Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

A Direct Assay for Measuring the Activity and Inhibition of Coactivator-Associated Arginine Methyltransferase 1.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a member of the family of protein arginine methyltransferases. CARM1 catalyzes methyl group transfer from the cofactor S-adenosyl-l-methionine (AdoMet) to both histone and nonhistone protein substrates. CARM1 is involved in a range of cellular processes, mainly involving RNA transcription and gene regulation. As the aberrant expression of CARM1 has been linked to tumorigenesis, the enzyme is a potential therapeutic target, leading to the development of inhibitors and tool compounds engaging with CARM1. To evaluate the effects of these compounds on the activity of CARM1, sensitive and specific analytical methods are needed. While different methods are currently available to assess the activity of methyltransferases, these assays mainly focus on either the measurement of the cofactor product S-adenosyl-l-homocysteine (AdoHcy) or employ radioactive or expensive reagents, each with their own advantages and limitations. To complement the tools currently available for the analysis of CARM1 activity, we here describe the development of a convenient assay employing peptide substrates derived from poly(A)-binding protein 1 (PABP1). This operationally straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach allows for the direct detection of substrate methylation with minimal workup. The method was validated, and its value in characterizing CARM1 activity and inhibition was demonstrated through a comparative analysis involving a set of established small molecules and peptide-based CARM1 inhibitors.

Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin.

Thyroid hormones are essential regulators of metabolism, development, and growth. They are formed from pairs of iodinated tyrosine residues within the precursor thyroglobulin (TG), a 660-kDa homodimer of the thyroid gland, by an oxidative coupling reaction. Tyrosine pairs that give rise to thyroid hormones have been assigned within the structure of human TG, but the process of hormone formation is poorly understood. Here we report a ~3.3-Å cryo-EM structure of native bovine TG with nascent thyroid hormone formed at one of the predicted hormonogenic sites. Local structural rearrangements provide insight into mechanisms underlying thyroid hormone formation and stabilization.

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction-reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors.

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys18 in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg17 was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys18 does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.

Hijacking DNA methyltransferase transition state analogues to produce chemical scaffolds for PRMT inhibitors.

DNA, RNA and histone methylation is implicated in various human diseases such as cancer or viral infections, playing a major role in cell process regulation, especially in modulation of gene expression. Here we developed a convergent synthetic pathway starting from a protected bromomethylcytosine derivative to synthesize transition state analogues of the DNA methyltransferases. This approach led to seven 5-methylcytosine-adenosine compounds that were, surprisingly, inactive against hDNMT1, hDNMT3Acat, TRDMT1 and other RNA human and viral methyltransferases. Interestingly, compound 4 and its derivative 2 showed an inhibitory activity against PRMT4 in the micromolar range. Crystal structures showed that compound 4 binds to the PRMT4 active site, displacing strongly the S-adenosyl-l-methionine cofactor, occupying its binding site, and interacting with the arginine substrate site through the cytosine moiety that probes the space filled by a substrate peptide methylation intermediate. Furthermore, the binding of the compounds induces important structural switches. These findings open new routes for the conception of new potent PRMT4 inhibitors based on the 5-methylcytosine-adenosine scaffold.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Visualizing Bacterial Colony Morphologies Using Time-Lapse Imaging Chamber MOCHA.

Capturing microbial growth on a macroscopic scale is of great importance to further our understanding of microbial life. However, methods for imaging microbial life on a scale of millimeters to centimeters are often limited by designs that have poor environmental control, resulting in dehydration of the agar plate within just a few days. Here, we created MOCHA (microbial chamber), a simple but effective chamber that allows users to study microbial growth for extended periods (weeks) in a stable environment. Agar hydration is maintained with a double-decker design, in which two glass petri dishes are connected by a wick, allowing the lower plate to keep the upper plate hydrated. This flexible chamber allows the observation of a variety of microbiological phenomena, such as the growth and development of single bacterial and fungal colonies, interspecies interactions, swarming motility, and pellicle formation.IMPORTANCE Detailed study of microbial life on the colony scale of millimeters to centimeters has been lagging considerably behind microscopic inspection of microbes. One major reason for this is the lack of inexpensive instrumentation that can reproducibly capture images in a controlled environment. In this study, we present the design and use of a unique chamber that was used to produce several time-lapse movies that aimed to capture the diversity of microbial colony phenotypes over long periods.

Transition state mimics are valuable mechanistic probes for structural studies with the arginine methyltransferase CARM1.

Coactivator associated arginine methyltransferase 1 (CARM1) is a member of the protein arginine methyltransferase (PRMT) family and methylates a range of proteins in eukaryotic cells. Overexpression of CARM1 is implicated in a number of cancers, and it is therefore seen as a potential therapeutic target. Peptide sequences derived from the well-defined CARM1 substrate poly(A)-binding protein 1 (PABP1) were covalently linked to an adenosine moiety as in the AdoMet cofactor to generate transition state mimics. These constructs were found to be potent CARM1 inhibitors and also formed stable complexes with the enzyme. High-resolution crystal structures of CARM1 in complex with these compounds confirm a mode of binding that is indeed reflective of the transition state at the CARM1 active site. Given the transient nature of PRMT-substrate complexes, such transition state mimics represent valuable chemical tools for structural studies aimed at deciphering the regulation of arginine methylation mediated by the family of arginine methyltransferases.

Structural studies of protein arginine methyltransferase 2 reveal its interactions with potential substrates and inhibitors.

PRMT2 is the less-characterized member of the protein arginine methyltransferase family in terms of structure, activity, and cellular functions. PRMT2 is a modular protein containing a catalytic Ado-Met-binding domain and unique Src homology 3 domain that binds proteins with proline-rich motifs. PRMT2 is involved in a variety of cellular processes and has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. PRMT2 has been demonstrated to have weak methyltransferase activity on a histone H4 substrate, but its optimal substrates have not yet been identified. To obtain insights into the function and activity of PRMT2, we solve several crystal structures of PRMT2 from two homologs (zebrafish and mouse) in complex with either the methylation product S-adenosyl-L-homocysteine or other compounds including the first synthetic PRMT2 inhibitor (Cp1) studied so far. We reveal that the N-terminal-containing SH3 module is disordered in the full-length crystal structures, and highlights idiosyncratic features of the PRMT2 active site. We identify a new nonhistone protein substrate belonging to the serine-/arginine-rich protein family which interacts with PRMT2 and we characterize six methylation sites by mass spectrometry. To better understand structural basis for Cp1 binding, we also solve the structure of the complex PRMT4:Cp1. We compare the inhibitor-protein interactions occurring in the PRMT2 and PRMT4 complex crystal structures and show that this compound inhibits efficiently PRMT2. These results are a first step toward a better understanding of PRMT2 substrate recognition and may accelerate the development of structure-based drug design of PRMT2 inhibitors. DATABASE: All coordinates and structure factors have been deposited in the Protein Data Bank: zPRMT21-408 -SFG = 5g02; zPRMT273-408 -SAH = 5fub; mPRMT21-445 -SAH = 5ful; mPRMT21-445 -Cp1 = 5fwa, mCARM1130-487 -Cp1 = 5k8v.

Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase.

AIMS: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. RESULTS: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. INNOVATION AND CONCLUSIONS: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization.