Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis

Vivian, J. P., Scoullar, J, Rimmer, K, Bushell, S. R., Beddoe, T, Wilce, M. C. J., Byres, E, Boyle, T. P., Doak, B, Simpson, J. S., Graham, B, Heras, B, Kahler, C. M., Rossjohn, J and Scanlon, M. J. (2009) Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis. Journal of Molecular Biology, 394 5: 931-943. doi:10.1016/j.jmb.2009.09.065


Author Vivian, J. P.
Scoullar, J
Rimmer, K
Bushell, S. R.
Beddoe, T
Wilce, M. C. J.
Byres, E
Boyle, T. P.
Doak, B
Simpson, J. S.
Graham, B
Heras, B
Kahler, C. M.
Rossjohn, J
Scanlon, M. J.
Title Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis
Formatted title
Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis
Journal name Journal of Molecular Biology   Check publisher's open access policy
ISSN 0022-2836
Publication date 2009-12-01
Year available 2009
Sub-type Article (original research)
DOI 10.1016/j.jmb.2009.09.065
Open Access Status DOI
Volume 394
Issue 5
Start page 931
End page 943
Total pages 13
Editor R. Huber
Place of publication London, United Kingdom
Publisher Academic Press (Elsevier Inc)
Language eng
Subject C1
970106 Expanding Knowledge in the Biological Sciences
060112 Structural Biology (incl. Macromolecular Modelling)
Abstract The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.
Formatted abstract
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-Å-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.
Keyword DsbAs
disulfide bonds
oxidoreductases
crystal structure
bacterial pathogen
DISULFIDE BOND FORMATION
ESCHERICHIA-COLI
VIBRIO-CHOLERAE
BIOCHEMICAL-CHARACTERIZATION
DESTABILIZING DISULFIDE
CRYSTAL-STRUCTURES
IN-VIVO
PROTEINS
THIOREDOXIN
SUBSTRATE
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID R01 AI055977
R01 AI044239
R56 AI114821
R37 AI033493
R01 AI033493
Institutional Status UQ
Additional Notes Available online 6 October 2009

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
Institute for Molecular Bioscience - Publications
 
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Created: Sun, 10 Jan 2010, 10:04:23 EST