ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

Abstract The knowledge on the binding of vanadium ions and complexes to serum proteins and how vanadium might be transported in blood and up-taken by cells has received much attention during the last decade, particularly as far as the transport of VIVO2+ is concerned. In this work we revise and discuss some relevant aspects of previous research, namely the two main types of binding proposed for transport of VIVO(carrier)2 complexes. New results, obtained by circular dichroism (CD), \{EPR\} and gel electrophoresis, regarding the binding of vanadium to hTF in the oxidation states +5 and +3 are also presented. Namely, evidences for the binding of VV-species to diferric-transferrin, designated by (FeIII)2hTF, as well as to (AlIII)2hTF, are presented and discussed, the possibility of up-take of vanadate by cells through (FeIII)2hTF endocytosis being suggested. It is also confirmed that \{VIII\} binds strongly to hTF, forming di-vanadium(III)-transferrin, designated by (VIII)2hTF, and gel electrophoresis experiments indicate that (VIII)2hTF corresponds to a ‘closed conformation’ similar to (FeIII)2hTF.

Abstract The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding \{GH81\} from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of  82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. \{TLC\} and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from \{DP2\} to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.
Methods and results
By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.
Conclusions
SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.
General Significance
This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conserved putative heme-binding proteins that contains a number of members in animal, plant andbacterial species. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL, have been determined in 2006 and 2011 respectively. In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme bound murine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic in nature, was thought to also involve electrostatic interactions between heme propionate groups and positively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1 variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting in Kd values in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMR relaxation time measurements for human HEBP1 describe a rigid globular protein with no change in motional regime upon heme binding. X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the new heme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1 Å). Results from a HSQC spectrum centred on the histidine side chain Nδ-proton region for HEBP2 confirm that HEBP2 does not bind heme via H42 as no chemical shift differences were observed upon heme addition for backbone NH and Nδ protons. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years span a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals and a potential link between heme release under oxidative stress and human HEBP1 is also examined using recent data. However, at the present moment, trying to relate function to the involvement of heme or tetrapyrrole binding, specifically, makes little sense with our current biological knowledge and can only be applied to HEBP1, as HEBP2 does not interact with heme. We suggest that it may not be justified to call this very small family of proteins, heme-binding proteins. The family may be more correctly called “the SOUL family of proteins related to cellular fate” as, even though only HEBP1 binds heme tightly, both proteins may be involved in cell survival and/or proliferation.

Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe–4S]1+ clusters, and full reduction requires Ti(III)–citrate. The redox potentials of the four [4Fe–4S]1+ centers range between −250 and −530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe–4S]1+ clusters, and full reduction requires Ti(III)–citrate. The redox potentials of the four [4Fe–4S]1+ centers range between −250 and −530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species-specific \{AOX\} isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, \{AOX3\} and AOX4). The physiological function of mammalian \{AOX\} isoenzymes is unknown, although human \{AOX1\} is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as \{AOX\} substrates is increasing. The recent crystallization and structure determination of human \{AOX1\} as well as mouse \{AOX3\} has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes.

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we disclosed the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAc?1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Gal?1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions is observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards novel strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be fully understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the O2 sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a new species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This new form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyze the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate. These results reveal that oxidative inactivation does not require reduction of the metal, as widely assumed, as it can also occur in the oxidized state in the presence of CO2.Competing Interest StatementThe authors have declared no competing interest.AORAldehyde Oxido-reductaseDTTDithiothreitolDvDesulfovibrio vulgarisEPRElectron Paramagnetic ResonanceFdhFormate dehydrogenaseHPHigh PressureMGDMolybdopterin Guanine DinucleotidesNDNew dropROSReactive Oxygen SpeciesSODSuperoxide dismutaseTSAThermal Shift Assay

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment. This article is protected by copyright. All rights reserved.