERCA type (subfamily P2A); (3) H^(+)-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation-transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na^(+)-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na^(+)-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na^(+) transport.

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl-substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane-cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b, 8 c, and 9 b preserved the ATPase function of BmrA, an ATP-binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a, 8 b, 8 f, 9 a, and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G-protein-coupled adenosine receptor A 2A R. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia , which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min -1  mg -1 ) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The RNA helicase DEAD-box protein Sub2 (yeast)/UAP56 (mammals) is conserved across eukaryotes and is essential for mRNA export in trypanosomes. Despite the high conservation of Sub2 in lower eukaryotes such as Trypanosoma cruzi, the low conservation of other mRNA export factors raises questions regarding whether the mode of action of TcSub2 is similar to that of orthologs from other eukaryotes. Mutation of the conserved K87 residue of TcSub2 abolishes ATPase activity, showing that its ATPase domain is functional. However, the Vmax of TcSub2 was much higher than the Vmax described for the human protein UAP56, which suggests that the TcSub2 enzyme hydrolyzes ATP faster than its human homolog. Furthermore, we demonstrate that RNA association is less important to the activity of TcSub2 compared to UAP56. Our results show differences in activity of this protein, even though the structure of TcSub2 is very similar to UAP56. Functional complementation assays indicate that these differences may be common to other trypanosomatids. Distinct features of RNA influence and ATPase efficiency between UAP56 and TcSub2 may reflect distinct structures for functional sites of TcSub2. For this reason, ligand-based or structure-based methodologies can be applied to investigate the potential of TcSub2 as a target for new drugs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses. They can therefore affect adhesion and infection of host cells and the parasite virulence. To further characterize these enzymes, in this work, we expressed LicNTPDase1 and -2 in the classical bacterial system Escherichia coli and mammalian cell system COS-7 cells. Our data demonstrate that changes in refolding after expression in bacteria can increase the activity of recombinant (r) rLicNTPDase2 up to 20 times but has no significant effect on rLicNTPDase1. Meanwhile, the expression in COS-7 led to a significant increase in activity for rLicNTPDase1., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli . The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains. gsiA was shown to be essential for GSI mediated glutathione uptake and gsiA deletion could decrease the virulence of Salmonella enterica . Interactions of glutathione import proteins GsiA, GsiB, GsiC, and GsiD were investigated by using bacterial two-hybrid system. GsiA could interact with itself and inner membrane proteins GsiC and GsiD. This report provides the first description of gsiA functions in Salmonella enterica . The results could help elucidating the glutathione uptake mechanism and glutathione functions in bacteria.

Candida albicans is an opportunistic human pathogen, and its pathogenicity is associated with hyphal formation. Previous studies have shown that at neutral-to-alkaline pH, hyphal growth is dependent on the Rim101 pathway whose activation requires Snf7, a member of the ESCRT system. In this work, we described the purification and characterization of the C. albicans Vps4, an AAA ATPase required for recycling of the ESCRTs. Its role on hyphal growth has been investigated. Our data suggest deletion of Vps4 decreases overall hyphal growth at pH 7 and increases the growth of multiple hyphae induced by serum, which indicates that the ESCRTs may make a Rim101-independent contribution to hyphal growth. Furthermore, DBeQ, an inhibitor of the AAA ATPase p97, was shown to inhibit the ATPase activity of Vps4 with an IC50 of about 11.5 μM. To a less degree, it also inhibits hyphal growth. Our work may provide a new strategy to control C. albicans infection.

Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals.

Endocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P-Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkörper from another Type 4 P-type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact-C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4-ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkörper in A. nidulans., (© 2015 John Wiley & Sons Ltd.)

The protein 26S proteasome regulatory subunit p27 is one of the four chaperones that help in the assembly of the 19S regulatory particle (RP) of the 26S proteasome. In the present work, the N-terminus of p27 (residues 1-128) from Mus musculus was cloned, expressed, purified and crystallized alone and in complex with the C-terminal ATPase domain of Rpt5 (residues 173-442). The crystals of p27((1-128)) diffracted to 1.7 Å resolution and belonged to space group P212121, with unit-cell parameters a = 26.79, b = 30.39, c = 145.06 Å. Resolution-dependent Matthews coefficient probability analysis suggested the presence of only one molecule per asymmetric unit, with 40.5% solvent content and a VM value of 2.02 Å(3) Da(-1). The crystal of the p27((1-128))-Rpt5((173-442)) complex diffracted to 4 Å resolution and belonged to space group P222, with unit-cell parameters a = 75.93, b = 76.08, c = 336.85 Å. The presence of four heterodimers in the asymmetric unit with 53.2% solvent content and a VM value of 2.63 Å(3) Da(-1) or five heterodimers in the asymmetric unit with 41.5% solvent content and a VM value of 2.10 Å(3) Da(-1) is assumed.

