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ISSN: 2053-230X

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

aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia, bAustralian Centre for Neutron Scattering, Australian Nuclear Science and Technology Organisation, New South Wales 2234, Australia, cSchool of Pathology and Laboratory Medicine, The University of Western Australia, Crawley, Western Australia 6009, Australia, and dMedicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia
*Correspondence e-mail: b.heras@latrobe.edu.au

Edited by J. Newman, Bio21 Collaborative Crystallisation Centre, Australia (Received 19 October 2017; accepted 12 December 2017)

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-NmDsbDOx, n-NmDsbDRed, c-NmDsbDOx and c-NmDsbDRed diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P213, P321, P41 and P1211, respectively.

1. Introduction

In bacteria, disulfide-bond formation occurs in the periplasm and is mediated by the disulfide-bond (Dsb) family of proteins (Heras et al., 2007[Heras, B., Kurz, M., Shouldice, S. R. & Martin, J. L. (2007). Curr. Opin. Struct. Biol. 17, 691-698.], 2009[Heras, B., Shouldice, S. R., Totsika, M., Scanlon, M. J., Schembri, M. A. & Martin, J. L. (2009). Nature Rev. Microbiol. 7, 215-225.]; Kadokura & Beckwith, 2010[Kadokura, H. & Beckwith, J. (2010). Antioxid. Redox Signal. 13, 1231-1246.]). In most Gammaproteobacteria, Dsb proteins form a two-pathway system. In the oxidation pathway, DsbA and its cognate oxidase DsbB introduce disulfide bonds into newly translocated proteins that are undergoing oxidative folding in the periplasm (Kamitani et al., 1992[Kamitani, S., Akiyama, Y. & Ito, K. (1992). EMBO J. 11, 57-62.]; Bardwell et al., 1993[Bardwell, J. C., Lee, J. O., Jander, G., Martin, N., Belin, D. & Beckwith, J. (1993). Proc. Natl Acad. Sci. USA, 90, 1038-1042.]; Missiakas et al., 1993[Missiakas, D., Georgopoulos, C. & Raina, S. (1993). Proc. Natl Acad. Sci. USA, 90, 7084-7088.]; Depuydt et al., 2011[Depuydt, M., Messens, J. & Collet, J.-F. (2011). Antioxid. Redox Signal. 15, 49-66.]). The isomerase pathway is composed of the isomerase DsbC and the reductase DsbD, which are responsible for reshuffling non-native disulfide bonds formed by the DsbA/DsbB system (Cho & Collet, 2013[Cho, S.-H. & Collet, J.-F. (2013). Antioxid. Redox Signal. 18, 1690-1698.]; Missiakas et al., 1994[Missiakas, D., Georgopoulos, C. & Raina, S. (1994). EMBO J. 13, 2013-2020.], 1995[Missiakas, D., Schwager, F. & Raina, S. (1995). EMBO J. 14, 3415-3424.]; Rietsch et al., 1996[Rietsch, A., Belin, D., Martin, N. & Beckwith, J. (1996). Proc. Natl Acad. Sci. USA, 93, 13048-13053.]).

