Analysis of the DDE Motif in the Mutator Superfamily

The eukaryotic Mutator family of transposable elements is widespread in plants. Active or potentially active copies are also found in fungi and protozoans, and sequences related to this family have been detected in metazoans as well. Members of this family are called Mutator-like elements (MULE s). They encode transposases, which contain a region conserved with transposases of the IS256 prokaryotic family, known to harbor a DDE catalytic domain. Different DDE or D34E motifs have been proposed in some groups of eukaryotic MULEs based on primary sequence conservation. On a large number of protein sequences related to, and representative of, all MULE families, we analyzed global conservation, the close environment of different acidic residues and the secondary structure. This allowed us to identify a potential DDE motif that is likely to be homologous to the one in IS256-like transposases. The characteristics of this motif are depicted in each known family of MULEs. Different hypotheses about the evolution of this triad are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

TcdA1 of Photorhabdus luminescens: Electrophysiological Analysis of Pore Formation and Effector Binding

Tc toxins are widely distributed among different gram-negative and gram-positive bacteria, where they act as pathogenicity factors. The toxins are composed of different components that form oligomers for biological activity. Lipid bilayer experiments were performed with the TcdA1 component of the Tc toxin from Photorhabdus luminescens, which preferentially kills insects by actin polymerization. TcdA1 was able to increase the specific conductance of artificial lipid bilayer membranes by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 125 pS in 150 mM KCl and were found to be cation selective. The single-channel conductance of the TcdA1-channels was only moderately dependent on the bulk aqueous KCl concentration, which indicated point-charge effects on the channel properties. Experiments to study the voltage dependence of the TcdA1 channel demonstrated that it is reconstituted in a fully oriented way when it is added to only one side of the lipid bilayer membrane. A combination of biologically active components (TccC3) and a possible chaperone (TcdB2) blocked the TcdA1-mediated conductance efficiently in a dose-dependent manner when they were added to the cis side of the membrane. The half-saturation constant for binding of TcdB2-TccC3 to TcdA1 is in the low nanomolar range.     

Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including l-carnosine and l-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD^CAT. The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an “extra” domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD^CAT exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD^CAT for GABAergic therapies or neuroprotection.     

Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a “helicase” strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k_cat), but not the affinity for ATP (K_m). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of γ-PO₄ of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP.     

The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily

NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical ‘lid’ region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.     

The Crystal Structure of Arabidopsis VSP1 Reveals the Plant Class C-Like Phosphatase Structure of the DDDD Superfamily of Phosphohydrolases

Arabidopsis thaliana vegetative storage proteins, VSP1 and VSP2, are acid phosphatases and belong to the haloacid dehalogenase (HAD) superfamily. In addition to their potential nutrient storage function, they were thought to be involved in plant defense and flower development. To gain insights into the architecture of the protein and obtain clues about its function, we have tested their substrate specificity and solved the structure of VSP1. The acid phosphatase activities of these two enzymes require divalent metal such as magnesium ion. Conversely, the activity of these two enzymes is inhibited by vanadate and molybdate, but is resistant to inorganic phosphate. Both VSP1 and VSP2 did not exhibit remarkable activities to any physiological substrates tested. In the current study, we presented the crystal structure of recombinant VSP1 at 1.8 Å resolution via the selenomethionine single-wavelength anomalous diffraction (SAD). Specifically, an α-helical cap domain on the top of the α/β core domain is found to be involved in dimerization. In addition, despite of the low sequence similarity between VSP1 and other HAD enzymes, the core domain of VSP1 containing conserved active site and catalytic machinery displays a classic haloacid dehalogenase fold. Furthermore, we found that VSP1 is distinguished from bacterial class C acid phosphatase P4 by several structural features. To our knowledge, this is the first study to reveal the crystal structure of plant vegetative storage proteins.     

Crystal and Solution Structure of the Bi-Loop Motif in Cyclic Octanucleotides

Abstract

The refined NMR structures of the cyclic octamers d?pATTCATTC?1 and d?pGCTCGCTT?2 are consistent with the reported Crystallographic data of 1 and the linear heptamer d(GCATGCT), respectively. The NMR study of four new cyclic octamers shows that in all cases a dimeric bi-loop structure is observed in equilibrium with a monomeric form.     

X-Ray Crystal Structure of the Full Length Human Chitotriosidase (CHIT1) Reveals Features of Its Chitin Binding Domain

Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1) is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD). This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family) and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase) comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL) at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBD_CHIT1) structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBD_CHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBD_CHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBD_CHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain.     

The Structure of High Molecular Weight Ribonucleic Acid in Solution: A Small-Angle X-Ray Scattering Study

Small-angle x-ray scattering studies on an absolute scale have been carried out on isotropic solutions of high molecular weight RNA obtained from ascites tumor cells, E. coli, and yeast. It was found that in all three cases the RNA is composed of short rigid rods (50 to 150Å in length) joined by small flexible regions. The rods account for almost the entire structure (at least 90 per cent); their radius of gyration about the axis and their mass per unit length are similar to those of DNA, suggesting a double-stranded helical structure. The rods are joined in an array forming the compact RNA molecule. On thermal degradation, the molecular superstructure disappears while the rods persist.     

