scholarly journals Role of the Intramolecular Disulfide Bond in FlgI, the Flagellar P-Ring Component of Escherichia coli

2006 ◽  
Vol 188 (12) ◽  
pp. 4190-4197 ◽  
Author(s):  
Yohei Hizukuri ◽  
Toshiharu Yakushi ◽  
Ikuro Kawagishi ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Self Assembly ◽  
Bacterial Species ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Wild Type ◽  
Ring Assembly ◽  
Intramolecular Disulfide Bond

ABSTRACT The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our ΔflgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of ΔdsbB cells. Additionally, the low level of FlgI observed in the ΔdsbB strain increased in the presence of l-cystine, an oxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient self-assembly into the P ring.

scholarly journals Disulfide Bond Formation in Secreton Component PulK Provides a Possible Explanation for the Role of DsbA in Pullulanase Secretion

2001 ◽  
Vol 183 (4) ◽  
pp. 1312-1319 ◽  
Author(s):  
Anthony P. Pugsley ◽  
Nicolas Bayan ◽  
Nathalie Sauvonnet
Keyword(s):  
Disulfide Bond ◽  
Klebsiella Oxytoca ◽  
Bond Formation ◽  
Type Ii Secretion ◽  
Disulfide Bond Formation ◽  
Gene Encoding ◽  
Intramolecular Disulfide Bond ◽  
Disulfide Oxidoreductase ◽  
Considerable Excess

ABSTRACT When expressed in Escherichia coli, the 15Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by adsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.

scholarly journals Role of disulfide bond formation in the folding and assembly of the envelope glycoproteins of a pestivirus

2002 ◽  
Vol 296 (2) ◽  
pp. 470-476 ◽  
Author(s):  
Norica Branza-Nichita ◽  
Catalin Lazar ◽  
David Durantel ◽  
Raymond A Dwek ◽  
Nicole Zitzmann
Keyword(s):  
Disulfide Bond ◽  
Bond Formation ◽  
Envelope Glycoproteins ◽  
Disulfide Bond Formation

scholarly journals The crystal structure of wild-type human brain neuroglobin reveals flexibility of the disulfide bond that regulates oxygen affinity

2014 ◽  
Vol 70 (4) ◽  
pp. 1005-1014 ◽  
Author(s):  
Beatriz G. Guimarães ◽  
Djemel Hamdane ◽  
Christophe Lechauve ◽  
Michael C. Marden ◽  
Béatrice Golinelli-Pimpaneau
Keyword(s):  
Disulfide Bond ◽  
Conformational Transition ◽  
Bond Formation ◽  
Oxygen Affinity ◽  
Disulfide Bridge ◽  
Wild Type ◽  
X Ray ◽  
Intramolecular Disulfide Bond ◽  
Internal Cavities ◽  
High Oxygen Affinity

Neuroglobin plays an important function in the supply of oxygen in nervous tissues. In human neuroglobin, a cysteine at position 46 in the loop connecting the C and D helices of the globin fold is presumed to form an intramolecular disulfide bond with Cys55. Rupture of this disulfide bridge stabilizes bi-histidyl haem hexacoordination, causing an overall decrease in the affinity for oxygen. Here, the first X-ray structure of wild-type human neuroglobin is reported at 1.74 Å resolution. This structure provides a direct observation of two distinct conformations of the CD region containing the intramolecular disulfide link and highlights internal cavities that could be involved in ligand migration and/or are necessary to enable the conformational transition between the low and high oxygen-affinity states following S—S bond formation.

scholarly journals The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Brian M. Meehan ◽  
Cristina Landeta ◽  
Dana Boyd ◽  
Jonathan Beckwith
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Anaerobic Growth ◽  
Strictly Anaerobic ◽  
Disulfide Bond Formation ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Laboratory Conditions

ABSTRACT Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited. IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

Expression of a Two Chain Gamma-Glutamyl Carboxylase: Importance of Disulfide Bond Formation and Transmembrane Domain Interactions.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1694-1694
Author(s):  
Jian-Kie Tie ◽  
Mei-Yan Zheng ◽  
Darrel W. Stafford ◽  
David L. Straight
Keyword(s):  
Vitamin K ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Active Enzyme ◽  
Chain Molecule ◽  
Disulfide Bond Formation ◽  
Wild Type ◽  
Single Chain ◽  
Disulfide Linkage ◽  
Terminal Fragment

Abstract The vitamin K-dependent carboxylase is an integral membrane protein with five transmembrane domains (TMDs). It catalyzes the post-translational modification of specific glutamic acid residues of vitamin K-dependent proteins to gamma-carboxyglutamic acid residues. This posttranslational modification is critical for the biological functions of blood coagulation. The native enzyme is a single chain molecule with one disulfide bond. In this study, we have expressed carboxylase as two chains: residues 1–345 and 346–758 in the same insect cells. Our results show that these two fragments are assembled into a fully active enzyme and are joined by a disulfide. Affinity purification of the carboxylase C-terminal fragment (346–758) results in co-purification of the N-terminal fragment (1–345) even under reducing condition. This indicates that, in addition to the disulfide linkage between these two fragments, they are also linked by non-covalent interactions. One possibility is that the hydrophobic interactions between the TMDs play a role. According to carboxylase membrane topology, there are four TMDs (1–4) in the N-terminal fragment and one TMD (fifth) in the C-terminal fragment. The C-terminal fragment contains all glycosylation sites. When we introduced two prolines to disrupt the transmembrane helix in the wild type carboxylase’s fifth TMD, glycosylation was eliminated. This indicates that the domain is not inserted into the lumen of the ER, but remains in the cytoplasm. Therefore, as our results demonstrate, in the two chain carboxylase with its fifth TMD disrupted, the two chains do not form a disulfide bound nor do they associate through essential non-covalent TMD interactions. While proline residues can disrupt membrane helices as described above, they often occur at the interface between the membrane and the lumenal surface of ER; these prolines appear to affect the chain orientation as it exits the membrane. There is a proline at residue 378 near the lumenal surface of the fifth TMD helix of carboxylase. To examine P378’s effect on disulfide bond formation, we mutated it to leucine. Results show that less disulfide bond formed in the two chain mutant carboxylase and the protein was significantly degraded when compared to the unmutated two chain molecule. Based on our results, we conclude the following: 1) the two chain carboxylase is assembled into a single molecule in vivo and the two chains are joined by a disulfide bond, the enzyme carboxylates gla-containing substrates and binds propeptide with affinity similar to that of wild type enzyme. Therefore, this molecule is a good model for structural studies of TMD interactions and disulfide bond formation; 2) TMD association in the membrane is important for the orientation of the N- and C-terminal portions of carboxylase to be assembled into the active enzyme; 3) and finally proline residue 378 at the lumenal interface of the fifth TMD plays a key role in the conformation which promotes disulfide formation.

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