DsbD is a 59 kDa integral membrane reductase that transfers electrons from the cytoplasm to periplasmic substrate proteins (Arts et al., 2015[Arts, I. S., Gennaris, A. & Collet, J.-F. (2015). FEBS Lett. 589, 1559-1568.]; Cho & Collet, 2013[Cho, S.-H. & Collet, J.-F. (2013). Antioxid. Redox Signal. 18, 1690-1698.]; Rietsch et al., 1997[Rietsch, A., Bessette, P., Georgiou, G. & Beckwith, J. (1997). J. Bacteriol. 179, 6602-6608.]). Escherichia coli DsbD (EcDsbD) consists of a membrane-spanning domain, t-DsbD, flanked by two periplasmic domains, n-DsbD and c-DsbD (Katzen et al., 2002[Katzen, F., Deshmukh, M., Daldal, F. & Beckwith, J. (2002). EMBO J. 21, 3960-3969.]; Cho et al., 2007[Cho, S.-H., Porat, A., Ye, J. & Beckwith, J. (2007). EMBO J. 26, 3509-3520.]). The N- and C-terminal domains of DsbD adopt an immunoglobulin-like and a thioredoxin-like fold, respectively (Rozhkova et al., 2004[Rozhkova, A., Stirnimann, C. U., Frei, P., Grauschopf, U., Brunisholz, R., Grütter, M. G., Capitani, G. & Glockshuber, R. (2004). EMBO J. 23, 1709-1719.]). t-DsbD is membrane-embedded and consists of eight α-helices (Cho & Beckwith, 2009[Cho, S.-H. & Beckwith, J. (2009). J. Biol. Chem. 284, 11416-11424.]). Each DsbD module harbours two catalytic cysteines, which allow efficient electron flow through the three DsbD domains via a sequential cascade of thiol–disulfide exchange reactions (Stewart et al., 1999[Stewart, E. J., Katzen, F. & Beckwith, J. (1999). EMBO J. 18, 5963-5971.]; Katzen & Beckwith, 2000[Katzen, F. & Beckwith, J. (2000). Cell, 103, 769-779.]). This process begins with oxidized t-DsbD, which accepts electrons from reduced cytoplasmic thioredoxin, leaving t-DsbD reduced and thioredoxin oxidized (Stewart et al., 1999[Stewart, E. J., Katzen, F. & Beckwith, J. (1999). EMBO J. 18, 5963-5971.]). Reduced t-DsbD undergoes a conformational change which allows interaction with and reduction of c-DsbD (Cho & Collet, 2013[Cho, S.-H. & Collet, J.-F. (2013). Antioxid. Redox Signal. 18, 1690-1698.]). The latter domain reduces n-DsbD, which then reduces disulfides in its periplasmic substrates (Arts et al., 2015[Arts, I. S., Gennaris, A. & Collet, J.-F. (2015). FEBS Lett. 589, 1559-1568.]) such as DsbC, CcmG and DsbG (Stirnimann et al., 2005[Stirnimann, C. U., Rozhkova, A., Grauschopf, U., Grütter, M. G., Glockshuber, R. & Capitani, G. (2005). Structure, 13, 985-993.]; Depuydt et al., 2009[Depuydt, M., Leonard, S. E., Vertommen, D., Denoncin, K., Morsomme, P., Wahni, K., Messens, J., Carroll, K. S. & Collet, J.-F. (2009). Science, 326, 1109-1111.]).

Dsb proteins promote multiple virulence phenotypes and regulate the redox balance in the cell envelope, but in general these proteins are not required for survival. However, this is not the case in Neisseria meningitidis, where DsbD (NmDsbD) was found to be essential for the viability of this obligate human pathogen (Kumar et al., 2011[Kumar, P., Sannigrahi, S., Scoullar, J., Kahler, C. M. & Tzeng, Y.-L. (2011). Mol. Microbiol. 79, 1557-1573.]). We are interested in investigating the structural and functional characteristics of NmDsbD in order to understand the molecular basis of this uncommon phenotype. Furthermore, this information could form the basis for the future development of antineisserial agents targeting DsbD (Smith et al., 2016[Smith, R. P., Paxman, J. J., Scanlon, M. J. & Heras, B. (2016). Molecules, 21, 811.]). Here, we report the expression, biophysical characterization, crystallization and preliminary X-ray diffraction data for the two periplasmic domains of NmDsbD in both redox states.

2. Materials and methods

2.1. Macromolecular production

For the expression of the N- and C-terminal domains of NmDsbD (residues 1–124 and 465–579, respectively, of the mature protein), the corresponding coding regions were PCR-amplified from N. meningitidis strain NMB genomic DNA. Ligation-independent cloning (LIC) was then used to insert the sequences into bacterial expression vector pMCSG7 (Eschenfeldt et al., 2009[Eschenfeldt, W. H., Lucy, S., Millard, C. S., Joachimiak, A. & Mark, I. D. (2009). Methods Mol. Biol. 498, 105-115.]). The final constructs contained an N-terminal His6 tag that preceded a Tobacco etch virus (TEV) protease cleavage site. This tag is used in the purification of the proteins and is cleaved during the process, leaving two additional residues (SN) on n-NmDsbD and three additional residues (SNA) on c-NmDsbD (Table 1[link]).

