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bioRxiv2017

Thermal Decomposition Of The Amino Acids Glycine, Cysteine, Aspartic Acid, Asparagine, Glutamic Acid, Glutamine, Arginine And Histidine

Calorimetry, thermogravimetry and mass spectrometry were used to follow the thermal decomposition of the eight amino acids G, C, D, N, E, Q, R and H between 185{degrees}C and 280{degrees}C. Endothermic heats of decomposition between 72 and 151 kJ/mol are needed to form 12 to 70 % volatile products. This process is neither melting nor sublimation. With exception of cysteine they emit mainly H2O, some NH3 and no CO2. Cysteine produces CO2 and little else. The reactions are described by polynomials, AA ^ a (NH3) + b (H2O) + c (CO2) + d (H2S) + e (residue), with integer or half integer coefficients. The solid monomolecular residues are rich in peptide bonds.

biochemistry↗

bioRxiv2017

Towards Water-Free Biobanks: Long-Term Dry-Preservation At Room Temperature Of Desiccation-Sensitive Enzyme Luciferase In Air-Dried Insect Cells

Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

biochemistry↗

bioRxiv2017

Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

The enzyme methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The subunit of this enzyme (McrA) contains several unusual post-translational modifications, including an exceptionally rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioamide-containing natural products, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA in a {Delta}ycaO-tfuA mutant of the methanogenic archaeon Methanosarcina acetivorans revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Physiological characterization of this mutant suggested a new role for the thioglycine modification in enhancing protein stability, as opposed to playing a direct catalytic role. The universal conservation of this modification suggests that MCR arose in a thermophilic ancestor.

biochemistry↗

bioRxiv2017

Biosynthesis of Isonitrile Lipopeptides by Conserved Non-ribosomal Peptide Synthetase Gene Clusters in Actinobacteria

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in intro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a new family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a non-heme iron(II)-dependent oxidase homologue, and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a non-ribosomal peptide synthetase (NRPS). In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.\n\nSignificance StatementMycobacterium tuberculosis is the leading causative agent of tuberculosis (TB), of which millions of deaths occur annually. A putative lipopeptide biosynthetic gene cluster has been shown to be essential for the survival of this pathogen in hosts, and homologous gene clusters have also been found in all pathogenic mycobacteria and other species of Actinobacteria. We have identified the function of these gene clusters in making a new family of isonitrile lipopeptides. The biosynthesis has several unique features, including an unprecedented mechanism for isonitrile synthesis. Our results have further suggested that these biosynthetic gene clusters play a role in metal transport, and thus have shed light on a new metal transport system that is crucial for virulence of pathogenic mycobacteria.

biochemistry↗

bioRxiv2017

A Shortened Version Of SecA (SecAN) Functions As The Protein-Conducting Channel For Nascent β-Barrel Outer Membrane Proteins

Many proteins are translocated across biomembranes via protein translocons in targeting to their subcellular destinations. Hitherto, the SecYEG/Sec61 translocon, existing in prokaryotes and eukaryotes, represents the most intensively studied one. According to the current perception, both periplasmic and {beta}-barrel outer membrane proteins ({beta}-barrel OMPs) are translocated via the SecYEG translocon in bacterial cells, although direct living cell evidences remain lacking. Here, mainly via in vivo protein photo-crosslinking analysis, we revealed that the never reported membrane-integrated SecAN protein apparently functions as the translocon for {beta}-barrel OMPs. Additionally, SecAN contains a GXXXG motif known for mediating protein interactions in biomembranes, and processing of {beta}-barrel OMP precursors was severely affected in cells producing an assembly-defective SecAN variant resulted from the GXXXG motif mutations. Furthermore, SecAN was demonstrated to directly interact with the Bam complex, thus likely be a part of the supercomplex that we revealed earlier to be responsible for {beta}-barrel OMP biogenesis.

biochemistry↗

bioRxiv2017

Disease-Associated Mutations In Human BICD2 Hyperactivate Motility Of Dynein-Dynactin

Bicaudal D2 (BICD2) joins dynein with dynactin into a ternary complex (termed DDB) capable of processive movement. Point mutations in the BICD2 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2s membrane vesicle partner, the GTPase Rab6a. Rab6a-GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein-dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus- versus plus-end-directed microtubule motility in neurons may underlie spinal muscular atrophy.

