Small-angle x-ray scattering investigation of the integration of free fatty acids in polysorbate 20 micelles.

A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 µg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 µg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.

Mechanism of activation and regulation of deubiquitinase activity in MINDY1 and MINDY2.

Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.

Structural Modeling Using Solution Small-Angle X-ray Scattering (SAXS).

Small-angle X-ray scattering (SAXS) offers a way to examine the overall shape and oligomerization state of biological macromolecules under quasi native conditions in solution. In the past decades, SAXS has become a standard tool for structure biologists due to the availability of high brilliance X-ray sources and the development of data analysis/interpretation methods. Sample handling robots and software pipelines have significantly reduced the time necessary to conduct SAXS experiments. Presently, most synchrotrons feature beamlines dedicated to biological SAXS, and the SAXS-derived models are deposited into dedicated and accessible databases. The size of macromolecules that may be analyzed ranges from small peptides or snippets of nucleic acids to gigadalton large complexes or even entire viruses. Compared to other structural methods, sample preparation is straightforward, and the risk of inducing preparation artefacts is minimal. Very importantly, SAXS is a method of choice to study flexible systems like unfolded or disordered proteins, providing the structural ensembles compatible with the data. Although it may be utilized stand-alone, SAXS profits a lot from available experimental or predicted high-resolution data and information from complementary biophysical methods. Here, we show the basic principles of SAXS and review latest developments in the fields of hybrid modeling and flexible systems.

Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering.

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Novel reverse thia-analogs of fosmidomycin: Synthesis and antiplasmodial activity.

Thia analogs of fosmidomycin are potent inhibitors of the non-mevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers displayed antiplasmodial in vitro activity in the low nanomolar range, without apparent cytotoxic effects in HeLa cells. The (S)-(+)-enantiomer of a typical representative selectively inhibited IspC and the growth of P. falciparum in continuous culture. The inhibitor was stable at pH 7.6 and room temperature, and no racemization was observed under these conditions during a period of up to two days. Oxidation of selected thioethers to sulfones reduced antiplasmodial activity and the inhibitory activity against Escherichia coli, Mycobacterium tuberculosis and P. falciparum IspC orthologs.

Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.

Characterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana, an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of AtIspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of AtIspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of AtIspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.

The complex isotopologue space of glucose as a framework for the study of human intermediary metabolism.

The positional distributions of stable isotopes in metabolites provide specific fingerprints of the pathways and fluxes that have occurred in the organisms under study. In particular, modern nuclear magnetic resonance (NMR) spectroscopy enables the detailed assignment of isotope patterns in natural products, for example, in metabolites obtained from labelling experiments using (13)C-enriched precursors, such as glucose, acetate or CO2. In this study, the transient (13)C-isotopologue composition of blood glucose from an adult human volunteer after intravenous supply of [U-(13)C6]glucose was determined by high-resolution (13)C NMR spectroscopy. The non-linear progression curves displaying the relative amounts of eight (13)C-glucose isotopologues reflected the contributions of glucose metabolism by glycolytic cycling, the pentose phosphate pathway and anaplerotic reactions involving the citric acid cycle. The pilot study suggests that the experimental setting can be useful in analysing under non-invasive conditions the impact of physiological and pharmacological constraints on glucose turnover in humans.

Prodrugs of reverse fosmidomycin analogues.

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drug's pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.

Inhibition of methyl-CoM Reductase from Methanobrevibacter ruminantium by 2-bromoethanesulfonate.

Cattle husbandry is a major contributor to atmospheric methane, which is considered as an important greenhouse gas. Moreover, the generation of methane in the intestine of domestic ruminants by methanogenic bacteria is a drag on feed efficacy. Studies on methanogenesis have typically implied model organisms that are, however, not relevant in the ruminant gut. This paper shows that methyl-CoM reductase catalyzing the final step of methanogenesis in Methanobrevibacter ruminantium, a major participant in methane production by cattle, is inhibited by 2-bromoethanesulfonate, a compound often used as a model in animal agriculture, with an apparent IC50 of 0.4 ± 0.04 µM.

Binding modes of reverse fosmidomycin analogs toward the antimalarial target IspC.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase of Plasmodium falciparum (PfIspC, PfDxr), believed to be the rate-limiting enzyme of the nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway), is a clinically validated antimalarial target. The enzyme is efficiently inhibited by the natural product fosmidomycin. To gain new insights into the structure activity relationships of reverse fosmidomycin analogs, several reverse analogs of fosmidomycin were synthesized and biologically evaluated. The 4-methoxyphenyl substituted derivative 2c showed potent inhibition of PfIspC as well as of P. falciparum growth and was more than one order of magnitude more active than fosmidomycin. The binding modes of three new derivatives in complex with PfIspC, reduced nicotinamide adenine dinucleotide phosphate, and Mg(2+) were determined by X-ray structure analysis. Notably, PfIspC selectively binds the S-enantiomers of the study compounds.

Recent advances in riboflavin biosynthesis.

Riboflavin is biosynthesized from GTP and ribulose 5-phosphate. Whereas the early reactions conducing to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate show significant taxonomic variation, the subsequent reaction steps are universal in all taxonomic kingdoms. With the exception of a hitherto elusive phosphatase, all enzymes of the pathway have been characterized in some detail at the structural and mechanistic level. Some of the pathway enzymes (GTP cycloyhdrolase II, 3,4-dihydroxy-2-butanone 4-phosphate synthase, riboflavin synthase) have exceptionally complex reaction mechanisms. The commercial production of the vitamin is now entirely based on highly productive fermentation processes. Due to their absence in animals, the pathway enzymes are potential targets for the development of novel anti-infective drugs.

