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Acinetobacter baumannii ( A. baumannii ) challenges clinical infection treatment due to its resistance to various antibiotics. Multiple resistance genes in the core genome or mobile elements contribute to multidrug resistance in A. baumannii . Macrolide phosphotransferase gene mphE has been identified in A. baumannii , which is particularly relevant to macrolide antibiotics. Here, we determined the structure of MphE protein in three states: the apo state, the complex state with erythromycin and guanosine triphosphate (GTP), and the complex state with azithromycin and guanosine. Interestingly, GTP and two magnesium ions were observed in the erythromycin-bound MphE complex. This structure captured the active state of MphE, in which the magnesium ions stabilized the active site and assisted the transfer of phosphoryl groups. Based on these structures, we verified that the conserved residues Asp29, Asp194, His199, and Asp213 play an important role in the catalytic phosphorylation of MphE leading to drug resistance. Our work helps to understand the molecular basis of drug resistance and provides reference targets for optimizing macrolide antibiotics.

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N 4 -modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase ( Dm dNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase ( Bs dCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type Dm dNK or Bs dCK.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation., Competing Interests: AA, VD No competing interests declared, (© 2024, Andreotti and Dötsch.)

U47 phosphorylation (U p 47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Drug discovery pipelines nowadays rely on machine learning models to explore and evaluate large chemical spaces. While including 3D structural information is considered beneficial, structural models are hindered by the availability of protein-ligand complex structures. Exemplified for kinase drug discovery, we address this issue by generating kinase-ligand complex data using template docking for the kinase compound subset of available ChEMBL assay data. To evaluate the benefit of the created complex data, we use it to train a structure-based E (3)-invariant graph neural network. Our evaluation shows that binding affinities can be predicted with significantly higher precision by models that take synthetic binding poses into account compared to ligand- or drug-target interaction models alone.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes., (© 2024. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

The bacterial cell wall consists of a three-dimensional peptidoglycan layer, composed of peptides linked to the sugars N-acetylmuramic acid (MurNAc) and GlcNAc. Unlike other bacteria, the pathogenic Tannerella forsythia, a member of the red complex group of bacteria associated with the late stages of periodontitis, lacks biosynthetic pathways for MurNAc production and therefore obtains MurNAc from the environment. Sugar kinases play a crucial role in the MurNAc recycling process, activating the sugar molecules by phosphorylation. In this study, we present the first crystal structures of a MurNAc kinase, called murein sugar kinase (MurK), in its unbound state as well as in complexes with the ATP analog β-γ-methylene adenosine triphosphate (AMP-PCP) and with MurNAc. We also determined the crystal structures of K1058, a paralogous MurNAc kinase of T. forsythia, in its unbound state and in complex with MurNAc. We identified the active site and residues crucial for MurNAc specificity as the less bulky side chains of S133, P134, and L135, which enlarge the binding cavity for the lactyl ether group, unlike the glutamate or histidine residues present in structural homologs. In establishing the apparent kinetic parameters for both enzymes, we showed a comparable affinity for MurNAc (K m 180 μM and 30 μM for MurK and K1058, respectively), with MurK being over two hundred times faster than K1058 (V max 80 and 0.34 μmol min -1  mg -1 , respectively). These data might support a structure-guided approach to development of inhibitory MurNAc analogs for pathogen MurK enzymes., Competing Interests: Conflicts of interest The authors declare that they no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Boronic acids are widely used for labeling catechols and carbohydrates in analytical (bio)chemistry due to their high binding affinities for diols. Here, we present two asymmetrically substituted Bodipy dyes with a boronic acid at the β-position (BBB). We present a green-emitting BBB, gBBB, and, by expanding the conjugated system of the Bodipy core at α-position, a red-emitting rBBB. Especially, gBBB shows a bathochromic shift of the electronic spectra upon binding to saccharides and polyols, whereas the fluorescence lifetime of rBBB is more sensitive to hydroxy-ligand binding. Moreover, gBBB constantly shows higher binding affinities than rBBB, reaching K b ≈ 10 3 M -1 at pH 8.5 for fructose. Finally, time-resolved fluorescence anisotropy allows to distinguish the number of saccharide units of oligosaccharides as the bond along the transition dipole moment ensures that the fluorescence anisotropy only decays due to the rotational diffusion of labeled carbohydrates. β-substituted BODIPY dyes are, thus, foreseen as fluorescence anisotropy labels for macromolecule sizing, where conventional fluorophores fail to discriminate due to the chemical similarity of recognition sites.

Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co-substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light-energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades., (© 2021 Wiley-VCH GmbH.)

The structures of intrinsically disordered proteins (IDPs) are highly dynamic. It is hard to characterize the structures of these proteins experimentally. Molecular dynamics (MD) simulation is a powerful tool in the understanding of protein dynamic structures and function. This chapter describes the application of metadynamics-based enhanced sampling methods in the study of phosphorylation regulation on the structure of kinase-inducible domains (KID). The structural properties of free pKID and KID were obtained by parallel tempering metadynamics combined with well-tempered ensemble (PTMetaD WTE) method, and the binding free energy surfaces of pKID/KID and KIX were characterized by bias-exchanged metadynamics (BE-MetaD) simulations., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The BarA/UvrY two-component signal transduction system is widely conserved in γ-proteobacteria and provides a link between the metabolic state of the cells and the Csr posttranscriptional regulatory system. In Escherichia coli, the BarA/UvrY system responds to the presence of acetate and other short-chain carboxylic acids by activating transcription of the noncoding RNAs, CsrB and CsrC, which sequester the RNA-binding protein CsrA, a global regulator of gene expression. However, the state of the carboxyl group in the acetate molecule, which serves as the BarA stimulus, and the signal reception site of BarA remain unknown. In this study, we show that the deletion or replacement of the periplasmic domain of BarA and also the substitution of certain hydroxylated and hydrophobic amino acid residues in this region, result in a sensor kinase that remains unresponsive to its physiological stimulus, demonstrating that the periplasmic region of BarA constitutes a functional detector domain. Moreover, we provide evidence that the protonated state of acetate or formate serves as the physiological stimulus of BarA. In addition, modeling of the BarA sensor domain and prediction of the signal-binding site, by blind molecular docking, revealed a calcium channels and chemotaxis receptors domain with a conserved binding pocket, which comprised uncharged polar and hydrophobic amino acid residues. Based on the comparative sequence and phylogenetic analyses, we propose that, at least, two types of BarA orthologues diverged and evolved separately to acquire distinct signal-binding properties, illustrating the wide adaptability of the bacterial sensor kinase proteins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The regulation of lipid kinases has remained elusive given the difficulties of assessing changes in lipid levels. Here, we describe the isolation of protein and lipid kinases to determine the regulation of lipid kinases in vitro . This can be followed by analysis of effects of regulators on lipid kinase-mediated changes in phospholipids without the use of radioactivity, with a specific focus on PI(5)P generation by the enzyme PIKfyve. For complete details on the use and execution of this protocol, please refer to Karabiyik et al. (2021)., Competing Interests: D.C.R. is a consultant for D.C.R. is a consultant for Alladdin Healthcare Technologies Ltd., Mindrank AI, Nido Biosciences, Drishti Discoveries, and PAQ Therapeutics., (© 2021 The Author(s).)

Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.