biochemistry↗

bioRxiv2017

Production and purification of human Hsp90β in Escherichia coli

The molecular chaperone Hsp90 is an essential member of the cellular proteostasis system. It plays an important role in the stabilisation and activation of a large number of client proteins and is involved in fatal disease processes e.g. Alzheimer disease, cancer and cystic fibrosis. This makes Hsp90 a crucial protein to study. Mechanistic studies require large amounts of protein but the production and purification of recombinant human Hsp90 in E. coli is challenging and laborious. Here we identified conditions that influence Hsp90 production and optimised a fast and efficient purification protocol. We found that the nutrient value of the culturing medium and the length of induction had significant effect on Hsp90 production in Escherichia coli. Our fast, single-day purification protocol resulted in a stable, well-folded and pure sample that was resistant to degradation in a reproducible manner. We anticipate that our results provide a useful tool to produce higher amount of pure, well-folded and stable recombinant human Hsp90{beta} in Escherichia coli in an efficient way.

biochemistry↗

bioRxiv2017

Discovery of BAZ2A Bromodomain Ligands

The bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) is implicated in aggressive prostate cancer. The BAZ2A bromodomain is a challenging target because of the shallow pocket of its natural ligand, the acetylated side chain of lysine. Here, we report the successful screening of a library of nearly 1500 small molecules by high-throughput docking and force field-based binding-energy evaluation. For seven of the 20 molecules selected in silico, evidence of binding to the BAZ2A bromodomain is provided by ligand-observed NMR spectroscopy. Two of these compounds show a favorable ligand efficiency of 0.42 kcal/mol per non-hydrogen atom in a competition-binding assay. The crystal structures of the BAZ2A bromodomain in complex with four fragment hits validate the predicted binding modes. The binding modes of compounds 1 and 3 are compatible with ligand growing for optimization of affinity for BAZ2A and selectivity against the close homologue BAZ2B.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC=\"FIGDIR/small/122317_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (36K):\norg.highwire.dtl.DTLVardef@aba498org.highwire.dtl.DTLVardef@c32ddeorg.highwire.dtl.DTLVardef@1f85fc0org.highwire.dtl.DTLVardef@15671bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

Structure Of The Cpf1 Endonuclease R-Loop Complex After Target DNA Cleavage

Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45{degrees} angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 [A] away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a \"rail shape\" and \"flap-on\" conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a \"septum\" that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNAase sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system2-4.

biochemistry↗

bioRxiv2017

The C-Terminal Domain Of ParB Is Critical For Dynamic DNA Binding And Bridging Interactions Which Condense The Bacterial Centromere

The ParB protein forms DNA bridging interactions around parS to form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring at parS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

biochemistry↗

bioRxiv2017

Characterisation Of The Biflavonoid Hinokiflavone As A Pre-mRNA Splicing Modulator That Inhibits SENP

Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of purified E. coli expressed SUMO protease, SENP1 in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.

biochemistry↗

bioRxiv2017

Sec17 (α-SNAP) And An SM-Tethering Complex Control The Outcome Of SNARE Zippering In Vitro And In Vivo

Zippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 catalysis, Sec17 either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, HOPS and Vps33 accelerate SNARE zippering, particularly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially-zipped, Sec17 promotes fusion in either the presence or absence of HOPS -- but with faster kinetics when HOPS is absent. Our data further indicate that Sec17 promotes fusion both through its direct penetration of the membrane and by enhancing C-terminal SNARE zippering. In a working model, the interplay among Sec17, Sec18, SMs, and SNARE zippering can explain why SM proteins are indispensable for SNARE-mediated fusion in vivo.\n\nImpact statementSec17 is shown to have divergent effects on pre-fusion SNARE complex activity, depending on the state of SNARE zippering. HOPS, an SM-tether complex, controls the outcome of Sec17-SNARE engagement. The results suggest a coherent working model for SM activity in vivo.

biochemistry↗

bioRxiv2017

Structural Models And Considerations On The COA6, COX18 And COX20 Factors That Assist Assembly Of Human Cytochrome C Oxidase Subunit II

The soluble domain of cytochrome c oxidase subunit II (COX2), located in the outer side of the inner mitochondrial membrane, contains a binuclear copper site (CuA) through which electrons flow from cytochrome c to the core of the oxidase where oxygen reduction takes place. Being COX2 encoded in the mitochondrial genome, newly synthesized protein undergoes maturation steps in which it is translocated through and inserted into the inner mitochondrial membrane, and copper ions are loaded to form the CuA site. These steps are ensured by several protein factors in a complex pathway that is not fully understood, including copper-loading and disulfide-reduction proteins plus chaperones that assist proper membrane insertion. While the structure and function of copper-loading and disulfide-reducing proteins Sco1 and Sco2 have been quite studied at atomistic level, the latest biological studies have uncovered roles for other proteins that are not yet much understood at the structural level. In particular, recent experiments showed that membrane protein COX18 is a membrane-protein insertase for COX2, whereas membrane protein COX20 is a chaperone that stabilizes COX2 during translocation through the inner mitochondrial membrane, and soluble protein COA6 is part of the copper-loading pathway in conjunction with Sco1 and Sco2. This work reports structural models for COX18, COX20 and COA6, built judiciously from homology modeling, contact prediction-based modeling and transmembrane helix predictions, while considering the underlying biology. Implications and limitations of the models are discussed, and possible experimental routes to pursue are proposed. All models are provided as PyMOL sessions in the Supporting Information and can be visualized online at http://lucianoabriata.altervista.org/modelshome.html.

