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Results for “Biochemistry”

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bioRxiv2018

High accuracy protein structures from minimal sparse paramagnetic solid-state NMR restraints

There is a pressing need for new computational tools to integrate data from diverse experimental approaches in structural biology. We present a strategy that combines sparse paramagnetic solid-state NMR restraints with physics-based atomistic simulations. Our approach explicitly accounts for uncertainty in the interpretation of experimental data through the use of a semi-quantitative mapping between the data and the restraint energy that is calibrated by extensive simulations. We apply our approach to solid-state NMR data for the model protein GB1 labeled with Cu2+-EDTA at six different sites. We are able to determine the structure to ca. 1 [A] accuracy within a single day of computation on a modest GPU cluster. We further show that in 4 of 6 cases, the data from only a single paramagnetic tag are sufficient to fold the protein to high accuracy.

biochemistry↗

bioRxiv2018

Structure of a bacterial ATP synthase

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit{varepsilon} shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

biochemistry↗

bioRxiv2018

Schistosoma mansoni does not and cannot oxidize fatty acids, but these are used for biosynthetic purposes instead

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and in their energy metabolism strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid {beta}-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [14C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidize fatty acids, as no 14CO2 production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding {beta}-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for S. japonicum. Absence of {beta}-oxidation, however, does not imply that fatty acids from the host are not metabolized by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate them in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid {beta}-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicates that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.

biochemistry↗

bioRxiv2018

A novel cell-based sensor detecting the activity of individual basic proprotein convertases

The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides and do not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. We report here PCific, a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. PCific consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in cis. Screening of selected candidate peptidomimetic inhibitors revealed that PCific can readily distinguish between general and selective PC inhibitors in a high-throughput screening format.

biochemistry↗

bioRxiv2018

Semi-in vitro Reconstitution of Roseocin, a Two-Component Lantibiotic from an Actinomycete

Lantibiotics are lanthionine containing peptide natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Recent expansion in the availability of microbial genomic data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic roseocin was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Post-translationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in E. coli, and were in vitro reconstituted to their bioactive form. The two lanthipeptides displayed antimicrobial activity against Gram-positive bacteria only synergistically, a property reminiscent of two-component lantibiotics. Structural analysis of the -component identified a disulfide bridge flanking two of its four thioether bridges and the {beta}-component having six thioether bridges with its C-terminus extended than the previously known two-component lantibiotics.

biochemistry↗

bioRxiv2018

Multimerization of Homo sapiens TRPA1 ion channel cytoplasmic domains

The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modulated by myriad noxious stimuli that interact with multiple regions of the channel, including cysteine-reactive natural extracts from onion and garlic which modify residues in the cytoplasmic domains. The way in which TRPA1 cytoplasmic domain modification is coupled to opening of the ion-conducting pore has yet to be elucidated. The cryo-EM structure of TRPA1 revealed a tetrameric C-terminal coiled-coil surrounded by N-terminal ankyrin repeat domains (ARDs), an architecture shared with the canonical transient receptor potential (TRPC) ion channel family. Similarly, structures of the TRP melastatin (TRPM) ion channel family also showed a C-terminal coiled-coil surrounded by N-terminal cytoplasmic domains. This conserved architecture may indicate a common gating mechanism by which modification of cytoplasmic domains can transduce conformational changes to open the ion-conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal coiled-coil domains can be expressed in bacteria and maintain the ability to interact. We tested three gating regulators: temperature; the polyphosphate compound IP6; and the covalent modifier allyl isothiocyanate to determine whether they alter N-and C-terminal interactions. We found that none of the modifiers tested abolished ARD-coiled-coil interactions, though there was a significant reduction at 37{degrees}C. We found that coiled-coils tetramerize in a concentration dependent manner, with monomers and trimers observed at lower concentrations. Our system provides a method for examining the mechanism of oligomerization of TRPA1 cytoplasmic domains as well as a system to study the transmission of conformational changes resulting from covalent modification.

