Penicillin-binding protein SpoVD disulphide is a target for StoA in Bacillus subtilis forespores

The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae , confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD , stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.     

Transpeptidase activity of penicillin‐binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation‐specific class B penicillin‐binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain – one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation‐specific thiol‐disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra‐cytoplasmic protein disulfides in bacterial cells.     

Bacillus subtilis StoA Is a thiol-disulfide oxidoreductase important for spore cortex synthesis

Bacillus subtilis is an endospore-forming bacterium. There are indications that protein disulfide linkages occur in spores, but the role of thiol-disulfide chemistry in spore synthesis is not understood. Thiol-disulfide oxidoreductases catalyze formation or breakage of disulfide bonds in proteins. CcdA is the only B. subtilis thiol-disulfide oxidoreductase that has previously been shown to play some role in endospore biogenesis. In this work we show that lack of the StoA (YkvV) protein results in spores sensitive to heat, lysozyme, and chloroform. Compared to CcdA deficiency, StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiency. StoA is a membrane-bound protein with a predicted thioredoxin-like domain probably localized in the intermembrane space of the forespore. Electron microscopy of spores of CcdA- and StoA-deficient strains showed that the spore cortex is absent in both cases. The BdbD protein catalyzes formation of disulfide bonds in proteins on the outer side of the cytoplasmic membrane but is not required for sporulation. Inactivation of bdbD was found to suppress the sporulation defect of a strain deficient in StoA. Our results indicate that StoA is a thiol-disulfide oxidoreductase that is involved in breaking disulfide bonds in cortex components or in proteins important for cortex synthesis.     

Cytosolic thioredoxin system facilitates the import of mitochondrial small Tim proteins

Thiol‐disulphide redox regulation has a key role during the biogenesis of mitochondrial intermembrane space (IMS) proteins. Only the Cys‐reduced form of precursor proteins can be imported into mitochondria, which is followed by disulphide bond formation in the mitochondrial IMS. In contrast to the wealth of knowledge on the oxidation process inside mitochondria, little is known about how precursors are maintained in an import‐competent form in the cytosol. Here we provide the first evidence that the cytosolic thioredoxin system is required to maintain the IMS small Tim proteins in reduced forms and facilitate their mitochondrial import during respiratory growth.     

Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40–substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40–substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.     

Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. A model for serine protease catalysis

Purified protein-disulphide isomerase has been examined for effects on the pathway and kinetics of the unfolding and refolding which accompanies disulphide bond breakage and reformation in bovine pancreatic trypsin inhibitor and bovine ribonuclease A. The intermediates of the pathways were not altered, although some interconversions which normally are not significant became so in the presence of the isomerase. The rate of every step involving both substantial protein conformational changes and protein disulphide bond formation, breakage or rearrangement was found to be increased significantly, but only when the conformational changes were rate-determining. The protein-disulphide isomerase appears to be a true catalyst of protein unfolding and refolding involving disulphide bond breakage, formation or rearrangement.     

Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically

Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In this study, we investigated the effects of the biologically relevant thiol-disulphide redox molecule, glutathione, and Zn2+-binding on the oxidative folding of yeast mitochondrial Tim10 using both biochemical and biophysical methods in vitro. We show that, whilst oxidized Tim10 cannot be reduced by reduced glutathione, reduced Tim10 is effectively oxidized at levels of glutathione comparable to those found in the cytosol. The oxidized Tim10 generated in the presence of glutathione is competent for complex formation with its partner protein Tim9, confirming it has a native fold. The standard redox potential of Tim10 at pH 7.4 was determined to be -0.32 V, confirming that Tim10 is a much stronger reductant than glutathione (-0.26 V, at pH 7.4) and could therefore be oxidized rapidly by oxidized glutathione in the cytosol. However, we found that Zn2+-binding can stabilize the reduced Tim10, decreasing the rate of the oxidative folding more than tenfold. In addition, we show that protein disulphide isomerase can catalyse the oxidative folding of Tim10 provided that Zn2+ was removed. We propose that Zn2+-binding is essential to maintain the protein in a reduced and import-competent state in the cytosol, and that zinc has to be removed after the protein is imported into mitochondria to initiate protein oxidative folding and assembly.     

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.     

Multiple interactions of a DNA-binding protein in vivo. II. Effects of host mutations on DNA replication of phage T4 gene 32 mutants.

