Molecular basis for transposase activation by a dedicated AAA+ ATPase.

Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1-3. 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.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication.

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis.

Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1-4, based on the polymorphisms of 3' external transcribed sequences. The variants are known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, obtaining mosaic mutational events on ~ 5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop.

Rearrangement hot spot (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module, and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, TreTu, which is delivered by the type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor Tu (EF-Tu). This modification is specifically placed on the side chain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that the TriTu immunity protein neutralizes TreTu activity by acting like a lid that closes the catalytic site and traps the NAD+.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

The P-Site Loop of the Universally Conserved Bacterial Ribosomal Protein L5 Is Required for Maintaining Both Translation Rate and Fidelity.

The bacterial ribosomal 5S rRNA-binding protein L5 is universally conserved (uL5). It contains the so-called P-site loop (PSL), which contacts the P-site tRNA in the ribosome. Certain PSL mutations in yeast are lethal, suggesting that the loop plays an important role in translation. In this work, for the first time, a viable Escherichia coli strain was obtained with the deletion of the major part of the PSL (residues 73-80) of the uL5 protein. The deletion conferred cold sensitivity and drastically reduced the growth rate and overall protein synthesizing capacity of the mutant. Translation rate is decreased in mutant cells as compared to the control. At the same time, the deletion causes increased levels of -1 frameshifting and readthrough of all three stop codons. In general, the results show that the PSL of the uL5 is required for maintaining both the accuracy and rate of protein synthesis in vivo.

Impacts of Mutations in the P-Loop on Conformational Alterations of KRAS Investigated with Gaussian Accelerated Molecular Dynamics Simulations.

G12 mutations heavily affect conformational transformation and activity of KRAS. In this study, Gaussian accelerated molecular dynamics (GaMD) simulations were performed on the GDP-bound wild-type (WT), G12A, G12D, and G12R KRAS to probe mutation-mediated impacts on conformational alterations of KRAS. The results indicate that three G12 mutations obviously affect the structural flexibility and internal dynamics of the switch domains. The analyses of the free energy landscapes (FELs) suggest that three G12 mutations induce more conformational states of KRAS and lead to more disordered switch domains. The principal component analysis shows that three G12 mutations change concerted motions and dynamics behavior of the switch domains. The switch domains mostly overlap with the binding region of KRAS to its effectors. Thus, the high disorder states and concerted motion changes of the switch domains induced by G12 mutations affect the activity of KRAS. The analysis of interaction network of GDP with KRAS signifies that the instability in the interactions of GDP and magnesium ion with the switch domain SW1 drives the high disordered state of the switch domains. This work is expected to provide theoretical aids for understanding the function of KRAS.

Yme2, a putative RNA recognition motif and AAA+ domain containing protein, genetically interacts with the mitochondrial protein export machinery.

The mitochondrial respiratory chain is composed of nuclear as well as mitochondrial-encoded subunits. A variety of factors mediate co-translational integration of mtDNA-encoded proteins into the inner membrane. In Saccharomyces cerevisiae, Mdm38 and Mba1 are ribosome acceptors that recruit the mitochondrial ribosome to the inner membrane, where the insertase Oxa1, facilitates membrane integration of client proteins. The protein Yme2 has previously been shown to be localized in the inner mitochondrial membrane and has been implicated in mitochondrial protein biogenesis, but its mode of action remains unclear. Here, we show that multiple copies of Yme2 assemble into a high molecular weight complex. Using a combination of bioinformatics and mutational analyses, we find that Yme2 possesses an RNA recognition motif (RRM), which faces the mitochondrial matrix and a AAA+ domain that is located in the intermembrane space. We further show that YME2 genetically interacts with MDM38, MBA1 and OXA1, which links the function of Yme2 to the mitochondrial protein biogenesis machinery.

Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation.

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.

Intraoperative Diagnosis of a Stuck Bioprosthetic Valve Leaflet Due to a Loop of Suture After Mitral Valve Replacement.

A stuck mechanical valve leaflet is a well-known cardiovascular complication; however, a stuck bioprosthetic valve is a rare but potentially fatal complication. Herein a case of stuck bioprosthetic mitral valve caused by a loop of suture, which was detected on intraoperative 3-dimensional (3D) transesophageal echocardiography immediately after cardiopulmonary bypass, is presented. Restricted motion of the 2 leaflets during diastole and incomplete coaptation during systole were observed clearly on 3D imaging. Thus, intraoperative 3D transesophageal echocardiography imaging is useful for detecting such complications immediately after cardiopulmonary bypass.