The degradation of cholesterol and related steroids by microbes follows fundamentally different strategies in aerobic and anaerobic environments. In anaerobic bacteria, the primary C26 of the isoprenoid side chain is hydroxylated without oxygen via a three-step cascade: (i) water-dependent hydroxylation at the tertiary C25, (ii) ATP-dependent dehydration to form a subterminal alkene, and (iii) water-dependent hydroxylation at the primary C26 to form an allylic alcohol. However, the enzymes involved in the ATP-dependent dehydration have remained unknown. Here, we isolated an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from the anaerobic bacterium Sterolibacterium denitrificans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OH-vitamin D 3 . Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of γ-( 18 O) 2 -ATP from ADP and the C25-( 18 O) 2 -phosphoester. The observed full reversibility of 25-HSK with an equilibrium constant below one can be rationalized by an unusual high phosphoryl transfer potential of tertiary steroid C25-phosphoesters, which is ≈20 kJ mol -1 higher than that of standard sugar phosphoesters and even slightly greater than the β,γ-phosphoanhydride of ATP. In summary, 25-HSK plays an essential role in anaerobic bacterial degradation of zoo- and phytosterols and shows only little similarity to known phosphotransferases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2'-PO4 to NAD+ yielding RNA 2'-OH and ADP-ribose-1',2'-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states. ITC measurements of RslTpt1 binding to NAD+ (KD ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD+-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP+, which contains an adenylate 2'-PO4 (mimicking the substrate RNA 2'-PO4), binds with lower affinity (KD ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD+ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD+ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3'-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5' tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Phosphorylation of RNA polymerase II (RNAP II) coordinates the temporal progression of eukaryotic transcription. The development and application of chemical genetic methods have enhanced our ability to investigate the intricate and intertwined pathways regulated by the kinases and phosphatases targeting RNAP II to ensure transcription accuracy and efficiency. Although identifying small molecules that modulate these enzymes has been challenging due to their highly conserved structures, powerful new chemical biology strategies such as targeted covalent inhibitors and small molecule degraders have significantly improved chemical probe specificity. The recent success in discovering phosphatase holoenzyme activators and inhibitors, which demonstrates the feasibility of selective targeting of individual phosphatase complexes, opens up new avenues into the study of transcription. Herein, we summarize how chemical biology is used to delineate kinases' identities involved in RNAP II regulation and new concepts in inhibitor/activator design implemented for kinases/phosphatases involved in modulating RNAP II-mediated transcription., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Light-induced stomatal opening stimulates CO2 uptake and transpiration in plants. Weak blue light under strong red light effectively induces stomatal opening. Blue light-dependent stomatal opening initiates light perception by phototropins, and the signal is transmitted to a plasma membrane H+-ATPase in guard cells via BLUE LIGHT SIGNALING 1 (BLUS1) kinase. However, it is unclear how BLUS1 transmits the signal to H+-ATPase. Here, we characterized BLUS1 signaling in Arabidopsis thaliana, and showed that the BLUS1 C-terminus acts as an auto-inhibitory domain and that phototropin-mediated Ser-348 phosphorylation within the domain removes auto-inhibition. C-Terminal truncation and phospho-mimic Ser-348 mutation caused H+-ATPase activation in the dark, but did not elicit stomatal opening. Unexpectedly, the plants exhibited stomatal opening under strong red light and stomatal closure under weak blue light. A decrease in intercellular CO2 concentration via red light-driven photosynthesis together with H+-ATPase activation caused stomatal opening. Furthermore, phototropins caused H+-ATPase dephosphorylation in guard cells expressing constitutive signaling variants of BLUS1 in response to blue light, possibly for fine-tuning stomatal opening. Overall, our findings provide mechanistic insights into the blue light regulation of stomatal opening., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) allows bacteria to use various carbohydrates as energy resources including mannitol. The mannitol-specific PTS transporter in Vibrio cholerae is encoded by the mtlADR operon. Expression of the mtl operon has been shown to be strictly regulated by CRP, MtlS, and MtlR. In the present study, we investigated the regulation of mtlADR by the ferric uptake regulator (Fur). The results showed that Fur binds to the promoter-proximal DNA region of mtlADR to repress its transcription independent of iron, in mannitol-containing growth medium. The capacity for mannitol fermentation was significantly increased in Δfur relative to that of WT for normal and iron-replete growth media. The level of organic acids produced by Δfur was significantly enhanced relative to that produced by the WT strain in the normal and iron-replete media but not in an iron-starved medium. The results provided for a deeper understanding of the regulation of mtlADR in V. cholerae., Competing Interests: Declaration of competing interest We declare that we have no conflicts of interest to this work., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Genome-wide protein-protein interaction (PPI) determination remains a significant unsolved problem in structural biology. The difficulty is twofold since high-throughput experiments (HTEs) have often a relatively high false-positive rate in assigning PPIs, and PPI quaternary structures are more difficult to solve than tertiary structures using traditional structural biology techniques. We proposed a uniform pipeline, Threpp, to address both problems. Starting from a pair of monomer sequences, Threpp first threads both sequences through a complex structure library, where the alignment score is combined with HTE data using a naïve Bayesian classifier model to predict the likelihood of two chains to interact with each other. Next, quaternary complex structures of the identified PPIs are constructed by reassembling monomeric alignments with dimeric threading frameworks through interface-specific structural alignments. The pipeline was applied to the Escherichia coli genome and created 35,125 confident PPIs which is 4.5-fold higher than HTE alone. Graphic analyses of the PPI networks show a scale-free cluster size distribution, consistent with previous studies, which was found critical to the robustness of genome evolution and the centrality of functionally important proteins that are essential to E. coli survival. Furthermore, complex structure models were constructed for all predicted E. coli PPIs based on the quaternary threading alignments, where 6771 of them were found to have a high confidence score that corresponds to the correct fold of the complexes with a TM-score >0.5, and 39 showed a close consistency with the later released experimental structures with an average TM-score = 0.73. These results demonstrated the significant usefulness of threading-based homologous modeling in both genome-wide PPI network detection and complex structural construction., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Enzymes with hydroxymethylpyrimidine/phosphomethylpyrimidine kinase activity (HMPPK) are essential in the vitamin B1 (thiamine pyrophosphate) biosynthesis and recycling pathways. In contrast, enzymes with pyridoxal kinase activity (PLK) produce pyridoxal phosphate (vitamin B6), an essential cofactor for various biochemical reactions. In the ATP-dependent vitamin kinases family, the members of PLK/HMPPK-like subfamily have both enzymatic activities. It has been proposed that the promiscuous PLK activity of ancestral HMPPK enzymes could have been the starting point for this activity. In earlier work, we reconstructed the ancestral sequences of this family and characterized the substrate specificity of the common ancestor between PLK/HMPPK-like and HMPPK enzymes (AncC). From these studies, the Gln45Met mutation was proposed as a critical event for the PLK activity emergence. Here, we crystallize and determine the AncC structure by X-ray crystallography and assess the role of the Gln45Met mutation by site-directed mutagenesis. Kinetic characterization of this mutant shows a significant increase in the PL affinity. Through molecular dynamics simulation and MM/PBSA calculations some residues, important for substrate interactions and catalysis, were identified in the wild type and in the mutated ancestor. Interestingly, a strong epistatic interaction responsible for the evolutionary pathway of the PLK activity in PLK/HMPPK-like enzymes was revealed. Also, other putative mutations relevant to PLK activity in modern PLK/HMPPK-like enzymes were identified., (© 2021 The Protein Society.)