A quantitative analysis has been made of the kinetics of disulphide bond formation, breakage, and rearrangement which occur during the folding and unfolding of the pancreatic trypsin inhibitor. The results have been used to infer the energetics of the protein conformational transitions which accompany each step.The folding transition is shown to be a co-operative process in which all intermediate states with one or two disulphide bonds are unstable relative to the unfolded, reduced protein and that in the fully folded conformation with three disulphide bonds. The approximate two-state nature of the transition at equilibrium was demonstrated experimentally. The folding transition of the inhibitor which has been determined kinetically is therefore analogous to that observed generally with other globular proteins.     

Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

The zinc-binding protein Hot13 promotes oxidation of the mitochondrial import receptor Mia40

A disulphide relay system mediates the import of cysteine-containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine-rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc-binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1-dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc-binding chelators. We propose that Hot13 maintains Mia40 in a zinc-free state, thereby facilitating its efficient oxidation by Erv1.     

Oxidative folding of small Tims is mediated by site-specific docking onto Mia40 in the mitochondrial intermembrane space

Oxidative folding in the mitochondrial intermembrane space (IMS) is crucial for the import of certain cysteine-rich IMS proteins. The essential proteins Mia40 and Erv1 are key components for this mechanism functioning as a disulphide protein cascade that is functionally linked to the respiratory chain by shuttling electrons onto CytC. The subunits of the chaperone complex Tim9-Tim10 require Mia40 for their biogenesis. Previously, it was shown that the four cysteines of Tim10 are crucial for folding and assembly, that they are connected intramolecularly into an inner and an outer disulphide bridge, and that the inner disulphide has a more prominent role in these processes. Here we show that interaction with Mia40 is a site-specific event: (i) the N-terminal first cysteine of the precursor is crucial for docking onto Mia40 via a mixed disulphide; (ii) release is triggered by disulphide pairing of the C-terminal cysteine onto the N-terminal one; and (iii) formation of the inner disulphide between the second and third cysteines apparently precedes the release reaction and is critical for assembly with Tim9. The Tim10-Mia40 interaction is independent of divalent cations, any other mitochondrial proteins or membranes, and is shown to occur efficiently in organello and in vitro.     

The MIA pathway: a tight bond between protein transport and oxidative folding in mitochondria

Many newly synthesized proteins obtain disulfide bonds in the bacterial periplasm, the endoplasmic reticulum (ER) and the mitochondrial intermembrane space. The acquisition of disulfide bonds is critical for the folding, assembly and activity of these proteins. Spontaneous oxidation of thiol groups is inefficient in vivo, therefore cells have developed machineries that catalyse the oxidation of substrate proteins. The identification of the machinery that mediates this process in the intermembrane space of mitochondria, known as MIA (mitochondrial intermembrane space assembly), provided a unique mechanism of protein transport. The MIA machinery introduces disulfide bonds into incoming intermembrane space precursors and thus tightly couples the process of precursor translocation to precursor oxidation. We discuss our current understanding of the MIA pathway and the mechanisms that oversee thiol-exchange reactions in mitochondria.     

Interactions between cysteine residues as probes of protein conformation: the disulphide bond between Cys-14 and Cys-38 of the pancreatic trypsin inhibitor.

The rate constants for the reversible reduction by dithiothreitol of the disulphide bond linking eysteines 14 and 38 of the native pancreatic trypsin inhibitor have been measured. The results are consistent with this disulphide bond being formed as the last step in refolding of the fully reduced inhibitor.The rates of reduction of several model linear disulphides have been measured under the same conditions. A linear relationship was found between the rate of reduction and the ionization tendency of the thiol group generated.The apparent pK value of the thiol groups of cysteines 14 and 38 of the selectively reduced inhibitor were measured by the pH-dependence of their rate of alkylation with iodoacetamide. The rate of reduction of the disulphide bond between these two residues was very close to that predicted from the model compounds.The kinetics and thermodynamics of disulphide bond formation and breakage are shown to be useful for experimental determination of conformational transitions in proteins and model peptides.     

Interactions Between Late-Acting Proteins Required for Peptidoglycan Synthesis during Sporulation

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the latter steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here, we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a nonessential, sporulation-specific homolog of the well-conserved and essential SEDS (shape elongation, division, and sporulation) proteins, and SpoVD, a nonessential class B penicillin binding protein. We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and fluorescence resonance energy transfer experiments indicated that SpoVE and SpoVD interact, and co-affinity purification in Escherichia coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of an SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in loss of function of the entire fusion that was not complemented by a wild-type protein. Thus, SpoVE has a direct and functional interaction with SpoVD, and this conclusion will facilitate understanding the essential function that SpoVE and related SEDS proteins, such as FtsW and RodA, play in bacterial growth and division.     

Cell polarization during monopolar cytokinesis

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。