biochemistry↗

bioRxiv2017

A Chiral Selectivity Relaxed Paralog Of DTD For Proofreading tRNA Mischarging In Animalia

D-aminoacyl-tRNA deacylase (DTD), a trans-editing factor found in bacteria and eukaryotes, removes D-amino acids mischarged on tRNAs as well as achiral glycine mischarged on tRNAAla. An invariant cross-subunit Gly-cisPro motif forms the mechanistic basis of strict L-amino acid rejection from the catalytic site. Here, we present the identification of a DTD variant, named ATD (Animalia-specific tRNA deacylase), that harbors a Gly-transPro motif. The cis-to-trans switch causes a \"gain of function\" through L-chiral selectivity in ATD resulting in the clearing of L-alanine mischarged on tRNAThr(G4*U69) by eukaryotic AlaRS. The biochemical proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNAThr(G4*U69) genes are in strict association with the presence of ATD, underlining the mandatory requirement of a dedicated factor to proofread tRNA misaminoacylation. The study highlights the emergence of ATD during genome expansion as a key event associated with the evolution of Animalia.

biochemistry↗

bioRxiv2017

Differential Effects of LIS1 On Processive Dynein-Dynactin Motility

Cytoplasmic dynein is the primary minus-end directed microtubule motor protein in cells. LIS1 is a highly conserved dynein regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor, and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein regulatory factor, acts to release dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we find that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bind up to two LIS1 dimers, and mutational analysis suggests LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhances DDB velocity in a concentration dependent manner, in contrast to observations of LIS1s effects on the motility of isolated dynein. Thus, LIS1 exerts concentration dependent effects on dynein motility, and can synergize with dynactin to enhance processive movement in the absence of load.

biochemistry↗

bioRxiv2017

Covalent Protein Labeling By SpyTag-SpyCatcher In Fixed Cells For Super-Resolution Microscopy

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. While the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We have also applied this method to endogenous proteins via gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.

biochemistry↗

bioRxiv2017

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

Mammalian TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs are targets of the enzyme OGT, which post-translationally modifies intracellular proteins in response to cellular nutrient status. The biological implications of the OGT-TET interaction have not been thoroughly explored. Here, we show for the first time that modification of TET1 by OGT enhances its activity in vitro. We identify a previously uncharacterized domain of TET1 responsible for binding to OGT and report a point mutation that disrupts the OGT-TET1 interaction. Finally, we show that the interaction between TET1 and OGT is necessary for TET1 to rescue tet mutant zebrafish hematopoetic stem cell formation, suggesting that OGT promotes TET1s function in development. Our results demonstrate regulation of TET activity by OGT in vitro and in vivo. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

biochemistry↗

bioRxiv2017

Functional Implications Of Disordered Terminal Regions Of Macrotyloma uniflorum Bowman-Birk Inhibitors: A Molecular Dynamics Study

Bowman-Birk Inhibitors (BBI) - a class of serine protease inhibitors is of considerable interest due to their anti-inflammatory and anti-carcinogenic properties. Recent efforts have focused on understanding the structure and dynamics of these inhibitors, and the molecular mechanism behind its bioactive properties. BBI derived from Horsegram seeds is an interesting member of the class that exists as a number of isoforms that differ in length at the C- and N-terminal disordered regions. Interestingly, the length (or conversely, truncation) of the terminal regions affect whether the protein exists as a dimer or monomer. Here, we have investigated the mechanism of dimerization in Horsegram BBI. A recent study has proposed that the dimerization occur via a C-terminal hook that forms a salt bridge with the opposite monomer and is pivotal to the dimerization process. We have employed long computational simulation methods to predict the stability of the proposed C-terminal hook; we show that the terminal regions are highly disordered and the salt bridges are significantly solvent exposed. Further, using Hamiltonian replica exchange method, we have sought to obtain the conformational ensemble of the disordered terminal regions and have identified a conformational state that provides an interaction hot-spot that aids in the dimerization of HGI. Our analysis predicts an alternate model of dimerization that largely agrees with previous experimental studies and yet again, highlights the importance of intrinsically disordered region in tailoring the protein function.

biochemistry↗