Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase.

The allosteric effectors of aspartate transcarbamoylase from Escherichia coli, CTP and ATP, associate with both the regulatory and the catalytic moieties of the enzyme. Studies with isolated, active subunits yield one binding site per regulatory dimer and one per catalytic trimer. Investigations of effector association with hybrid enzymes, containing either the three regulatory dimers or the two catalytic trimers in inactivated forms, indicate that the data obtained with isolated subunits can be used to analyze the binding patterns of these ligands to the native hexamer. Thus, the nonlinear Scatchard plots, characteristic of the binding of CTP and ATP to the native enzyme, can be interpreted in terms of three effector molecules associating with the regulatory subunits, and two binding to the catalytic moiety of the enzyme. Results with native protein in the presence of saturating concentrations of active site ligands support these assignments. The differences between the binding isotherms of CTP and ATP to the enzyme are due to their different affinities to the two types of subunits. The apparent half-of-the-site saturation of the regulatory moiety of aspartate transcarbamoylase supports the concept that this protein has a tendency to exist in an asymmetric state.     

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.     

Intramolecular signaling in immunoglobulins -- new evidence emerging from the use of supramolecular protein ligands.

The postulated intramolecular signaling in immunoglobulins generated by antigen binding has been controversial for years. The high heterogeneity of immune complexes as signaling systems and the requirement of the immobilized antigen form for efficient triggering of effector activity is likely the reason for the lack of clarity. Here we present new evidence supporting the notion of intramolecular signaling, based on the use of supramolecular dyes that bind to signal-derived specific sites in immunoglobulins.     

Replication from oriP of Epstein-Barr virus requires exact spacing of two bound dimers of EBNA1 which bend DNA

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on oriP are replicated once per cell cycle by cellular factors. The replicator of oriP is an approximately 120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from oriP requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.     

Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules.

The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.     

Ribonucleotide reductase from Escherichia coli. Identification of allosteric effector sites by chromatography on immobilized effectors.

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.     

Hierarchical delivery of an essential host colonization factor in enteropathogenic Escherichia coli

Many significant bacterial pathogens use a type III secretion system to inject effector proteins into host cells to disrupt specific cellular functions, enabling disease progression. The injection of these effectors into host cells is often dependent on dedicated chaperones within the bacterial cell. In this report, we demonstrate that the enteropathogenic Escherichia coli (EPEC) chaperone CesT interacts with a variety of known and putative type III effector proteins. Using pull-down and secretion assays, a degenerate CesT binding domain was identified within multiple type III effectors. Domain exchange experiments between selected type III effector proteins revealed a modular nature for the CesT binding domain, as demonstrated by secretion, chaperone binding, and infection assays. The CesT-interacting type III effector Tir, which is crucial for in vivo intestinal colonization, had to be expressed and secreted for efficient secretion of other type III effectors. In contrast, the absence of other CesT-interacting type III effectors did not abrogate effector secretion, indicating an unexpected hierarchy with respect to Tir for type III effector delivery. Coordinating the expression of other type III effectors with cesT in the absence of tir partially restored total type III effector secretion, thereby implicating CesT in secretion events. Collectively, the results suggest a coordinated mechanism involving both Tir and CesT for type III effector injection into host cells.     

Functional dissection of SseF, a membrane-integral effector protein of intracellular Salmonella enterica

During intracellular life, the bacterial pathogen Salmonella enterica translocates a complex cocktail of effector proteins by means of the SPI2-encoded type III secretions system. The effectors jointly modify the endosomal system and vesicular transport in host cells. SseF and SseG are two effectors encoded by genes within Salmonella Pathogenicity Island 2 and both effector associate with endosomal membranes and microtubules and are involved in the formation of Salmonella-induced filaments. Our previous deletional analyses identified protein domains of SseF required for the effector function. Here we present a detailed mutational analysis that identifies a short hydrophobic motif as functionally essential. We demonstrate that SseF and SseG are still functional if translocated as a single fusion protein, but also mediate effector function if translocated in cells co-infected with sseF and sseG strains. SseF has characteristics of an integral membrane protein after translocation into host cells.     

Effective selective modification of a single-stranded fragment of DNA with alkylating derivatives of short oligodeoxyribonucleotides in the presence of reaction effectors N-(2-hydroxyethyl)phenazine derivatives of oligodeoxyribonucleotides

Tri-, tetra-, penta- and hexanucleotides bearing a reactive 4-(N-methylamino-N-2-chloroethyl)benzylamide group can effectively and selectively modify a single-stranded DNA fragment (302 nucleotides) in the presence of effectors, N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides complementary to DNA sequences adjacent to the binding site of the reagent. The reagents investigated modify not only single-stranded but also secondary-structured DNA regions. The modification extent depends on the length of oligonucleotide parts of the reagent and effector. A gap between the two stretches associated with the target DNA prevents the effector from functioning. The substitution of an octanucleotide effector by two tetranucleotide ones only slightly reduces the modification extent with a hexanucleotide reagent. A very efficient and specific modification can be achieved by using two effectors flanking the reactive oligonucleotide derivative. The approach leads to the modification extent of up to 89% with a hexanucleotide reagent.     

The Association of BAG6 with SGTA and Tail-Anchored Proteins

Background

The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate.

Methodology and Principal Findings

BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61β, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6.

Significance

On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates.     

Decoupling of melting domains in immobilized ribonuclease A

Ribonuclease A has been immobilized on silica beads through glutaraldeyde-mediated chemical coupling in order to improve the stability of the protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared with those of the free enzyme. The parameters describing the binding of the inhibitor 3′ -CMP (K_a and ΔH) as monitored by spectrophotometry and calorimetry were not significantly affected after immobilization. Conversely both the stability and unfolding mechanism drastically changed. Thermodynamic analysis of the DSC data suggests that uncoupling of protein domains has occurred as a consequence of the immobilization. The two state approximation of the protein unfolding process is not longer valid for the immobilized RNase. Protein stability strongly depends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobilization on a highly hydrophobic surface, the enzyme is partially in the unfolded state. The binding of a ligand is able to reorganize the protein structure into a native-like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3′-CMP. © 1994 Wiley-Liss, Inc.     

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation

Many gram-negative pathogens use a type IV secretion system (T4SS) to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.     

The Salmonella effector SteA binds phosphatidylinositol 4‐phosphate for subcellular targeting within host cells

Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4‐phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella‐containing vacuole (SCV) and to Salmonella‐induced tubules; using the PI(4)P‐binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N‐terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.