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.

A novel peroxisome-related gene signature predicts clinical prognosis and is associated with immune microenvironment in low-grade glioma.

Low-grade glioma (LGG), a common primary tumor, mainly originates from astrocytes and oligodendrocytes. Increasing evidence has shown that peroxisomes function in the regulation of tumorigenesis and development of cancer. However, the prognostic value of peroxisome-related genes (PRGs) in LGG has not been reported. Therefore, it is necessary to construct a prognostic risk model for LGG patients based on the expression profiles of peroxisome-related genes. Our study mainly concentrated on developing a peroxisome-related gene signature for overall survival (OS) prediction in LGG patients. First, according to these peroxisome-related genes, all LGG patients from The Cancer Genome Atlas (TCGA) database could be divided into two subtypes. Univariate Cox regression analysis was used to find prognostic peroxisome-related genes in TCGA_LGG dataset, and least absolute shrinkage and selection operator Cox regression analysis was employed to establish a 14-gene signature. The risk score based on the signature was positively associated with unfavorable prognosis. Then, multivariate Cox regression incorporating additional clinical characteristics showed that the 14-gene signature was an independent predictor of LGG. Time-dependent ROC curves revealed good performance of this prognostic signature in LGG patients. The performance about predicting OS of LGG was validated using the GSE107850 dataset derived from the Gene Expression Omnibus (GEO) database. Furethermore, we constructed a nomogram model based on the gene signature and age, which showed a better prognostic power. Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analyses showed that neuroactive ligand-receptor interaction and phagosome were enriched and that the immune status was decreased in the high-risk group. Finally, cell counting kit-8 (CCK8) were used to detect cell proliferation of U251 and A172 cells. Inhibition of ATAD1 (ATPase family AAA domain-containing 1) and ACBD5 (Acyl-CoA binding-domain-containing-5) expression led to significant inhibition of U251 and A172 cell proliferation. Flow cytometry detection showed that ATAD1 and ACBD5 could induce apoptosis of U251 and A172 cells. Therefore, through bioinformatics methods and cell experiments, our study developed a new peroxisome-related gene signature that migh t help improve personalized OS prediction in LGG patients.

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.

Activation Loop Plasticity and Active Site Coupling in the MAP Kinase, ERK2.

Previous studies of the protein kinase, ERK2, using NMR and hydrogen-exchange measurements have shown changes in dynamics accompanying its activation by phosphorylation. However, knowledge about the conformational motions involved is incomplete. Here, we examined ERK2 using long conventional molecular dynamics (MD) simulations starting from crystal structures of phosphorylated (2P) and unphosphorylated (0P) forms. Individual trajectories were run for (5 to 25) µs, totaling 727 µs. The results show unexpected flexibility of the A-loop, with multiple long-lived (>5 µs) conformational states in both 2P- and 0P-ERK2. Differential contact network and principal component analyses reveal coupling between the A-loop fold and active site dynamics, with evidence for conformational selection in the kinase core of 2P-ERK2 but not 0P-ERK2. Simulations of 2P-ERK2 show A-loop states corresponding to restrained dynamics within the N-lobe, including regions around catalytic residues. One A-loop conformer forms lasting interactions with the L16 segment, leading to reduced RMSF and greater compaction in the active site. By contrast, simulations of 0P-ERK2 reveal excursions of A-loop residues away from the C-lobe, leading to greater active site mobility. Thus, the A-loop in ERK2 switches between distinct conformations that reflect coupling with the active site, possibly via the L16 segment. Crystal packing interactions suggest that lattice contacts with the A-loop may restrain its structural variation in X-ray structures of ERK2. The novel conformational states identified by MD expand our understanding of ERK2 regulation, by linking the activated state of the kinase to reduced dynamics and greater compaction surrounding the catalytic 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.

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family.

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.

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.

ATPase family AAA domain-containing protein 2 (ATAD2): From an epigenetic modulator to cancer therapeutic target.