Table 1
Macromolecule-production information

  n-NmDsbD c-NmDsbD
Source organism N. meningitidis strain NMB N. meningitidis strain NMB
DNA source N. meningitidis strain NMB N. meningitidis strain NMB
Forward primer (5′–3′) TACTTCCAATCCAATGCGAACGATCTGCTGCCGC TACTTCCAATCCAATGCGATGTTTGCCGATACTGCCGCGC
Reverse primer (5′–3′) TTATCCACTTCCAATGTCAGGTTTGCGGATGGTAAGTGC TTATCCACTTCCAATGTCAGCGGTTTTGTTCATACCACTCG
Cloning vector pMCSG7 pMCSG7
Expression vector pMCSG7 pMCSG7
Expression host E. coli BL21 (DE3) pLysS E. coli BL21 (DE3) pLysS
Complete amino-acid sequence of the construct produced MHHHHHHSSGVDLGTENLYFQSNANDLLPPEKAFVPELAVADDGVNVRFRIADGYYMYQAKIVGKTDPADLLGQPSFSKGEEKEDEFFGRQTVYHHEAQVAFPYAKAVGEPYKLVLTYQGCAEVGVCYPPVDTEFDISGNGTYHPQT MHHHHHHSSGVDLGTENLYFQSNAMFADTAALKAAMDTALKEHPDKPVVLDFYADWCISCKEMAAYTLNQPEVHQAVDMERFFQIDVTANKPEHQALLKEYGLFGPPGVFVVRSDGSRSEPLLGFVKADKFIEWYEQN

Plasmid constructs pMCSG7::n-NmdsbD and pMCSG7::c-NmdsbD were transformed into E. coli BL21 (DE3) pLysS cells. n-NmDsbD and c-NmDsbD were expressed using the autoinduction method (Studier, 2005[Studier, F. W. (2005). Protein Expr. Purif. 41, 207-234.]). Briefly, E. coli BL21 (DE3) pLysS cells harbouring n-NmDsbD- and c-NmDsbD-containing plasmids were grown at 303 K for 24 h with agitation at 180 rev min−1 in rich (ZY) medium supplemented with 100 µg ml−1 ampicillin and 34 µg ml−1 chloramphenicol (typically to a final optical density at 600 nm in the range between 4 and 5). Bacterial cultures were harvested by centrifugation (7500g, 20 min, 277 K) and flash-frozen before storage at 183 K.

For purification, harvested pellets were resuspended in 25 mM Tris pH 8.0, 150 mM NaCl supplemented with EDTA-free protease-inhibitor cocktail tablets and 400 units of DNase I per litre of culture. Cells were lysed using a bench-top cell disruptor (one cycle at 241 MPa; TS series, Constant Systems Ltd). Cellular debris was removed from the lysate via centrifugation (30 000g, 20 min, 277 K) and the lysate was loaded onto a 5 ml HisTrap FF column (GE Healthcare). Proteins were eluted from the column in 25 mM Tris pH 8.0, 150 mM NaCl and a gradient of 0–500 mM imidazole using a Bio-Rad NGC system. Recombinant His-tagged TEV protease (Cabrita et al., 2007[Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. & Bottomley, S. P. (2007). Protein Sci. 16, 2360-2367.]) was added to fractions containing His6-n-NmDsbD and His6-c-NmDsbD (0.2 and 0.4 mg TEV protease per 10 mg of protein, respectively) supplemented with 5 mM reduced dithiothreitol (DTTRed) and samples were dialyzed overnight against 25 mM Tris pH 8.0, 150 mM NaCl. The samples were further purified using a 5 ml HisTrap FF column followed by size-exclusion chromatography (SEC) using a Superdex S-75 16/60 column (GE Healthcare) pre-equilibrated in 25 mM HEPES pH 6.7, 150 mM NaCl. The target proteins were oxidized using 1.7 mM copper phenanthro­lene or reduced using 40 molar equivalents of reduced DTT and incubated for 1 h at 277 K. Oxidized and reduced proteins were buffer-exchanged into 25 mM HEPES pH 6.7, 50 mM NaCl and the same buffer supplemented with 1 mM EDTA using a 10 ml PD10 column (GE Healthcare) prior to crystallization experiments. The redox state of the NmDsbD domains was confirmed by Ellman's assay (Evans & Ellman, 1959[Evans, J. C. & Ellman, G. L. (1959). Biochim. Biophys. Acta, 33, 574-576.]).

