Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, a-DsbA1 and a-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein a-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of a-DsbA1 for both dithiol oxidants(E. coli DsbA, 12%) and disulfide isomerases(E. coli DsbC, 14%) is similar. We therefore sought to establish whether a-DsbA1 is an oxidant or an isomerase based on functional activity. The purified a-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that a-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified a-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins. Copyright © 2008 Elsevier In

Characterization of the DsbA oxidative folding catalyst from pseudomonas aerugionsa reveals a highly oxidizing protein that binds small molecules

Bacterial antibiotic resistance is an emerging global crisis, and treatment of multidrug-resistant gram-negative infections, particularly those caused by the opportunistic human pathogen Pseudomonas aeruginosa, remains a major challenge. This problem is compounded by a lack of new antibiotics in the development pipeline: only two new classes have been developed since the 1960s, and both are indicated for multidrug-resistant gram-positive infections. A promising new approach to combat antibiotic resistance is by targeting bacterial virulence, rather than bacterial viability. The bacterial periplasmic protein DsbA represents a central point for antivirulence intervention because its oxidoreductase activity is essential for the folding and function of almost all exported virulence factors. Here we describe the three-dimensional structure of this DsbA target from P. aeruginosa, and we establish for the first time that a member of this enzyme family is capable of binding small molecules. We also describe biochemical assays that validate the redox activity of PaDsbA. Together, the structural and functional characterization of PaDsbA provides the basis for future studies aimed at designing a new class of antivirulence compounds to combat antibiotic-resistant P. aeruginosa infection

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone

The nature of the Syntaxin4 C-terminus affects Munc18c-supported SNARE assembly

Vesicular transport of cellular cargo requires targeted membrane fusion and formation of a SNARE protein complex that draws the two apposing fusing membranes together. Insulin-regulated delivery and fusion of glucose transporter-4 storage vesicles at the cell surface is dependent on two key proteins: the SNARE integral membrane protein Syntaxin4 (Sx4) and the soluble regulatory protein Munc18c. Many reported in vitro studies of Munc18c:Sx4 interactions and of SNARE complex formation have used soluble Sx4 constructs lacking the native transmembrane domain. As a consequence, the importance of the Sx4 C-terminal anchor remains poorly understood. Here we show that soluble C-terminally truncated Sx4 dissociates more rapidly from Munc18c than Sx4 where the C-terminal transmembrane domain is replaced with a T4-lysozyme fusion. We also show that Munc18c appears to inhibit SNARE complex formation when soluble C-terminally truncated Sx4 is used but does not inhibit SNARE complex formation when Sx4 is C-terminally anchored (by a C-terminal His-tag bound to resin, by a C-terminal T4L fusion or by the native C-terminal transmembrane domain in detergent micelles). We conclude that the C-terminus of Sx4 is critical for its interaction with Munc18c, and that the reported inhibitory role of Munc18c may be an artifact of experimental design. These results support the notion that a primary role of Munc18c is to support SNARE complex formation and membrane fusion

Low-resolution solution structures of Munc18: Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode

Isothermal titration calorimetry data.

<p>The raw data (upper part of each panel) and integrated normalized data (lower part of each panel) are shown from ITC experiments between HMunc18c or Munc18a-His and cognate/non-cognate Sx partners.</p

Reported Munc18c expression and purification.

<p><b>NR*</b>- Not reported, <b>FL</b>- full-length, <b>PDA</b>-pull down assays; <b>IP-</b> immunoprecipitation; <b>ITC</b>- isothermal titration calorimetry; <b>SAXS</b>-small angle X-ray scattering.</p

Purified HMunc18c is monomeric in solution.

<p><b>A</b>. Elution profile of purified HMunc18c on a calibrated analytical size exclusion chromatography column (S200 10/300 GL). HMunc18c eluted at a volume consistent with a ∼70 kDa protein. Peak fractions were analysed on 4–12% gradient SDS-PAGE (inset). <b>B</b>. Elution profile of HMunc18c examined by SEC-MALS. The horizontal blue line corresponds to the SEC-MALS calculated mass (right axis) plotted with the refractive index indicating the peak (left axis) of the protein in the sample (68,200 Da ±0.5%).</p