biochemistry↗

bioRxiv2018

A structure-based approach towards identification of inhibitory fragments for eleven-nineteen-leukemia protein (ENL) YEATS domain

Lysine acetylation is an epigenetic mark that is principally recognized by bromodomains and recently structurally diverse YEATS domains also emerged as readers of lysine acetyl/acylations. Here we present a crystallography-based strategy and the discovery of fragments binding to the ENL YEATS domain, a potential drug target. Crystal structures combined with synthetic efforts led to the identification of a sub-micromolar binder, providing first starting points for the development of chemical probes for this reader domain family.

biochemistry↗

bioRxiv2018

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

biochemistry↗

bioRxiv2018

Divergent interactions maintain the quaternary octameric structure of a new family of esterases

Protein oligomerization contributes significantly to the stability and function of enzymes, and the interacting interfaces that create the oligomers are expected to be conserved. The acetyl-xylo-oligosaccharide esterase, Axe2, from the thermophilic bacterium Geobacillus stearothermophilus represents a new family of esterases belonging to the SGNH superfamily of hydrolytic enzymes, and has a unique doughnut-like homo-octameric configuration, composed of four homo-dimers. The dimers of Axe2 are held together mainly by clusters of hydrogen bonds involving Tyr184 and Arg192, as was demonstrated by site directed mutagenesis. Dimeric mutants obtained by single amino acid replacements were inactive towards 2-naphthyl acetate, indicating the necessity of the octameric assembly for catalysis. The crystal structure of two homologous proteins (PDB 3RJT and 5JD3) reveal the same tertiary fold and octameric ring structure as of Axe2. Surprisingly, these octameric structures appear to be maintained by different sets of amino acids involving Asn183 in 3RJT and His185 in 5JD3 instead of Tyr184 in Axe2. These findings prompt us to investigate five more homologues proteins, which were found to have similar octameric structures, despite significant changes in their key residues. We revealed a conserved quaternary structure, which is maintained via non-conserved interactions.

biochemistry↗

bioRxiv2018

The distinct biochemical property enables thymidylate kinase as a drug target and participates in pyrimidine drug sensitivity in Candida albicans

The ability to overcome drug resistance in outbreaks of Candida albicans infection is an unmet need in health management. Here, we investigated CDC8, which encodes thymidylate kinase (TMPK), as a potential drug target for the treatment of C. albicans infection. In this study, we found that the specific region spanning amino acids 106-123, namely, the Ca-loop of C. albicans TMPK (CaTMPK) contributes to the hyperactivity of this enzyme compared to the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/ deoxy-5-Fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, CaTMPK but not hTMPK enables dUTP/5-FdUTP-mediated DNA toxicity in yeast. CRISPR-mediated deletion of this Ca-loop in C. albicans demonstrated the critical role of this Ca-loop in fungal growth and susceptibility to 5-Fluorouridine (5-FUrd). Moreover, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd. Thus, this study not only identified a target site for the development of CaTMPK-selective drugs but also revealed 5-FUrd to be a potential drug for the treatment of C. albicans infection.\n\nAuthor summaryThe emergence of drug-resistant C. albicans strains is a serious medical concern that may be addressed by targeting an essential fungal enzyme. CDC8 encodes thymidylate kinase (TMPK), which is the key enzyme required for dTTP synthesis and is an essential gene for yeast growth. Therefore, the differences of TMPK between human and C. albicans can be a potential drug targeting site. This study defines a specific Ca-loop unique to CaTMPK from C. albicans, contributing to hyper-activity over human enzyme (hTMPK). CRSPR-edited deletion of this loop also suppressed the growth of C. albicans. Moreover, we present evidence that this loop enables dUMP utilization by CaTMPK, but not hTMPK. CaTMPK is also capable of using 5-FdUMP as a substrate, which contributes to 5-FUrd-mediated toxicity. Importantly, we found that many drug resistant pathogenic C. albicans isolates from patients are sensitive to 5-FUrd, which has not been used as a drug against fungal infection.