Mutations in kinases are abundant and critical to study signaling pathways and regulatory roles in human disease, especially in cancer. Somatic mutations in kinase genes can affect drug treatment, both sensitivity and resistance, to clinically used kinase inhibitors. Here, we present a newly constructed database, KinaseMD (kinase mutations and drug response), to structurally and functionally annotate kinase mutations. KinaseMD integrates 679 374 somatic mutations, 251 522 network-rewiring events, and 390 460 drug response records curated from various sources for 547 kinases. We uniquely annotate the mutations and kinase inhibitor response in four types of protein substructures (gatekeeper, A-loop, G-loop and αC-helix) that are linked to kinase inhibitor resistance in literature. In addition, we annotate functional mutations that may rewire kinase regulatory network and report four phosphorylation signals (gain, loss, up-regulation and down-regulation). Overall, KinaseMD provides the most updated information on mutations, unique annotations of drug response especially drug resistance and functional sites of kinases. KinaseMD is accessible at https://bioinfo.uth.edu/kmd/, having functions for searching, browsing and downloading data. To our knowledge, there has been no systematic annotation of these structural mutations linking to kinase inhibitor response. In summary, KinaseMD is a centralized database for kinase mutations and drug response., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington's disease (HD). In this paper, we describe a new and simple methodology for producing milligram quantities of highly pure wild-type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK), and 2) the development of an efficient methodology for producing recombinant native Httex1 proteins by using a SUMO-fusion expression and purification strategy. [26] As a proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site-specifically phosphorylated and fluorescently or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 or HTT structure, aggregation, interactome, and function(s) in health and disease., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Kinases and phosphatases are major players in a variety of cellular events, including cell signaling. Aberrant activity or mutations in kinases and phosphatases can lead to diseases such as cancer, diabetes, and Alzheimer's. Compared to kinases, phosphatases are understudied; this is partly a result of the limited methods for identifying substrates. As a solution, we developed a proteomics-based method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) that previously identified substrates of Ser/Thr phosphatases using small molecule inhibitors. Here, for the first time, K-BIPS was applied to identify substrates of a tyrosine phosphatase, protein tyrosine phosphatase 1B (PTP1B), under siRNA knockdown conditions. Eight possible substrates of PTP1B were discovered in HEK293 cells, including the known substrate pyruvate kinase. In addition, l-lactate dehydrogenase (LDHA) was validated as a novel PTP1B substrate. With the ability to use knockdown conditions with Ser/Thr or Tyr phosphatases, K-BIPS represents a general discovery tool to explore phosphatases biology by identifying unanticipated substrates., (© 2020 Wiley-VCH GmbH.)