Computational Analysis of the Crystal and Cryo-EM Structures of P-Loop Channels with Drugs.

The superfamily of P-loop channels includes various potassium channels, voltage-gated sodium and calcium channels, transient receptor potential channels, and ionotropic glutamate receptors. Despite huge structural and functional diversity of the channels, their pore-forming domain has a conserved folding. In the past two decades, scores of atomic-scale structures of P-loop channels with medically important drugs in the inner pore have been published. High structural diversity of these complexes complicates the comparative analysis of these structures. Here we 3D-aligned structures of drug-bound P-loop channels, compared their geometric characteristics, and analyzed the energetics of ligand-channel interactions. In the superimposed structures drugs occupy most of the sterically available space in the inner pore and subunit/repeat interfaces. Cationic groups of some drugs occupy vacant binding sites of permeant ions in the inner pore and selectivity-filter region. Various electroneutral drugs, lipids, and detergent molecules are seen in the interfaces between subunits/repeats. In many structures the drugs strongly interact with lipid and detergent molecules, but physiological relevance of such interactions is unclear. Some eukaryotic sodium and calcium channels have state-dependent or drug-induced π-bulges in the inner helices, which would be difficult to predict. The drug-induced π-bulges may represent a novel mechanism of gating modulation.

Moyamoya disease patient mutations in the RING domain of RNF213 reduce its ubiquitin ligase activity and enhance NFκB activation and apoptosis in an AAA+ domain-dependent manner.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries. Genetic studies originally identified RNF213 as an MMD susceptibility gene that encodes a large 591 kDa protein with a functional RING domain and dual AAA+ ATPase domains. As the functions of RNF213 and its relationship to MMD onset are unknown, we set out to characterize the ubiquitin ligase activity of RNF213, and the effects of MMD patient mutations on these activities and on other cellular processes. In vitro ubiquitination assays, using the RNF213 RING domain, identified Ubc13/Uev1A as a key ubiquitin conjugating enzyme that together generate K63-linked polyubiquitin chains. However, nearly all MMD patient mutations in the RING domain greatly reduced this activity. When full-length proteins were overexpressed in HEK293T cells, patient mutations that abolished the ubiquitin ligase activities conversely enhanced nuclear factor κB (NFκB) activation and induced apoptosis accompanied with Caspase-3 activation. These induced activities were dependent on the RNF213 AAA+ domain. Our results suggest that the NFκB- and apoptosis-inducing functions of RNF213 may be negatively regulated by its ubiquitin ligase activity and that disruption of this regulation could contribute towards MMD onset.

Fungal Kti12 proteins display unusual linker regions and unique ATPase p-loops.

Kti12 (Kluyveromyces lactis toxin insensitive 12) is an evolutionary highly conserved ATPase, crucial for the tRNA-modification activity of the eukaryotic Elongator complex. The protein consists of an N-terminal ATPase and a C-terminal tRNA-binding domain, which are connected by a flexible linker. The precise role of the linker region and its involvement in the communication between the two domains and their activities remain elusive. Here, we analyzed all available Kti12 protein sequences and report the discovery of a subset of Kti12 proteins with abnormally long linker regions. These Kti12 proteins are characterized by a co-occurring lysine to leucine substitution in their Walker A motif, previously thought to be invariable. We show that the K14L substitution lowers the affinity to ATP, but does not affect the catalytic activity of Kti12 at high ATP concentrations. We compare the activity of mutated variants of Kti12 in vitro with complementation assays in vivo in yeast. Ultimately, we compared Kti12 to other known p-loop ATPase family members known to carry a similar deviant Walker A motif. Our data establish Kti12 of Eurotiomycetes as an example of eukaryotic ATPase harboring a significantly deviating but still functional Walker A motif.

A Loop-Wiring Technique for Volarly Displaced Distal Radius Fractures With Small Thin Volar Marginal Fragments.

Volarly displaced distal radius fractures (VD-DRFs) with small thin volar marginal fragments (VMFs) are challenging to treat with conventional volar locking plates (VLPs) alone. Several alternative surgical options have been reported to treat these fractures, including wire-loop fixation, spring-wire fixation, hook plates, headless compression screws, and tension-band wiring, but the optimal method is yet to be determined. We have devised a surgical treatment for these fractures, entitled the "loop-wiring method" in which VMFs are anatomically reduced and 2 0.043-inch K-wires are drilled through the volar tip of the fragment and introduced into the dorsal cortex. Two bone tunnels are then made with a 0.039-inch K-wire, before a 0.028-inch soft wire is passed from the volar to the dorsal side and tightened over the dorsal cortex. After a fracture at the volar rim, even if properly reduced, compression stress is continuously generated on the volar cortical side of the fracture site and tension is generated on the articular side, which leads to VMF displacement, articular surface widening, and carpal subluxation. The loop-wiring technique applies compressive pressure securely to the articular side of the fracture site as well as to the whole fracture site and may be advantageous in terms of secure fixation of small thin volar marginal fragments, stability of the carpus, and bone healing.

[Detection methods for results of a loop-mediated isothermal amplification of DNA.]

The loop mediated isothermal amplification (LAMP) was developed by T. Notomi et al. in 2000. It has become one of the most promising methods for point-of-care diagnostics due to its accuracy, sensitivity and ease of execution. In this review, various methods for detecting the results of the LAMP reaction are considered; their advantages and disadvantages are revealed. Methods for detecting LAMP results can be divided into indirect and direct. Indirect methods aimed at detecting changes in the chemical composition of the reaction mixture include real-time turbidimetry, fluorescence detection with calcein, colorimetric detection with hydroxynaphthol blue, and detection using modified gold nanoparticles. Direct methods based on the detection of accumulation amplicons during the reaction include fluorimetric detection with intercalating dyes, resonance fluorescence energy transfer, enzyme immunoassay, immunochromatography, using cationic polymers and gold nanoparticles. The development in the field of point-of-care diagnostics is characterized by a pronounced tendency to miniaturization, the LAMP reaction on microchips and microfluidic devices with an electrochemical or optical detection method. The most promising for the diagnosis of infectious diseases are turbidimetry methods and the use of intercalating dyes. The development of portable domestic instruments for detecting of LAMP results based on real-time fluorescence detection or turbidimetry will contribute to the widespread introduction of the method into clinical laboratory diagnostic practice. A literature research was conducted in the Pubmed ncbi based on keywords.

The relationship between IFNL4 genotype and the rate of fibrosis in hepatitis C patients.

Introduction: IFNL4 rs12979860 genotype CC is associated with increased ALT activity and liver stiffness in hepatitis C virus (HCV) genotype (G) 3 infection but not in G1. The primary aim of this study is to assess an interaction between IFNL4 genotype, viral genotype and the stage of liver fibrosis. Secondary aims are to study the potential interactions between IFNL4 genotype, viral genotype and viral load as well as ALT levels. Methods: We performed a cross sectional study of patients with untreated chronic hepatitis C. Inflammation and liver fibrosis were scored using METAVIR. DNA was extracted from serum samples and the rs12979860 was genotyped using a custom made Taqman assay. Results: About 304 consecutive patients with chronic Hepatitis C were included. 52% had G1 infection and 48% had G3. Among patients with G3, advanced fibrosis or cirrhosis (F3F4) was present in 35% of the patients with IFNL4 CC and 28% with CT/TT (p = 0.24). Among patients with G1, F3F4 was present in 20% of the patients with IFNL4 CC and 19% with CT/TT (p = 0.52). IFNL4 CC was associated with higher mean value of normalized (n)ALT both in HCV G1 and G3 infection. Conclusions: IFNL4 genotype was not a predictor of advanced liver fibrosis in G3 or G1 infected patients. IFNL4 CC predicted a higher mean value of ALT among both G1 and G3 infected patients.

Development of a loop-mediated isothermal amplification assay targeting lmo0753 gene for detection of Listeria monocytogenes in wastewater.

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L.  monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L.  monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl-1 of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L.  monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L.  monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L.  monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L.  monocytogenes in wastewater from treatment plants. The LAMP assay detects L.  monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L.  monocytogenes detection.

Functional characterization of lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p).

Mutational analyses of axonemal dyneins are useful for elucidating the molecular mechanism of ciliary motility. This study demonstrates a mutation system for characterizing lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p). The viable DYH3-knockout (vKO-DYH3) cells isolated in this study enabled the examination of lethal mutations in P-loops 1 and 2. The P1 mutant dynein localized in the oral apparatus and the proximal region of the cilia, and the P2 mutant dynein localized only in the oral apparatus. Both results are different from the localization of wild-type Dyh3p. In addition, a co-precipitation assay showed that the P1 and P2 mutant dyneins did not dissociate from microtubules in ATP plus vanadate or in no-ATP conditions, in contrast to wild-type Dyh3p. This mutation system is useful for further molecular studies of axonemal dyneins and ciliary motility.