X-Ray Analysis of New Crystal Forms of the Sweet Protein Thaumatin

Abstract

Thaumatin is a plant protein that in the mature form contains 8 disulfide bonds and 207 amino acids. Several forms of this protein occur naturally and each elicits an intense sweetness sensation when tasted in microgram quantities. The two major forms of thaumatin are easily separable by ion exchange chromatography. Crystals of the two proteins (designated here A and B) have been grown by vapor equilibration from solutions containing polyethylene glycol and examined by X-ray diffraction. The thaumatin A crystals are of space group P2₁2₁2₁ with a=44.3Å, b=63.7Åand c=72.7A. The crystals of thaumatin B are of space group C2 with a = 117.7Å, b=44.9Å, and c=38.0Å and β=94.0°. Both crystals diffract to well beyond 2.3Å and appear suitable for high resolution structure analysis. Four heavy atom derivatives of thaumatin B have been generated and diffraction data to 4Å resolution have been collected. This work is designed to provide a basis for studying the 3-dimensional structure of more than 100 genetically generated thaumatin derivatives, several of which show enhanced stability and improved taste characteristics.     

X-Ray Crystal Structure of a Highly Functionalized Thiophene as a New Backbone Amide Linker for Solid-phase Peptide Synthesis. Relationship between Crystal Structure and Reactivity

The development of new linkers (handles) for solid-phase synthesis provides new chemical opportunities for peptide synthesis. To understand the chemical properties of a recently developed backbone amide linker from a structural perspective, the crystal structure of S-((5-formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropionic acid (T-BAL2) was studied. Specifically, we wished to address whether this highly substituted thiophene retained planarity in the aromatic ring as well as between the aromatic ring and the aldehyde carbonyl. Furthermore, we sought an explanation for the relatively low reactivity in reductive aminations of the thienylaldehyde with amines in solution and on solid phase. Based on the crystal structure of T-BAL2, the thienyl-C (aldehyde) and C–O (aldehyde) bond lengths were applied as measures for the electron-deficiency (electrophilicity) of the aldehyde and compared to similar bond lengths found in previously reported formylated homo- and hetero-aromatic systems, which show significantly higher reactivity towards imine formation. The bond lengths found in the present structure are in accordance with normal C–C single bond and C–O double bond lengths. The high similarity in aldehyde bond lengths in the present system and in the reported systems indicates similar electron distribution in these systems. The lower reactivity of the present system may therefore not be attributed to electronic factors.     

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution,Obtained Using Small-Angle X-Ray Scattering

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed.     

Crystal Structures of Mite Allergens Der f 1 and Der p 1 Reveal Differences in Surface-Exposed Residues that May Influence Antibody Binding

The group 1 mite allergens Der f 1 and Der p 1 are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. The human immunoglobulin E antibody responses to the group 1 allergens show more cross-reactivity than the murine immunoglobulin G antibody responses, which are largely species specific. Here, we report the crystal structure of the mature form of Der f 1, which was isolated from its natural source, and a new high-resolution structure of mature recombinant Der p 1. Unlike Der p 1, Der f 1 is monomeric both in the crystalline state and in solution. Moreover, no metal binding is observed in the structure of Der f 1 despite the fact that all amino acids involved in Ca²⁺ binding in Der p 1 are completely conserved in Der f 1. Although Der p 1 and Der f 1 share an extensive sequence identity, comparison of the crystal structures of both allergens revealed structural features that could explain the differences in murine IgG and human IgE antibody responses to these allergens. There are structural differences between Der f 1 and Der p 1 that are unevenly distributed on the allergens' surfaces. This uneven spatial arrangement of conserved versus altered residues could explain both the specificity and cross-reactivity of antibodies against Der f 1 and Der p 1.     

Characterization of the Binding of Gallium, Platinum, and Uranium to Pseudomonas fluorescens by Small-Angle X-Ray Scattering and Transmission Electron Microscopy

Small-angle X-ray scattering (SAXS) was used to determine the binding of Ga, U, and Pt to Pseudomonas fluorescens in aqueous buffer. Atomic absorption spectrophotometry was used to quantify the heavy metals during bulk analysis, whereas transmission electron microscopy of whole mounts and thin sections was used to determine the locations of the cell-bound metal precipitates, as well as their sizes and physical structures. Energy-dispersive X-ray spectroscopy confirmed the compositions and identities of the precipitates and helped show that they were associated primarily with the envelope layers of the bacteria. Unlike Ga and Pt, which were located only at the cell surface, U was also found intracellularly in ~ 10% of the cells. This cytoplasmic location ultimately killed and lysed the cells. Surface-bound Ga and U were spread over the entire cell envelope (outer membrane-peptidoglycan-plasma membrane complex), whereas Pt was associated only with the lipopolysaccharide-rich, external face of the outer membrane. SAXS confirmed these data and showed that the bacteria were metal-enshrouded particles that were 1.0 to 1.5 μm in diameter. SAXS also provided a statistically significant representation of the bound metal precipitates, which ranged in size from 10 nm to 1 μm. The correlation between the microscopic data and the scattering data was extremely good. Since SAXS is performed in an aqueous milieu, it yields a more representative picture of the physical state of the metal bound to cell surfaces.     

Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily

Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.     

The Crystal Structure and Activity of a Putative Trypanosomal Nucleoside Phosphorylase Reveal It to be a Homodimeric Uridine Phosphorylase

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.     

The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH₂ in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.