ATPase family AAA domain-containing protein 2 (ATAD2) has been widely reported to be a new emerging oncogene that is closely associated with epigenetic modifications in human cancers. As a coactivator of transcription factors, ATAD2 can participate in epigenetic modifications and regulate the expression of downstream oncogenes or tumor suppressors, which may be supported by the enhancer of zeste homologue 2. Moreover, the dominant structure (AAA + ATPase and bromine domains) can make ATAD2 a potential therapeutic target in cancer, and some relevant small-molecule inhibitors, such as GSK8814 and AZ13824374, have also been discovered. Thus, in this review, we focus on summarizing the structural features and biological functions of ATAD2 from an epigenetic modulator to a cancer therapeutic target, and further discuss the existing small-molecule inhibitors targeting ATAD2 to improve potential cancer therapy. Together, these inspiring findings would shed new light on ATAD2 as a promising druggable target in cancer and provide a clue on the development of candidate anticancer drugs.

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+.

Common Patterns of Hydrolysis Initiation in P-loop Fold Nucleoside Triphosphatases.

The P-loop fold nucleoside triphosphate (NTP) hydrolases (also known as Walker NTPases) function as ATPases, GTPases, and ATP synthases, are often of medical importance, and represent one of the largest and evolutionarily oldest families of enzymes. There is still no consensus on their catalytic mechanism. To clarify this, we performed the first comparative structural analysis of more than 3100 structures of P-loop NTPases that contain bound substrate Mg-NTPs or their analogues. We proceeded on the assumption that structural features common to these P-loop NTPases may be essential for catalysis. Our results are presented in two articles. Here, in the first, we consider the structural elements that stimulate hydrolysis. Upon interaction of P-loop NTPases with their cognate activating partners (RNA/DNA/protein domains), specific stimulatory moieties, usually Arg or Lys residues, are inserted into the catalytic site and initiate the cleavage of gamma phosphate. By analyzing a plethora of structures, we found that the only shared feature was the mechanistic interaction of stimulators with the oxygen atoms of gamma-phosphate group, capable of causing its rotation. One of the oxygen atoms of gamma phosphate coordinates the cofactor Mg ion. The rotation must pull this oxygen atom away from the Mg ion. This rearrangement should affect the properties of the other Mg ligands and may initiate hydrolysis according to the mechanism elaborated in the second article.

Common Mechanism of Activated Catalysis in P-loop Fold Nucleoside Triphosphatases-United in Diversity.

To clarify the obscure hydrolysis mechanism of ubiquitous P-loop-fold nucleoside triphosphatases (Walker NTPases), we analysed the structures of 3136 catalytic sites with bound Mg-NTP complexes or their analogues. Our results are presented in two articles; here, in the second of them, we elucidated whether the Walker A and Walker B sequence motifs-common to all P-loop NTPases-could be directly involved in catalysis. We found that the hydrogen bonds (H-bonds) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr]WA) and aspartate of the Walker B motif (AspWB) are particularly short (even as short as 2.4 ångströms) in the structures with bound transition state (TS) analogues. Given that a short H-bond implies parity in the pKa values of the H-bond partners, we suggest that, in response to the interactions of a P-loop NTPase with its cognate activating partner, a proton relocates from [Ser/Thr]WA to AspWB. The resulting anionic [Ser/Thr]WA alkoxide withdraws a proton from the catalytic water molecule, and the nascent hydroxyl attacks the gamma phosphate of NTP. When the gamma-phosphate breaks away, the trapped proton at AspWB passes by the Grotthuss relay via [Ser/Thr]WA to beta-phosphate and compensates for its developing negative charge that is thought to be responsible for the activation barrier of hydrolysis.

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.

Caught in a Loop with Advance Care Planning and Advance Directives: How to Move Forward?

Completion of an advance care planning (ACP) process and/or an advance directive should result in patients receiving the care they desire at the end of life. However, three decades of research have shown that is just not the case. ACP has been a front runner in developing the science within palliative care. Some positive outcomes such as lowering levels of surrogate grief may be associated with ACP. Yet, it does not appear that further ACP research will ensure that seriously ill patients will get goal-concordant care. An unfortunate consequence of palliative care research and advocacy so far is the misguided notion of many hospital systems trying to solve their palliative care problems by only implementing an ACP initiative. At best, ACP is but one tool in the collective palliative care toolbox. New tools are needed. Given that we have finite resources, future research should focus more on tools to improve symptom management, better models of care, and systems that will ensure goal-concordant care that meet the needs of the population that the health care system is designed to meet.

