Structural and Functional Studies of the Phage Sf6 Terminase Small Subunit Reveal a DNA-Spooling Device Facilitated by Structural Plasticity

In many DNA viruses, genome packaging is initiated by the small subunit of the packaging terminase, which specifically binds to the packaging signal on viral DNA and directs assembly of the terminase holoenzyme. We have experimentally mapped the DNA-interacting region on Shigella virus Sf6 terminase small subunit gp1, which occupies extended surface areas encircling the gp1 octamer, indicating that DNA wraps around gp1 through extensive contacts. High‐resolution structures reveal large-scale motions of the gp1 DNA-binding domain mediated by the curved helix formed by residues 54–81 and an intermolecular salt bridge formed by residues Arg67 and Glu73, indicating remarkable structural plasticity underlying multivalent, pleomorphic gp1:DNA interactions. These results provide spatial restraints for protein:DNA interactions, which enable construction of a three-dimensional pseudo-atomic model for a DNA-packaging initiation complex assembled from the terminase small subunit and the packaging region on viral DNA. Our results suggest that gp1 functions as a DNA-spooling device, which may transform DNA into a specific architecture appropriate for interaction with and cleavage by the terminase large subunit prior to DNA translocation into viral procapsid. This may represent a common mechanism for the initiation step of DNA packaging in tailed double‐stranded DNA bacterial viruses.     

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8α-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8α-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8α-histidylation (either to N1 or to N3), 8α-O-tyrosylation, 8α-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX)⁴ from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8α-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7–9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12) and optimal binding of substrate molecules (11, 13) are also positively influenced by covalent tethering of the flavin, one might speculate that no generally applicable explanation for the covalent attachment of flavins to proteins exists. Therefore, it seems likely that the large variety of systems operating with one of the above mentioned modes of cofactor tethering might have evolved to also adapt to a diversity of enzymatic challenges.Berberine bridge enzyme (BBE) from Eschscholzia californica is a plant enzyme involved in alkaloid biosynthesis, catalyzing the challenging oxidative cyclization of (S)-reticuline to (S)-scoulerine (Scheme 1). This enzyme was recently shown to belong to the group of flavoenzymes with a bicovalently attached FAD (14). After the discovery of this unusual mode of linkage in the crystal structure of GOOX (4), several members of this group, all belonging to the vanillyl-alcohol oxidase family (15), were identified by biochemical methods (16–18) and also structural studies (19). Because some of the suggested benefits of a covalent cofactor attachment can easily be brought about by a single linkage, e.g. prevention of cofactor dissociation or stabilization of the tertiary structure, the two amino acids attached to FAD might have different and individual functions as well as an additive effect on physicochemical properties such as redox potentials or substrate binding and oxidation. To elucidate the relative importance for the overall enzymatic functioning of members of this group, more detailed studies have been performed on GOOX (11), chito-oligosaccharide oxidase (ChitO) from Fusarium graminearum (17), and BBE (20). Common results of these analyses show that the bicovalent FAD has a redox potential of about +130 mV, which is among the highest potentials reported for flavoenzymes. Replacement of one of the amino acids involved in anchoring of the cofactor generally reduces the rate of cofactor reduction and the steady-state turnover rate, but whether this can be directly linked to reduced redox potentials of these mutant proteins has been under debate (11).Open in a separate windowSCHEME 1.Overall reaction catalyzed by BBE.To address these issues further, we report the expression of the H104A mutant protein of BBE. A biochemical characterization of this protein variant with respect to the redox potential, transient kinetics, and steady-state analysis is combined with the structural analysis of both the H104A and the C166A mutant proteins. In addition, a structure of wild type (WT) BBE in complex with the product of the enzyme-catalyzed reaction is presented, which provides further insights toward the involvement of active site amino acids during the course of the reaction. Together with the recently reported x-ray crystal structure of WT BBE with and without substrate bound (21) and the biochemical characterization of the C166A mutant protein (20), these results provide interesting insights into the role of bicovalent FAD attachment in enzymes.     

Role of the Two Structural Domains from the Periplasmic Escherichia coli Histidine-binding Protein HisJ

Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.     

Structural and Functional Studies of the RBPJ-SHARP Complex Reveal a Conserved Corepressor Binding Site

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Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10⁹ to 10¹⁰ PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.     

Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein,ZapC

In Escherichia coli cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multiprotein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins is the Z-ring-associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8-kDa ZapC in particular shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Å and performed genetic and biochemical studies. ZapC is a monomer composed of two domains, an N-terminal α/β region and a C-terminal twisted β barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail, and the presence of two adjacent pockets suggests possible mechanisms for ZapC-mediated FtsZ bundling.     

Transduction of Various R Factors by Phage P1 in Escherichia coli and by Phage P22 in Salmonella typhimurium

R factors fi(+) and fi(-), with various combinations of drug-resistance markers and isolated from independent sources, were transduced by phage P1kc in Escherichia coli and by phage P22 in Salmonella typhimurium. Usually the entire R factor was transduced by P1kc in E. coli, as indicated by the absence of segregation of the drug-resistance markers from their conjugal transferability. In contrast, the patterns of segregation of the drug-resistance markers and their conjugal transferability differed considerably among various R factors after transduction by P22 in S. typhimurium. Transduction frequencies varied among R factors in both transduction systems.     

Structural and Functional Studies of the Biotin Protein Ligase from Aquifex aeolicus Reveal a Critical Role for a Conserved Residue in Target Specificity

Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5′-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 Å resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the α- and β-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg²⁺, and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPΔ67 fragment and BSA, and is subject to self-biotinylation.     

Comprehensive Analysis and Identification of the Human STIM1 Domains for Structural and Functional Studies

STIM1 is a Ca²⁺ sensor within the ER membrane known to activate the plasma membrane store-operated Ca²⁺ channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca²⁺ signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca²⁺ sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies.     

Structural and Functional Features of the Escherichia coli F 1 -ATPase

The structural organization and overall dimensions of the Escherichia coli F₁-ATPase in solutionhas been analyzed by synchroton X-ray scattering. Using an independent ab initio approach,the low-resolution shape of the hydrated enzyme was determined at 3.2 nm resolution. Theshape permitted unequivocal identification of the volume occupied by the _{3 3} complex ofthe atomic model of the ECF₁-ATPase. The position of the ^ and subunits were found byinteractive fitting of the solution scattering data and by cross-linking studies. Laser-inducedcovalent incorporation of 2-azido-ATP established a direct relationship between nucleotidebinding affinity and the different interactions between the stalk subunits and with the threecatalytic subunits () of the F₁-ATPase. Mutants of the ECF₁-ATPase with the introductionof Trp-for-Tyr replacement in the catalytic site of the complex made it possible to monitorthe activated state for ATP synthesis (ATP conformation) in which the and subunits arein close proximity to the subunits and the ADP conformation, with the stalk subunits arelinked to the subunit.     

Structural and Functional Characterization of Escherichia coli Toxin-Antitoxin Complex DinJ-YafQ

Toxin YafQ functions as a ribonuclease in the dinJ-yafQ toxin-antitoxin system of Escherichia coli. Antitoxin DinJ neutralizes YafQ-mediated toxicity by forming a stable protein complex. Here, crystal structures of the (DinJ)₂-(YafQ)₂ complex and the isolated YafQ toxin have been determined. The structure of the heterotetrameric complex (DinJ)₂-(YafQ)₂ revealed that the N-terminal region of DinJ folds into a ribbon-helix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule. Upon incorporation into the heterotetrameric complex, a conformational change of YafQ in close proximity to the catalytic site of the typical microbial ribonuclease fold was observed and validated. Mutagenesis experiments revealed that a DinJ mutant restored YafQ RNase activity in a tetramer complex in vitro but not in vivo. An electrophoretic mobility shift assay showed that one of the palindromic sequences present in the upstream intergenic region of DinJ served as a binding sequences for both the DinJ-YafQ complex and the antitoxin DinJ alone. Based on structure-guided and site-directed mutagenesis of DinJ-YafQ, we showed that two pairs of amino acids in DinJ were important for DNA binding; the R8A and K16A substitutions and the S31A and R35A substitutions in DinJ abolished the DNA binding ability of the DinJ-YafQ complex.     

Structural and Functional Analysis of BipA,a Regulator of Virulence in Enteropathogenic Escherichia coli

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3′, 5′-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.     

Comparative Survival of Free Shiga Toxin 2-Encoding Phages and Escherichia coli Strains outside the Gut

The behavior outside the gut of seeded Escherichia coli O157:H7, naturally occurring E. coli, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of E. coli and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment.     

X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions

Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.     

Structural and Functional Studies of a Klebsiella Phage Capsule Depolymerase Tailspike: Mechanistic Insights into Capsular Degradation

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