IspC as target for antiinfective drug discovery: synthesis, enantiomeric separation, and structural biology of fosmidomycin thia isosters.

The emergence and spread of multidrug-resistant pathogens are widely believed to endanger human health. New drug targets and lead compounds exempt from cross-resistance with existing drugs are urgently needed. We report on the synthesis and properties of "reverse" thia analogs of fosmidomycin, which inhibit the first committed enzyme of a metabolic pathway that is essential for the causative agents of tuberculosis and malaria but is absent in the human host. Notably, IspC displays a high level of enantioselectivity for an α-substituted fosmidomycin derivative.

Structures and reaction mechanisms of GTP cyclohydrolases.

GTP cyclohydrolases generate the first committed intermediates for the biosynthesis of certain vitamins/cofactors (folic acid, riboflavin, deazaflavin, and tetrahydrobiopterin), deazapurine antibiotics, some t-RNA bases (queuosine, archaeosine), and the phytotoxin, toxoflavin. They depend on divalent cations for hydrolytic opening of the imidazole ring of the substrate, guanosine triphosphate (GTP). Surprisingly, the ring opening reaction is not the rate-limiting step for GTP cyclohydrolases I and II whose mechanism have been studied in some detail. GTP cyclohydrolase I, Ib, and II are potential targets for novel anti-infectives. Genetic factors modulating the activity of human GTP cyclohydrolase are highly pleiotropic, since the signal transponders whose biosyntheses require their participation (nitric oxide, catecholamines) impact a very wide range of physiological phenomena. Recent studies suggest that human GTP cyclohydrolase may become an oncology target.

α-Substituted ß-oxa isosteres of fosmidomycin: synthesis and biological evaluation.

Specific inhibition of enzymes of the non-mevalonate pathway is a promising strategy for the development of novel antiplasmodial drugs. α-Aryl-substituted ß-oxa isosteres of fosmidomycin with a reverse orientation of the hydroxamic acid group were synthesized and evaluated for their inhibitory activity against recombinant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) of Plasmodium falciparum and for their in vitro antiplasmodial activity against chloroquine-sensitive and resistant strains of P. falciparum . The most active derivative inhibits IspC protein of P. falciparum (PfIspC) with an IC(50) value of 12 nM and shows potent in vitro antiplasmodial activity. In addition, lipophilic ester prodrugs demonstrated improved P. falciparum growth inhibition in vitro.

Crystal structures of mutant IspH proteins reveal a rotation of the substrate's hydroxymethyl group during catalysis.

Isoprenoids derive from two universal precursors, isopentenyl diphosphate and dimethylallyl diphosphate, which in most human pathogens are synthesized in the deoxyxylulose phosphate pathway. The last step of this pathway is the conversion of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate catalyzed by the iron-sulfur protein IspH. The crystal structures reported here of the IspH mutant proteins T167C, E126D and E126Q reveal an alternative substrate conformation compared to the wild-type structure. Thus, the previously observed alkoxide complex decomposes, and the substrate's hydroxymethyl group rotates to interact with Glu126. The carboxyl group of Glu126 then donates a proton to the hydroxyl group to enable water elimination. The structural and functional studies provide further knowledge of the IspH reaction mechanism, which opens up new routes to inhibitor design against malaria and tuberculosis.

Reverse fosmidomycin derivatives against the antimalarial drug target IspC (Dxr).

Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray analysis. Pilot experiments have demonstrated in vivo potential.

Biochemistry of the non-mevalonate isoprenoid pathway.

The non-mevalonate pathway of isoprenoid (terpenoid) biosynthesis is essential in many eubacteria including the major human pathogen, Mycobacterium tuberculosis, in apicomplexan protozoa including the Plasmodium spp. causing malaria, and in the plastids of plants. The metabolic route is absent in humans and is therefore qualified as a promising target for new anti-infective drugs and herbicides. Biochemical and structural knowledge about all enzymes involved in the pathway established the basis for discovery and development of inhibitors by high-throughput screening of compound libraries and/or structure-based rational design.

Biosynthesis of isoprenoids: crystal structure of the [4Fe-4S] cluster protein IspG.

IspG protein serves as the penultimate enzyme of the recently discovered non-mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The enzyme catalyzes the reductive ring opening of 2C-methyl-D-erythritol 2,4-cyclodiphosphate, which affords 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. The protein was crystallized under anaerobic conditions, and its three-dimensional structure was determined to a resolution of 2.7 Å. Each subunit of the c(2) symmetric homodimer folds into two domains connected by a short linker sequence. The N-terminal domain (N domain) is an eight-stranded ß barrel that belongs to the large TIM-barrel superfamily. The C-terminal domain (C domain) consists of a ß sheet that is flanked on both sides by helices. One glutamate and three cysteine residues of the C domain coordinate a [4Fe-4S] cluster. Homodimer formation involves an extended contact area (about 1100 Å(2)) between helices 8 and 9 of each respective ß barrel. Moreover, each C domain contacts the N domain of the partner subunit, but the interface regions are small (about 430 Å(2)). We propose that the enzyme substrate binds to the positively charged surface area at the C-terminal pole of the ß barrel. The C domain carrying the iron-sulfur cluster could then move over to form a closed conformation where the substrate is sandwiched between the N domain and the C domain. This article completes the set of three-dimensional structures of the non-mevalonate pathway enzymes, which are of specific interest as potential targets for tuberculostatic and antimalarial drugs.