Objective: The adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3.;ATPase) is a membrane -bound enzyme which transport protons across the plasma membrane using ATP as an energy source., Methods: The adenosine triphosphatase (ATPase ; EC: 3.6.1.3) was extracted from membrane preparations of adult Fasciola hepatica by chloroform treatment and purified by means of ammonium sulphate fractionation, gel filtration on sephadex G-200 and DEAE- Cellulose chromatography., Results: The molecular weight was calculated to be 305.000 dalton by gel filtration. Kinetic experiments demonstrated a biphasic linear lineweaver - burk relationship (km=0.142 and 1.66 mM) thus revealing the existence of two substrate binding enzyme sites., Conclusion: In our study revealed that partial inhibition of Mg²⁺ dependent purified enzyme by oligomycin suggest the absence of mitochondrial ATPase in F. hepatica.

ClpB is a molecular chaperone from the AAA+ superfamily of ATPases, which reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB is essential for infectivity and in-host survival of a number of pathogenic microorganisms, but systematic studies on ClpB from pathogens have not been reported yet. We purified and characterized one of the two ClpB isoforms from the malaria parasite Plasmodium falciparum, PfClpB1. PfClpB1 is targeted to the apicoplast, an essential plastid organelle that is a promising anti-malaria drug target. PfClpB1 contains all characteristic AAA+ sequence motifs, but the middle domain of PfClpB1 includes a 52-residue long non-conserved insert. Like in most AAA+ ATPases, ATP induces self-association of PfClpB1 into hexamers. PfClpB1 catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and poly-lysine. Similar to Escherichia coli ClpB, PfClpB1 reactivates aggregated firefly luciferase, but the PfClpB1-mediated aggregate reactivation is inhibited in the presence of E. coli DnaK, DnaJ, and GrpE. The lack of effective cooperation between PfClpB1 and the bacterial DnaK system may arise from the Plasmodium-specific sequence of the ClpB middle domain. Our results indicate that the chaperone activity of PfClpB1 may support survival of Plasmodium falciparum by maintaining the folding status and activity of apicoplast proteins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70.

Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47(C1) and Mtb-GyrB47(C2), have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P21 and diffracted to resolutions of 2.9 and 3.3 Å, respectively.

Homologous recombination (HR) is a major pathway for the repair of double-strand DNA breaks, a highly deleterious form of DNA damage. The main catalytic protein in HR is the essential RecA-family recombinase, which is conserved across all three domains of life. Eukaryotes and archaea encode varying numbers of proteins paralogous to their main recombinase. Although there is increasing evidence for the functions of some of these paralog proteins, overall their mechanism of action remains largely unclear. Here we present the first biochemical characterization of one of the paralog proteins, SsoRal3, from the crenarchaeaon Sulfolobus solfataricus. The SsoRal3 protein is a ssDNA-dependent ATPase that can catalyze strand invasion at both saturating and subsaturating concentrations. It can bind both ssDNA and dsDNA, but its binding preference is altered by the presence or absence of ATP. Addition of SsoRal3 to SsoRadA nucleoprotein filaments reduces total ATPase activity. Subsaturating concentrations of SsoRal3 increase the ssDNA binding activity of SsoRadA approximately 9-fold and also increase the persistence of SsoRadA catalyzed strand invasion products. Overall, these results suggest that SsoRal3 functions to stabilize the SsoRadA presynaptic filament., (Copyright © 2012 Elsevier B.V. All rights reserved.)

ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314 bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg(2+) and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).

The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain., (© 2012 The Authors Genes to Cells © 2012 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

P(IB)-type ATPases transport heavy metals (Cu(2+), Cu(+), Ag(+), Zn(2+), Cd(2+), Co(2+)) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative P(IB)-type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn(2+)/Cd(2+)-ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p-nitrophenyl phosphate (pNPP) as substrates. CopA was found to have greater activity in the presence of Cu(+), while CopB was found to have greater activity in the presence of Cu(2+). The putative Zn(2+)/Cd(2+)-ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli, however, the putative Zn(2+)/Cd(2+)-ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn(2+) and Cd(2+) as well as by Cu(+). Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.

ATAD3 is a mitochondrial integral inner membrane ATPase with unknown function. ATAD3 is absent in yeast and protozoan and present in all pluricellular eucaryotes where its expression is essential for development. To date, bacterial-based expression of full-length ATAD3 has been unsuccessful because of very high levels of endogenous degradation. Based on Saccharomyces cerevisiae as a heterogeneous expression system, we engineered a high copy strain expressing human ATAD3A-Myc-HIS at a relative high level (2.5mg/l of yeast culture) without significantly affecting yeast growth. Most of the expressed human ATAD3A-Myc-HIS co-purified with the yeast mitochondrial fraction thus suggesting that targeting to this organelle is preserved in yeast. Like the endogenous protein in human cells, ATAD3A-Myc-HIS expressed in yeast is found resistant to extraction with salt and certain detergents, suggesting membrane insertion. Sarkosyl, C13-DAO, C12-DAO and ONMG efficiently solubilized ATAD3A-Myc-HIS from yeast extracts, but these soluble species did not bind to agarose-nickel matrix. By contrast, urea-denaturated ATAD3A-Myc-HIS bound to agarose-nickel beads and could be renatured and eluted to obtain highly pure ATAD3A-Myc-HIS. As the native protein in vivo, this recombinant, renatured species specifically bound in vitro to S100B and S100A1 in Far-Western assays., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP-Mg(2+), mimicking the product-bound state; (iii) in complex with vanadate-ADP-Mg(2+), mimicking the ATP-bound state; and (iv) in complex with azide-ADP-Mg(2+), also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 Å. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6(2), with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported.

