Combination of two separate binding domains defines stoichiometry between type III secretion system chaperone IpgC and translocator protein IpaB

Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.     

The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri

Invasion of epithelial cells by Shigella flexneri involves entry and dissemination. The main effectors of entry, IpaB and IpaC, are also required for contact haemolytic activity and escape from the phagosome in infected macrophages. These proteins are stored in the cytoplasm in association with the chaperone IpgC, before their secretion by a type III secretion apparatus is activated by host cells. We used a His-tagged IpgC protein to purify IpgC-containing complexes and showed that only IpaB and IpaC are associated with IpgC. Plasmids expressing His6-IpgC either alone or together with IpaB or IpaC under the control of an IPTG-inducible lac promoter were introduced into ipgC , ipaB or ipaC mutants. Induction of expression of the recombinant plasmid-encoded proteins by IPTG allowed bacteria to enter epithelial cells, and the role of these proteins in dissemination was investigated by incubating infected cells in either the absence or the presence of IPTG. The size of plaques produced by recombinant strains on cell monolayers was regulated by IPTG, indicating that IpgC, IpaB and IpaC were each required for efficient dissemination. Electron microscopy analysis of infected cells indicated that these proteins were necessary for lysis of the membrane of the protrusions during cell-to-cell spread.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Using disruptive insertional mutagenesis to identify the in situ structure‐function landscape of the Shigella translocator protein IpaB

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle “tip complex” (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N‐terminal half of IpaB were most likely to result in a loss of steady‐state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild‐type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C‐terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post‐secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.     

Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.     

Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa

Pseudomonas aeruginosa, a Gram‐negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein–chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N‐terminal domain. However, disordered and flexible residues located both at the N‐ and C‐terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a “disorder‐to‐order” transition of PopB protein might take place upon PcrH binding. The long helical coiled‐coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. Proteins 2014; 82:3273–3285. © 2014 Wiley Periodicals, Inc.     

Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri

Shigella flexneri causes a severe form of bacillary dysentery also known as shigellosis. Onset of shigellosis requires bacterial invasion of colonic epithelial cells which is initiated by the delivery of translocator and effector proteins to the host cell membrane and cytoplasm, respectively, by the Shigella type III secretion system (TTSS). The Shigella translocator proteins, IpaB and IpaC, form a pore complex in the host cell membrane to facilitate effector delivery; however, prior to their secretion IpaB and IpaC are partitioned in the bacterial cytoplasm by association with the cytoplasmic chaperone IpgC. To determine their structural and biophysical properties, recombinant IpaB/IpgC and IpaC/IpgC complexes were prepared for their first detailed in vitro analysis. Both IpaB/IpgC and IpaC/IpgC complexes are highly stable and soluble heterodimers whose formation prevents IpaB-IpaC interaction as well as Ipa-dependent disruption of phospholipid membranes. Circular dichroism spectroscopy shows that IpgC binding has a detectable influence on IpaC secondary/tertiary structure and stability. In contrast, IpaB structure is not as dramatically affected by chaperone binding. To more precisely ascertain the influence of chaperone binding on IpaC structure and stability, single tryptophan mutants were generated for detailed fluorescence spectroscopy analysis. These mutants provide a low-resolution picture of how IpaC exists in the Shigella cytoplasm with chaperone binding possibly involving distinct regions within the N- and C-terminal halves of IpaC. This preliminary assessment of the IpaC-IpgC interaction is supported by initial deletion mutagenesis studies. The data provide the first structural analysis of IpgC association with IpaB and IpaC.     

Structure-function analysis of the Shigella virulence factor IpaB

Infection by the gram-negative bacterium Shigella flexneri results in dysentery, an acute inflammatory disease of the colon. Essential events in the pathogenesis of Shigella infections include bacterial invasion of epithelial cells, escape from the phagosome, and induction of apoptosis in macrophages. The Shigella virulence factor invasion plasmid antigen B (IpaB) is required for all of these processes. Induction of apoptosis is dependent on IpaB binding to the cysteine protease caspase-1 (Casp-1). The activation of this enzyme triggers both apoptosis and release of the proinflammatory cytokine interleukin-1beta. Several IpaB mutants were generated to correlate function with protein subdomains. We determined that the N-terminal portion of IpaB is necessary for stable expression of IpaB. A putative amphipathic alpha-helical domain preserves the structure of IpaB. We found 10 consecutive residues within the amino terminus of the hydrophobic region that play a critical role in invasion, phagosomal escape, and cytotoxicity. An IpaB mutant carrying a mutation in this region binds to Casp-1 yet is not cytotoxic, even following direct delivery to the macrophage cytoplasm. These results indicate that the association between IpaB and Casp-1 is only a step in the activation of macrophage apoptosis.     

CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells

Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid-phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half- maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella . Pretreatment of cells with an anti-CD44 monoclonal antibody resulted in inhibition of Shigella -induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB–CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.     

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from (1)H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using (1)H-(15)N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.     

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from ¹H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using ¹H-¹⁵N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.     

Binding of copper(I) by the Wilson disease protein and its copper chaperone

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K(a)) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are approximately 10(5) to 10(6) m(-1), suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K(a) values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.     

Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.     

Glycoprotein tertiary and quaternary structures are monitored by the same quality control mechanism

Folding of glycoproteins entering the secretory pathway is strictly surveyed in the endoplasmic reticulum by a quality control system. Folding intermediates and proteins irreparably misfolded are marked via glucosylation by the UDPglucose:glycoprotein glucosyltransferase, an enzyme that acts as a folding sensor by exclusively labeling glycoproteins not displaying their native structures. Here we show that this sensing mechanism also applies to the oligomerization of protein complexes, as the glucosyltransferase appeared to be able to glucosylate folded complex subunits lacking the full complement of oligomer components.     

Hydrophobic moments of tertiary protein structures

The helical hydrophobic moment is a measure of the amphiphilicity of a segment of protein secondary structure. Such measure yields information of potential relevance for issues relating to cell surface binding and secondary structure function. The present article describes a global analog of the helical hydrophobic moment. The global moment provides a concise measure of the degree and direction of the amphiphilicity or hydrophobic imbalance across the entire protein tertiary structure. Therefore, this measure is a succinct representation of the spatial organization of residue hydrophobicity for each protein. With this measure, a simple comparison of the hydrophobic imbalance or segregation of different protein structures can be made. For example, two structures having the same fold and close in root-mean-square deviation may exhibit very different overall hydrophobic organization. Such difference is classified simply by the global moment. Furthermore, the direction of the global moment may point to regions of functional interest. Certain formal issues in the development of such moment are described, and a number of applications to particular protein structures are discussed.     

Secondary and quaternary structures of the (+)-pinoresinol-forming dirigent protein

The (+)-pinoresinol-forming dirigent protein is the first protein capable of stereoselectively coupling two coniferyl alcohol derived radical species, in this case to give the 8-8' linked (+)-pinoresinol. Only dimeric cross-linked dirigent protein structures were isolated when 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide was used as cross-linking agent, whereas the associated oxidase, presumed to generate the corresponding free radical substrate, was not detected. Native Forsythia intermedia dirigent protein isoforms were additionally subjected to MALDI-TOF and ESI-MS analyses, which established the presence of both monomeric masses of 23-25 kDa and dimeric dirigent protein species ranging from 46 to 49 kDa. Analytical ultracentrifugation, sedimentation velocity, and sedimentation equilibrium analyses of the native dirigent protein in open solution confirmed further its dimeric nature as well as a propensity to aggregate, with the latter being dependent upon both temperature and solution ionic strength. Circular dichroism analysis suggested that the dirigent protein was primarily composed of beta-sheet and loop structures.     

The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.     

Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD

Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 Å resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely α-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.     

Probabilistic approach to the design of symmetric protein quaternary structures

Probabilistic methods have been developed that estimate the site-specific probabilities of the amino acids in sequences likely to fold to a particular target structure, and such information can be used to guide the de novo design of proteins and to probe sequence variability. An extension of these methods for the design of symmetric homo-oligomeric quaternary structures is presented. The theory is in excellent agreement with the results of studies on exactly solvable lattice models. Application to an atomically detailed representation of proteins verifies the utility of a symmetry assumption, which greatly simplifies and accelerates the calculations. The method may be applied to a wide variety of symmetric and periodic protein structures.     

Binding of the Shigella protein IpaA to vinculin induces F-actin depolymerization.

Shigella flexneri, the causative agent of bacillary dysentery, enters into epithelial cells by a macropinocytic process. IpaA, a Shigella protein secreted upon cell contact, binds to the focal adhesion protein vinculin and is required for efficient bacterial uptake. IpaA was shown here to bind with high affinity to the N-terminal residues 1-265 of vinculin. Using co-sedimentation and solid-phase assays, we demonstrated that binding of IpaA to vinculin strongly increases the association of vinculin with F-actin. We also characterized a depolymerizing activity on actin filaments associated with the vinculin-IpaA complex both in vitro and in microinjected cells. We propose that the conformational change of vinculin induced by IpaA binding allows interaction of the vinculin-IpaA complex with F-actin and subsequent depolymerization of actin filaments.