The concept of liquid-liquid phase separation (LLPS) has emerged as an intriguing mechanism for the organization of membraneless compartments in cells. The alcohol 1,6-hexanediol is widely used as a control to dissolve LLPS assemblies in phase separation studies in diverse fields. However, little is known about potential side effects of 1,6-hexanediol, which could compromise data interpretation and mislead the scientific debate. To examine this issue, we analyzed the effect of 1,6-hexanediol on the activities of various enzymes in vitro. Already at 1% volume concentration, 1,6-hexanediol strongly impaired kinases and phosphatases and partly blocked DNA polymerases, while it had no effect on DNase activity. At concentrations that are usually used to dissolve LLPS droplets (5-10%), both kinases and phosphatases were virtually inactive. Given the widespread function of protein phosphorylation in cells, our data argue for a careful review of 1,6-hexanediol in phase separation studies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Malaria control relies heavily on the small number of existing antimalarial drugs. However, recurring antimalarial drug resistance necessitates the continual generation of new antimalarial drugs with novel modes of action. In order to shift the focus from only controlling this disease towards elimination and eradication, next-generation antimalarial agents need to address the gaps in the malaria drug arsenal. This includes developing drugs for chemoprotection, treating severe malaria and blocking transmission. Plasmodial kinases are promising targets for next-generation antimalarial drug development as they mediate critical cellular processes and some are active across multiple stages of the parasite's life cycle. This review gives an update on the progress made thus far with regards to plasmodial kinase small-molecule inhibitor development.

Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Protein phosphorylation is one of the most important post-translational modifications, and many biological processes are related to phosphorylation, such as DNA repair, transcriptional regulation and signal transduction and, therefore, abnormal regulation of phosphorylation usually causes diseases. If we can accurately predict human phosphorylation sites, this could help to solve human diseases. Therefore, we developed a kinase-specific phosphorylation prediction system, GasPhos, and proposed a new feature selection approach, called Gas, based on the ant colony system and a genetic algorithm and used performance evaluation strategies focused on different kinases to choose the best learning model. Gas uses the mean decrease Gini index (MDGI) as a heuristic value for path selection and adopts binary transformation strategies and new state transition rules. GasPhos can predict phosphorylation sites for six kinases and showed better performance than other phosphorylation prediction tools. The disease-related phosphorylated proteins that were predicted with GasPhos are also discussed. Finally, Gas can be applied to other issues that require feature selection, which could help to improve prediction performance. GasPhos is available at http://predictor.nchu.edu.tw/GasPhos.

Streptococcus pneumoniae is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. S. pneumoniae lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of S. pneumoniae mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against Streptococcus pneumoniae .

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs., Competing Interests: Declaration of Interests The authors declare no competing financial interests. Prozomix is a commercial enzyme producer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment., Competing Interests: Declaration of Interests M.A. is an employee of AstraZeneca and has stock ownership and/or stock options or interests in the company., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The selenocysteine (Sec) incorporation is a co-translational event taking place at an in-frame UGA-codon and dependent on an organized molecular machinery. Selenium delivery requires mainly two enzymes, the selenocysteine lyase (CsdB) is essential for Sec recycling and conversion to selenide, further used by the selenophosphate synthetase (SelD), responsible for the conversion of selenide in selenophosphate. Therefore, understanding the catalytic mechanism involved in selenium compounds delivery, such as the interaction between SelD and CsdB (EcCsdB.EcSelD), is fundamental for the further comprehension of the selenocysteine synthesis pathway and its control. In Escherichia coli, EcCsdB.EcSelD interaction must occur to prevent cell death from the release of the toxic intermediate selenide. Here, we demonstrate and characterize the in vitro EcSelD.EcCsdB interaction by biophysical methods. The EcSelD.EcCsdB interaction occurs with a stoichiometry of 1:1 in presence of selenocysteine and at a low-nanomolar affinity (~1.8 nM). The data is in agreement with the small angle X-ray scattering model fitted using available structures. Moreover, yeast-2-hybrid assays supported the macromolecular interaction in the cellular environment. This is the first report that demonstrates the interaction between EcCsdB and EcSelD supporting the hypothesis that EcSelD.EcCsdB interaction is necessary to sequester the selenide during the selenocysteine incorporation pathway in Bacteria., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fluorinated carbohydrates, where one (or more) fluorine atom(s) have been introduced into a carbohydrate structure, typically through deoxyfluorination chemistry, have a wide range of applications in the glycosciences. Fluorinated derivatives of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, talose, fucose and sialic acid have been employed as either donor or acceptor substrates in glycosylation reactions. Fluorinated donors can be synthesised by synthetic methods or produced enzymatically from chemically fluorinated sugars. The latter process is mediated by enzymes such as kinases, phosphorylases and nucleotidyltransferases. Fluorinated donors produced by either method can subsequently be used in glycosylation reactions mediated by glycosyltransferases, or phosphorylases yielding fluorinated oligosaccharide or glycoconjugate products. Fluorinated acceptor substrates are typically synthesised chemically. Glycosyltransferases are most commonly used in conjunction with natural donors to further elaborate fluorinated acceptor substrates. Glycoside hydrolases are used with either fluorinated donors or acceptors. The activity of enzymes towards fluorinated sugars is often lower than towards the natural sugar substrates irrespective of donor or acceptor. This may be in part attributed to elimination of the contribution of the hydroxyl group to the binding of the substrate to enzymes. However, in many cases, enzymes still maintain a significant activity, and reactions may be optimised where necessary, enabling enzymes to be used more successfully in the production of fluorinated carbohydrates. This review describes the current state of the art regarding chemoenzymatic production of fluorinated carbohydrates, focusing specifically on examples of the enzymatic production of activated fluorinated donors and enzymatic glycosylation involving fluorinated sugars as either glycosyl donors or acceptors.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested., (© 2019 Wiley Periodicals, Inc.)

