ABSTRACT
Proper periplasmic disulfide bond formation is important for folding and stability of many secreted and membrane proteins, and is catalysed by three DsbA oxidoreductases in Neisseria meningitidis. DsbD provides reducing power to DsbC that shuffles incorrect disulfide bond in misfolded proteins as well as to the periplasmic enzymes that reduce apo-cytochrome c (CcsX) or repair oxidative protein damages (MrsAB). The expression of dsbD, but not other dsb genes, is positively regulated by the MisR/S two-component system. Quantitative real-time PCR analyses showed significantly reduced dsbD expression in all misR/S mutants, which was rescued by genetic complementation. The direct and specific interaction of MisR with the upstream region of the dsbD promoter was demonstrated by electrophoretic mobility shift assay, and the MisR binding sequences were mapped. Further, the expression of dsbD was found to be induced by dithiothrietol (DTT), through the MisR/S regulatory system. Surprisingly, we revealed that inactivation of dsbD can only be achieved in a strain carrying an ectopically located dsbD, in the dsbA1A2 double mutant or in the dsbA1A2A3 triple mutant, thus DsbD is indispensable for DsbA-catalysed oxidative protein folding in N. meningitidis. The defects of the meningococcal dsbA1A2 mutant in transformation and resistance to oxidative stress were more severe in the absence of dsbD.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Neisseria meningitidis/metabolism , Base Sequence , Molecular Sequence Data , Neisseria meningitidis/genetics , Oxidative Stress , Promoter Regions, Genetic , Protein BindingABSTRACT
Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-A-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.
Subject(s)
Neisseria meningitidis/enzymology , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Escherichia coli Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Binding , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.
