Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis

The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallographic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbD_Ox, n-NmDsbD_Red, c-NmDsbD_Ox and c-NmDsbD_Red diffracted to 2.3, 1.6, 2.3 and 1.7 resolution and belonged to space groups P2₁3, P321, P4₁ and P12₁1, respectively.The cloning, purification, biophysical characterization, crystallization and X-ray diffraction analysis of the two periplasmic domains of Neisseria meningitidis DsbD, a disulfide reductase that is essential for the viability of this human pathogen, are reported.