Dynamical features of cell signaling are the essence of living organisms. To understand animal development, it is fundamental to investigate signaling dynamics in vivo. Robust reporters are required to visualize spatial and temporal dynamics of enzyme activities and protein-protein interactions involved in signaling pathways. In this review, we summarize recent development in the design of new classes of fluorescent reporters for imaging dynamic activities of proteases, kinases, and protein-protein interactions. These reporters operate on new physical and/or chemical principles; achieve large dynamic range, high brightness, and fast kinetics; and reveal spatiotemporal dynamics of signaling that is correlated with developmental events such as embryonic morphogenesis in live animals including Drosophila and zebrafish. Therefore, many of these reporters are great tools for biological discovery and mechanistic understanding of animal development and disease progression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Genetically encoded fluorescent protein-based kinase biosensors are a central tool for illumination of the kinome. The adaptability and versatility of biosensors have allowed for spatiotemporal observation of real-time kinase activity in living cells and organisms. In this review, we highlight various types of kinase biosensors, along with their burgeoning applications in complex biological systems. Specifically, we focus on kinase activity reporters used in neuronal systems and whole animal settings. Genetically encoded kinase biosensors are key for elucidation of the spatiotemporal regulation of protein kinases, with broader applications beyond the Petri dish., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily., Competing Interests: The authors declare no competing interest.

The ratio between carbon (C) and nitrogen (N) utilization must be precisely coordinated to enable plant growth. Although numerous physiological studies have examined carbon/nitrogen (C/N) ratios, the mechanisms of sensing the C/N balance and C/N signaling remain elusive. Here, we report that a mutation of FERONIA (FER), a receptor kinase that plays versatile roles in plant cell growth and stress responses, caused hypersensitivity to a high C/N ratio in Arabidopsis. In contrast, FER-overexpressing plants displayed more resistant phenotypes. FER can interact with and phosphorylate ATL6, an E3 ubiquitin ligase that has been shown to regulate plant C/N responses. FER-mediated ATL6 phosphorylation enhanced the interaction between ATL6 and its previously identified target 14-3-3 proteins, thus decreasing 14-3-3 protein levels, leading to an increased insensitivity to high C/N ratios. Further analyses showed that the rapid alkalinization factor peptide (RALF1), which is a ligand of FER, also influenced the stability of 14-3-3 proteins via a FER-ATL6-mediated pathway. These findings reveal a novel regulatory mechanism that links the RALF1/FER-ATL6 pathway to whole-plant C/N responses and growth., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS activity for import of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides., Competing Interests: The authors have declared that no competing interests exist.

Selenophosphate synthetase 2 (SEPHS2) synthesizes selenide and ATP into selenophosphate, the selenium donor for selenocysteine (Sec), which is cotranslationally incorporated into selenoproteins. The action and regulatory mechanisms of SEPHS2 as well as its role in carcinogenesis (especially breast cancer) remain ambiguous and need further clarification. Therefore, lacking an experimentally determined structure for SEPHS2, we first analyzed the physicochemical properties of its sequence, modeled its three-dimensional structure and studied its conformational behavior to identify the key residues (named HUB nodes) responsible for protein stability and to clarify the molecular mechanisms by which it induced its function. Bioinformatics analysis evidenced higher amplification frequencies of SEPHS2 in breast cancer than in other cancer types. Therefore, because triple negative breast cancer (TNBC) is biologically the most aggressive breast cancer subtype and its treatment represents a challenge due to the absence of well-defined molecular targets, we evaluated SEPHS2 expression in two TNBC cell lines and patient samples. We demonstrated mRNA and protein overexpression to be correlated with aggressiveness and malignant tumor grade, suggesting that this protein could potentially be considered a prognostic marker and/or therapeutic target for TNBC.

