Search

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H + /e − stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living β-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts β-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

Escherichia coli contains two cytochrome bd oxidases, bd-I and bd-II. The structure of both enzymes is highly similar, but they exhibit subtle differences such as the accessibility of the active site through a putative proton channel. Here, we demonstrate that the duroquinol:dioxygen oxidoreductase activity of bd-I increased with alkaline pH, whereas bd-II showed a broad activity maximum around pH 7. Likewise, the pH dependence of NO release from the reduced active site, an essential property of bd oxidases, differed between the two oxidases as detected by UV/vis spectroscopy. Both findings may be attributed to differences in the proton channel leading to the active site heme d. The channel comprises a titratable residue (Asp58

Cytochrome

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

Cytochrome bd-I oxidase is a terminal reductase of bacterial respiratory chains produced under low oxygen concentrations, oxidative stress, and during pathogenicity. While the bulk of the protein forms transmembrane helices, a periplasmic domain, the Q-loop, is expected to be involved in binding and oxidation of (ubi)quinol. According to the length of the Q-loop, bd oxidases are classified into the S (short)- and the L (long)-subfamilies. Here, we show that either shortening the Q-loop of the Escherichia coli oxidase from the L-subfamily or replacing it by one from the S-subfamily leads to the production of labile and inactive variants, indicating a role for the extended Q-loop in the stability of the enzyme.

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.

Summary Respiratory complex I drives proton translocation across energy-transducing membranes by NADH oxidation coupled with (ubi)quinone reduction. In humans, its dysfunction is associated with neurodegenerative diseases. The Escherichia coli complex represents the structural minimal form of an energy-converting NADH:ubiquinone oxidoreductase. Here, we report the structure of the peripheral arm of the E. coli complex I consisting of six subunits, the FMN cofactor, and nine iron-sulfur clusters at 2.7 A resolution obtained by cryo electron microscopy. While the cofactors are in equivalent positions as in the complex from other species, individual subunits are adapted to the absence of supernumerary proteins to guarantee structural stability. The catalytically important subunits NuoC and D are fused resulting in a specific architecture of functional importance. Striking features of the E. coli complex are scrutinized by mutagenesis and biochemical characterization of the variants. Moreover, the arrangement of the subunits sheds light on the unknown assembly of the complex.

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.

Various malignant human diseases show disturbed signaling pathways due to increased activity of proteins within the epigenetic machinery. Recently, various novel inhibitors for epigenetic regulation have been introduced which promise a great therapeutic benefit. Inhibitors for the bromo- and extra-terminal domain (BET) family were of particular interest after inhibitors had shown a strong antiproliferative effect. More recently, the focus has increasingly shifted to bromodomains (BDs) outside the BET family. Based on previously developed inhibitors, we have optimized a small series of 4-acyl pyrroles, which we further analyzed by ITC, X-ray crystallography, selectivity studies, the NCI60 cell-panel, and GI50 determinations for several cancer cell lines. The inhibitors address both, BET and BRD7/9 BDs, with very high affinity and show a strong antiproliferative effect on various cancer cell lines that could not be observed for BD family selective inhibitors. Furthermore, a synergistic effect on breast cancer (MCF-7) and melanoma (SK-MEL-5) was proven.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP

Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energyrich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailedmechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementarybiochemical analyses, these define the molecular basis of nucleotidespecificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.

Bromodomain and extra-terminal domain (BET) inhibitors are widely used both as chemical tools to study the biological role of their targets in living organisms and as candidates for drug development against several cancer variants and human disorders. However, non-BET bromodomains such as those in p300 and CBP are less studied. XDM-CBP is a highly potent and selective inhibitor for the bromodomains of CBP and p300 derived from a pan-selective BET BRD-binding fragment. Along with X-ray crystal-structure analysis and thermodynamic profiling, XDM-CBP was used in screenings of several cancer cell lines in vitro to study its inhibitory potential on cancer cell proliferation. XDM-CBP is demonstrated to be a potent and selective CBP/p300 inhibitor that acts on specific cancer cell lines, in particular malignant melanoma, breast cancer, and leukemia.

Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism., Cytochrome bd oxidases couple quinol oxidation and the release of protons to the periplasmic side with proton uptake from the cytoplasmic side to reduce dioxygen to water and they are the terminal reductases in bacterial and archaeal respiratory chains. Here the authors present the cryo-EM structure of Escherichia coli bd oxidase and discuss mechanistic implications.

