Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents

Protein phosphatase 1c associated with the cardiac sodium calcium exchanger1 regulates its activity by dephosphorylating serine 68 phosphorylated phospholemman

The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na+/K+-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca2+ binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5–8Φ1Φ2-X8–9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation

An intact C-terminal end of albumin is required for its long half-life in humans.

Albumin has an average plasma half-life of three weeks and is thus an attractive carrier to improve the pharmacokinetics of fused therapeutics. The half-life is regulated by FcRn, a cellular receptor that protects against intracellular degradation. To tailor-design the therapeutic use of albumin, it is crucial to understand how structural alterations in albumin affect FcRn binding and transport properties. In the blood, the last C-terminal residue (L585) of albumin may be enzymatically cleaved. Here we demonstrate that removal of the L585 residue causes structural stabilization in regions of the principal FcRn binding domain and reduces receptor binding. In line with this, a short half-life of only 3.5 days was measured for cleaved albumin lacking L585 in a patient with acute pancreatitis. Thus, we reveal the structural requirement of an intact C-terminal end of albumin for a long plasma half-life, which has implications for design of albumin-based therapeutics

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1–4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability

Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats

3-methyladenine DNA glycosylases initiate repair of cytotoxic and promutagenic alkylated bases in DNA. We demonstrate by comparative modelling that Bacillus cereus AlkD belongs to a new, fifth, structural superfamily of DNA glycosylases with an alpha–alpha superhelix fold comprising six HEAT-like repeats. The structure reveals a wide, positively charged groove, including a putative base recognition pocket. This groove appears to be suitable for the accommodation of double-stranded DNA with a flipped-out alkylated base. Site-specific mutagenesis within the recognition pocket identified several residues essential for enzyme activity. The results suggest that the aromatic side chain of a tryptophan residue recognizes electron-deficient alkylated bases through stacking interactions, while an interacting aspartate–arginine pair is essential for removal of the damaged base. A structural model of AlkD bound to DNA with a flipped-out purine moiety gives insight into the catalytic machinery for this new class of DNA glycosylases

Structural features of a Bacteroidetes-affiliated cellulase linked with a polysaccharide utilization locus

Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A, and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family, and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft

Pseudoracemic amino acid complexes: blind predictions for flexible two-component crystals

Ab initio prediction of the crystal packing in complexes between two flexible molecules is a particularly challenging computational chemistry problem. In this work we present results of single crystal structure determinations as well as theoretical predictions for three 1 ? 1 complexes between hydrophobic L- and D-amino acids (pseudoracemates), known from previous crystallographic work to form structures with one of two alternative hydrogen bonding arrangements. These are accurately reproduced in the theoretical predictions together with a series of patterns that have never been observed experimentally. In this bewildering forest of potential polymorphs, hydrogen bonding arrangements and molecular conformations, the theoretical predictions succeeded, for all three complexes, in finding the correct hydrogen bonding pattern. For two of the complexes, the calculations also reproduce the exact space group and side chain orientations in the best ranked predicted structure. This includes one complex for which the observed crystal packing clearly contradicted previous experience based on experimental data for a substantial number of related amino acid complexes. The results highlight the significant recent advances that have been made in computational methods for crystal structure prediction.<br/

Improved One-Pot Synthesis of Mixed Methyl−Aryl Platinum(II) Diimine Complexes

A general one-pot synthetic route for mixed methyl−aryl Pt(II) diimine complexes is described. Performing the alkylation in neat Me_2S instead of ether or THF greatly reduces the amount of comproportionation products otherwise formed, diminishes separation problems, and improves yields. Treatment of the intermediate methyl−aryl complexes (Me_2S)_2Pt(Me)(Ar) with diimines (N−N) furnishes the methyl−aryl Pt(II) diimine complexes (N−N)Pt(Me)(Ar) in 76−84% yields. The Pt methyl−phenyl complex [p-Tol-N ═ VC(Me)C(Me) ═ N-p-Tol]Pt(Me)(Ph) has been characterized by X-ray diffraction

Single Transmembrane Peptide DinQ Modulates Membrane-Dependent Activities

The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membranedependent activities such as nucleoid compaction and recombination. Copyright: 2013 Weel-Sneve et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Synthesis and Evaluation of Fused Pyrimidines as E. coli Thymidylate Monophosphate Kinase Inhibitors

To front emergence of antibiotic resistance there is an urgent need for new therapeutics, and one seemingly relevant target is thymidylate monophosphate kinase (TMPK). Serendipitously, we discovered a naphthyl substituted pyrrolopyrimidine possessing activity towards E. coli TMPK. Based on this hit, synthesis, and screening of 61 fused pyrimidines were undertaken. The most potent derivatives were also counter assayed towards the human variant of the enzyme. Two of the inhibitors possessed promising drug-like properties and selectivity for E. coli TMPK. Although the initial pyrrolopyrimidine hit failed to have cellular activity, two alternative scaffolds were discovered providing starting points for further work