We have developed an enhanced conformational sampling method combining replica-exchange umbrella sampling (REUS) with Gaussian accelerated molecular dynamics (GaMD). REUS enhances the sampling along predefined reaction coordinates, while GaMD accelerates the conformational dynamics by adding a boost potential to the system energy. The method, which we call GaREUS (Gaussian accelerated replica-exchange umbrella sampling), enhances the sampling more efficiently than REUS or GaMD, while the computational resource for GaREUS is the same as that required for REUS. The two-step reweighting procedure using the multistate Bennett acceptance ratio method and the cumulant expansion for the exponential average is applied to the simulation trajectories for obtaining the unbiased free-energy landscapes. We apply GaREUS to the calculations of free-energy landscapes for three different cases: conformational equilibria of N -glycan, folding of chignolin, and conformational change of adenyl kinase. We show that GaREUS speeds up the convergences of free-energy calculations using the same amount of computational resources as REUS. The free-energy landscapes reweighted from the trajectories of GaREUS agree with previously reported ones. GaREUS is applicable to free-energy calculations of various biomolecular dynamics and functions with reasonable computational costs.

Reversible covalency, achieved with, for instance, highly electron-deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass-spectrometry-based platform that integrates gel filtration with activity-based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small-molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical-probe discovery., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polymyxins are considered to be the last-line antibiotics that are used to treat infections caused by multidrug-resistant (MDR) gram-negative bacteria; however, the plasmid-mediated transferable colistin resistance gene ( mcr-1 ) has rendered polymyxins ineffective. Therefore, the protein encoded by mcr-1 , MCR-1, could be a target for structure-based design of inhibitors to tackle polymyxins resistance. Here, we identified racemic compound 3 as a potential MCR-1 inhibitor by virtual screening, and 26 compound 3 derivatives were synthesized and evaluated in vitro. In the cell-based assay, compound 6g , 6h , 6i , 6n , 6p , 6q , and 6r displayed more potent activity than compound 3 . Notably, 25 μΜ of compound 6p or 6q combined with 2 μg·mL-1 colistin could completely inhibit the growth of BL21(DE3) expressing mcr-1 , which exhibited the most potent activity. In the enzymatic assay, we elucidate that 6p and 6q could target the MCR-1 to inhibit the activity of the protein. Additionally, a molecular docking study showed that 6p and 6q could interact with Glu246 and Thr285 via hydrogen bonds and occupy well the cavity of the MCR-1 protein. These results may provide a potential avenue to overcome colistin resistance, and provide some valuable information for further investigation on MCR-1 inhibitors.

The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 μM and 102 μM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs., Competing Interests: The authors have declared that no competing interests exist.

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS sugar ) and one for nitrogen regulation (PTS Ntr ), that utilize proteins enzyme I sugar (EI sugar ) and HPr, and enzyme I Ntr (EI Ntr ) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein-protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI Ntr :NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI Ntr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI Ntr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI Ntr . The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes., (Published by Elsevier Ltd.)

Class labels are required for supervised learning but may be corrupted or missing in various applications. In binary classification, for example, when only a subset of positive instances is labeled whereas the remaining are unlabeled, positive-unlabeled (PU) learning is required to model from both positive and unlabeled data. Similarly, when class labels are corrupted by mislabeled instances, methods are needed for learning in the presence of class label noise (LN). Here we propose adaptive sampling (AdaSampling), a framework for both PU learning and learning with class LN. By iteratively estimating the class mislabeling probability with an adaptive sampling procedure, the proposed method progressively reduces the risk of selecting mislabeled instances for model training and subsequently constructs highly generalizable models even when a large proportion of mislabeled instances is present in the data. We demonstrate the utilities of proposed methods using simulation and benchmark data, and compare them to alternative approaches that are commonly used for PU learning and/or learning with LN. We then introduce two novel bioinformatics applications where AdaSampling is used to: 1) identify kinase-substrates from mass spectrometry-based phosphoproteomics data and 2) predict transcription factor target genes by integrating various next-generation sequencing data.