Ca(2+)-ATPases are members of a large family of membrane proteins that maintain the selective movement of cations across biological membranes. A putative Listeria monocytogenes Ca(2+)-ATPase (Lmo0818) was crystallized in an unknown functional state. The crystal belonged to space group P2(1)2(1)2(1) and a complete data set was collected to 3.2 Å resolution. The molecular-replacement solution obtained revealed that Lmo0818 is likely to adopt an E2-like state mimicking the phosphorylated intermediate in the functional cycle of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a stacked bilayer `type I' packing in the crystal., (© 2012 International Union of Crystallography. All rights reserved.)

Calix[4]arene C-97 (code is shown) is the macrocyclic compound which has lipophilic intramolecular higly-structured cavity formed by four aromatic cycles, one of which on the upper rim is modified by methylene bisphosphonic group. It was shown that calix[4]arene C-97 (100 microM) efficiently inhibits ATPase activity of myosin subfragment-1 from pig myometrium, the inhibition coefficient I(0.5) being 83 +/- 7 microM. At the same time, this compound at 100 microM concentration significantly increases the effective hydrodynamic diameter of myosin subfragment-1, that may be indicative of intermolecular complexation between the calix[4]arene and myosin head. Computer simulation methods (docking, molecular dynamics, involving the Grid) have been used to clarify structural basis of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of the myometrium; participation of hydrophobic, electrostatic and pi-pi (stacking) interactions between calix[4]arene C-97 and amino acid residues of myosin subfragment-1, some of them being located near the active site of the ATPase has been found out.

Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

The evolutionarily conserved histone variant H2A.Z has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. Saccharomyces cerevisiae Swr1, a Swi2/Snf2-related ATPase, is the catalytic core of a multisubunit chromatin remodeling enzyme, called the SWR1 complex, that efficiently replaces conventional histone H2A in nucleosomes with histone H2A.Z. Swr1 is required for the deposition of histone H2A.Z at stereotypical promoter locations in vivo, and Swr1 and H2A.Z commonly regulate a subset of yeast genes. Here, we describe an integrated nucleosome assembly-histone replacement system whereby histone exchange by chromatin remodeling activities may be analyzed in vitro. The system demonstrates ATP- and SWR1-complex-dependent replacement of histone H2A for histone H2A.Z on a preassembled nucleosome array. This system may also be adapted to analyze dynamic interactions between chromatin remodeling and modifying enzymes, histone chaperones, and nucleosome substrates containing canonical, variant, or covalently modified histones., (Copyright © 2012 Elsevier Inc. All rights reserved.)

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJ⋅mol(-1)) over the whole concentration range while it increased from 17 to 42kJ⋅mol(-1) with rising copper concentration and decreased from 39 to 23kJ⋅mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We investigated ATP-ase and peptide-binding activity of recombinant human heat shock protein HSP70(A1B), HSC70, and two hybrid proteins derived from those. The UV-spectral recorded data was used to characterize conformational rearrangements, which were induced by domain replacement or HSP70-peptide interaction. We have shown that N-terminal domain dramatically affect substrate specificity of C-terminal peptide-binding domain. This proposes new hypothesis for HSP70 chaperone machinery. The linear dependence between ATP-ase activity and peptide complex ratio was found. This relationship could be used for unlabeled peptide-HSP70 complex quantification.

Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. The proteins appear distantly related to MukBEF, have a similar operon organization and similar predicted secondary structures albeit with notably shorter coiled-coils. All three subunits of MksBEF exhibit significant sequence variation and can be divided into a series of overlapping subfamilies. MksBEF often coexists with the SMC-ScpAB, MukBEF and, sometimes, other MksBEFs. In Pseudomonas aeruginosa, both SMC and MksB contribute to faithful chromosome partitioning, with their inactivation leading to increased frequencies of anucleate cells. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Purified PaMksB showed activities typical for condensins including ATP-modulated DNA binding and condensation. Notably, DNA binding by MksB is negatively regulated by ATP, which sets it apart from other known SMC proteins. Thus, several specialized condensins might be involved in organization of bacterial chromosomes., (© 2011 Blackwell Publishing Ltd.)

Background: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation., Methodology/principal Findings: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein., Conclusion/significance: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.