biochemistry↗

bioRxiv2018

The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2

The EGFR-adaptor protein CIN85 has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill-defined. Here, we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SH3 domains of CIN85 interact with the proline-arginine rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF-degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9 edited cells affected growth and migration properties as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2, and uncovered an essential survival function in tumor cells linking growth factor adaptors with hypoxia signaling.

biochemistry↗

bioRxiv2018

Global analysis of methionine oxidation provides a census of folding stabilities for the human proteome

The stability of proteins influences their tendency to aggregate, undergo degradation or become modified in cells. Despite their significance to understanding protein folding and function, quantitative analyses of thermodynamic stabilities have been mostly limited to soluble proteins in purified systems. We have used a highly multiplexed proteomics approach, based on analyses of methionine oxidation rates, to quantify stabilities of ~10,000 unique regions within ~3,000 proteins in human cell extracts. The data identify lysosomal and extracellular proteins as the most stable ontological subsets of the proteome. We show that the stability of proteins impacts their tendency to become oxidized and is globally altered by the osmolyte trimethylamine-N-oxide (TMAO). We also show that most proteins designated as intrinsically disordered retain their unfolded structure in the complex environment of the cell. Together, the data provide a census of the stability of the human proteome and validate a methodology for global quantitation of folding thermodynamics.

biochemistry↗

bioRxiv2018

Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and phosphate release

Plasmodium actins form very short filaments and have a non-canonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths are currently unknown. Using high-resolution crystallography, we show that magnesium binding activates the Plasmodium actin I monomer before polymerization by a slight flattening, which is reversed upon phosphate release. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A-loop. Asp180 interacts with either Lys270 or His74, depending on protonation, while Arg178 links the inner and outer domains. Hence, the A-loop is a switch between stable and non-stable filament conformations. Our data provide a comprehensive model for polymerization, phosphate release, and the inherent instability of parasite microfilaments.

biochemistry↗

bioRxiv2018

Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

The Rickettsia ~1,800 amino acid autotransporter protein Sca2 promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C-terminal fragments of Sca2 and their contributions to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers, but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 (FH2) domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three WH2 domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions Sca2 does not contain WH2 domains, and that the corresponding region is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.

biochemistry↗

bioRxiv2018

Polycomb Cbx2 Condensates Assemble through Phase Separation

Polycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-separated condensates.

biochemistry↗

bioRxiv2018

Enzymatic and structural characterization of the Naegleria fowleri glucokinase

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited and the mortality rate is very high (~98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The genome of the parasite encodes a single glucose phosphorylating enzyme, a glucokinase (Glck). The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (HsGlck), suggesting that parasite-specific inhibitors with anti-amoeba activity could be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 {+/-} 7.3 M and 141.6 {+/-} 9.9 M for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2 [A] resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose phosphorylating enzymes and identified several small molecules with IC50 values < 1 M that may prove useful as hit chemotypes for further lead and therapeutic development against N. fowleri.

biochemistry↗

bioRxiv2018

A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly-found plasticity lies in the >400-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.

biochemistry↗

bioRxiv2018

Covalent Aurora A regulation by the metabolic integrator coenzyme A

Aurora A is a cell cycle protein kinase implicated in multiple human cancers, and several Aurora A-specific kinase inhibitors have progressed into clinical trials. In this study, we report structural and cellular analysis of a novel biochemical mode of Aurora A inhibition, which occurs through reversible covalent interaction with the universal metabolic integrator coenzyme A (CoA). Mechanistically, the CoA 3-phospho ADP moiety interacts with Thr 217, an Aurora A selectivity filter, which permits the formation of an unprecedented covalent bond with Cys 290 in the kinase activation segment, lying some 15 [A] away. CoA modification (CoAlation) of endogenous Aurora A is rapidly induced by oxidative stresses at Cys 290 in human cells, and microinjection of CoA into mouse embryos perturbs meitoic spindle formation and chromosome alignment. Aurora A regulation by CoA reveals how targeting of Aurora A might be accomplished in the future by development of a double-anchored covalent inhibitor.

biochemistry↗