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.

P-Loop mutations-Negative prognosticators in tyrosine kinase inhibitors resistant chronic myeloid leukemia patients.

INTRODUCTION: P-Loop mutations in CML patients prevent the conformational change in BCR-ABL1 necessary for drug binding. The present study aimed to evaluate the impact of mutations in this domain on the prognosis of the disease and also to associate the baseline Sokal relative risk score with the overall survival in non-responding CML patients. METHODS: Blood samples were analyzed using ARMS-PCR and then an association was assessed between presence/absence of mutations, hematological and molecular response, disease progression, overall survival, and Sokal score. RESULTS: Of the total 250 CML patients, 102 were found to be treatment-resistant. Fifty-three patients harbored P-Loop mutations with G250E (12.7%) being most frequent. Complete hematological response and major molecular response were achieved by only 27.7% and 5.7 patients, respectively. Worst survival (57.1%) was observed in Y253H positive patients while according to Sokal score in high-risk patients harboring Y253F (50%) and E255V (50%). CONCLUSION: The presence of P-Loop domain mutations negatively impacted the prognosis of the disease in terms of disease advancement and overall survival. So, the timely performance of the BCR-ABL1 mutational analysis and the modifications in the treatment plan based on the mutation identified would help in a better outcome of the disease.

In silico comparative analysis of KRAS mutations at codons 12 and 13: Structural modifications of P-Loop, switch I&II regions preventing GTP hydrolysis.

KRAS mutation is prevalent in around 30% of all cancers and is an undruggable molecular target. Of seven mutations at codon 12 and 13, only one, the G12C mutant is finally proven to be druggable, as evidenced by the recent USFDA approval of sotorasib. Investigation of other small molecules targeting G12C and G12D are undergoing clinical trials. Understanding the fine structural details is a prerequisite to design specific inhibitors which also requires in depth molecular exploration. We used bioinformatics as a tool to analyze the KRAS protein's GTP (guanosine triphosphate) binding dynamics when mutated. KRAS undergoes significant conformational changes, affecting GTP binding conformation within the active site pocket of KRAS due to high torsional strains, hydrophobicity, and altered Switch I and II regions. GTP molecule for wildtype had a low torsional strain of 10.71, and is the only molecule, in comparison to KRAS mutant bound GTP, to have a glycine at position 10 interacting with its nitrogenous base. All mutant KRAS proteins lacked the interaction of glycine with the nitrogenous base. The binding affinity of wildtype (WT) KRAS for the gamma-phosphate was lower in scoring compared to the mutated KRAS protein in multiple analyses. This study provides an insight to the GTP-KRAS protein binding details that are important to define parameters required to be explored to design the appropriate inhibitor for each different type of mutant KRAS protein.

Structure-Activity Studies of Novel Di-substituted [1,2,5]oxadiazolo [3,4-b]pyrazine Analogs Targeting the A-loop Regulatory Site of p38 MAP Kinase.

INTRODUCTION: In the quest for novel allosteric inhibitors of the p38 MAP kinase, we recently described the A-loop regulatory site, identified by means of molecular modeling studies together with the disclosure of a small molecule hit with a moderate inhibitory profile. Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, analysis of their structures permitted to conclude about the suitability of the [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[ 3,4-b]pyrazine) scaffold for the development of potent A-loop regulatory site p38 MAP kinase inhibitors. Accordingly, we report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1ß secretion in human monocyte-derived macrophages. OBJECTIVE: To find small molecule potent inhibitors of the p38 MAP kinase A-loop regulatory site. METHODS: Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, we carried out a hit-tolead optimization process guided by molecular modeling using a [1,2,5]oxadiazolo[3,4- b]pyrazine (furazano[3,4-b]pyrazine) scaffold. RESULTS: We report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1ß secretion in human monocyte-derived macrophages. CONCLUSION: We describe in the present work a series of [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine), which are potent inhibitors of IL-1ß secretion in human monocytederived macrophages allosteric modulators of the p38 MAP kinase A-loop regulatory site.

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.