Several human diseases, including cancer, show altered signaling pathways resulting from changes in the activity levels of epigenetic modulators. In the past few years, small-molecule inhibitors against specific modulators, including the bromodomain and extra-terminal (BET) bromodomain family of acetylation readers, have shown early promise in the treatment of the genetically defined midline carcinoma and hematopoietic malignancies. We have recently developed a novel potent inhibitor of BET proteins, 1 (XD14[ Angew. Chem., Int. Ed. 2013, 52, 14055]), which exerts a strong inhibitory potential on the proliferation of specific leukemia cell lines. In the study presented here, we designed analogues of 1 to study the potential of substitutions on the 4-acyl pyrrole backbone to occupy additional sites within the substrate recognition site of BRD4(1). The compounds were profiled using ITC, DSF, and X-ray crystallography. We could introduce several substitutions that address previously untargeted areas of the substrate recognition site. This work may substantially contribute to the development of therapeutics with increased target specificity against BRD4-related malignancies.

Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.

The nuclear import of H1 linker histones is mediated by a heterodimer of transport receptors, known as importinbeta and importin7. Interestingly, both importins separately interact with H1, but only as a dimer they facilitate the translocation through the nuclear pore. We identified the H1 binding site of importin7, comprising two extended acidic loops near the C terminus of importin7. The analysis of the H1 import complex assembly by means of isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven. Furthermore, we show that the importinbeta binding domain of importin7 plays a key role in the activation of importin7 by importinbeta. This process is allosterically regulated by importinbeta and accounts for a specific tuning of the activity of the importinbeta.importin7 heterodimer. The results presented here provide new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importinbeta-related nuclear import processes.

c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin beta superfamily, importin beta itself and transportin, promote import of c-Fos in vitro. Under conditions where importin beta-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin beta were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin beta for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin beta in recognizing different nuclear import cargoes.

D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of alpha-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1 angstrom) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach. (C) 2014 Elsevier Inc. All rights reserved.

Bacteria have evolved mechanisms for the hydrogenation of unsaturated fatty acids. Hydroxy fatty acid formation may be the first step in such a process; however, knowledge of the structural and mechanistic aspects of this reaction is scarce. Recently, myosin cross-reactive antigen was shown to be a bacterial FAD-containing hydratase which acts on the 9Z and 12Z double bonds of C16 and C18 non-esterified fatty acids, with the formation of 10-hydroxy and 10,13-dihydroxy fatty acids. These fatty acid hydratases form a large protein family which is conserved across Gram-positive and Gram-negative bacteria with no sequence similarity to any known protein apart from the FAD-binding motif. In order to shed light on the substrate recognition and the mechanism of the hydratase reaction, the crystal structure of the hydratase from Lactobacillus acidophilus (LAH) was determined by single-wavelength anomalous dispersion. Crystal structures of apo LAH and of LAH with bound linoleic acid were refined at resolutions of 2.3 and 1.8 A, respectively. LAH is a homodimer; each protomer consists of four intricately connected domains. Three of them form the FAD-binding and substrate-binding sites and reveal structural similarity to three domains of several flavin-dependent enzymes, including amine oxidoreductases. The additional fourth domain of LAH is located at the C-terminus and consists of three α-helices. It covers the entrance to the hydrophobic substrate channel leading from the protein surface to the active site. In the presence of linoleic acid, the fourth domain of one protomer undergoes conformational changes and opens the entrance to the substrate-binding channel of the other protomer of the LAH homodimer. The linoleic acid molecule is bound at the entrance to the substrate channel, suggesting movement of the lid domain triggered by substrate recognition.

The nuclear import of assembled spliceosomal subunits, the uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), is mediated by a nuclear import receptor adaptor couple of importin beta (Imp beta) and snurportin1 (SPN1). In contrast to any other characterized active nuclear import, the Imp beta/SPN1/U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex (NPC). The crystal structure of Imp beta (127-876) in complex with the Imp beta-binding (IBB) domain of SPN1 (1-65) at 2.8-A resolution reveals that Imp beta adopts an open conformation, which is unique for a functional Imp beta/cargo complex, and rather surprisingly, it resembles the conformation of the Imp beta/RanGTP complex. As binding of RanGTP to Imp beta usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Imp beta/RanGTP the independent dissociation of Imp beta/SPN1 from the nuclear basket is energetically aided.

c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin beta superfamily, importin beta itself and transportin, promote import of c-Fos in vitro. Under conditions where importin beta-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin beta were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin beta for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin beta in recognizing different nuclear import cargoes.