2.2. Small-angle X-ray scattering (SAXS) studies

SAXS data for n-NmDsbD and c-NmDsbD were collected on the SAXS-WAXS beamline at the Australian Synchrotron (Kirby et al., 2013[Kirby, N. M., Mudie, S. T., Hawley, A. M., Cookson, D. J., Mertens, H. D. T., Cowieson, N. & Samardzic-Boban, V. (2013). J. Appl. Cryst. 46, 1670-1680.]; Table 2[link]). Serial dilutions of an ∼5 mg ml−1 stock were loaded into a 96-well plate. The estimated molecular mass was calculated using contrast and partial specific volumes determined from the protein sequences (Whitten et al., 2008[Whitten, A. E., Cai, S. & Trewhella, J. (2008). J. Appl. Cryst. 41, 222-226.]). The pair-distance distribution function [p(r)] was generated from the experimental data using GNOM (Svergun, 1992[Svergun, D. I. (1992). J. Appl. Cryst. 25, 495-503.]), from which I(0), Rg and Dmax were determined (Figs. 1[link]a and 1[link]b). The program DAMMIN (Svergun, 1999[Svergun, D. I. (1999). Biophys. J. 76, 2879-2886.]) was used to generate 16 molecular envelopes for each protein, which were averaged using the program DAMAVER (Volkov & Svergun, 2003[Volkov, V. V. & Svergun, D. I. (2003). J. Appl. Cryst. 36, 860-864.]), and the resolutions of the averaged structures were estimated using SASRES (Tuukkanen et al., 2016[Tuukkanen, A. T., Kleywegt, G. J. & Svergun, D. I. (2016). IUCrJ, 3, 440-447.]). The experimental scattering data were compared with scattering curves calculated from the crystal structures of the corresponding domains from E. coli [n-EcDsbD, PDB entry 1l6p (Goulding et al., 2002[Goulding, C. W., Sawaya, M. R., Parseghian, A., Lim, V., Eisenberg, D. & Missiakas, D. (2002). Biochemistry, 41, 6920-6927.]), and c-EcDsbD, PDB entry 1uc7 (Kim et al., 2003[Kim, J. H., Kim, S. J., Jeong, D. G., Son, J. H. & Ryu, S. E. (2003). FEBS Lett. 543, 164-169.])] using CRYSOL (Svergun et al., 1995[Svergun, D., Barberato, C. & Koch, M. H. J. (1995). J. Appl. Cryst. 28, 768-773.]) (Fig. 1[link]).

Table 2
SAXS data-collection and analysis details

  n-NmDsbD c-NmDsbD
Data-collection parameters
 Instrument SAXS-WAXS (Australian Synchrotron)
 Beam geometry Point
 Wavelength (Å) 1.127
 Sample-to-detector distance (m) 1.480
q-range (Å−1) 0.01–0.60
 Exposure time (s) 60 (30 × 2 s exposures)
 Protein concentration range (mg ml−1) ∼0.3–5.0
 Temperature (K) 285
 Standard Water
Structural parameters    
I(0) (cm−1) (from Guinier) 0.00776 ± 0.00003 0.00997 ± 0.00002
Rg (Å) (from Guinier) 17.6 ± 0.2 15.3 ± 0.1
I(0) (cm−1) [from p(r)] 0.007688 ± 0.000012 0.009905 ± 0.000011
Rg (Å) [from p(r)] 17.3 ± 0.1 14.8 ± 0.1
Dmax (Å) 57 ± 3 45 ± 3
Molecular-mass determination
 Protein concentration (mg ml−1) 0.74 1.12
 Partial specific volume (cm3 g−1) 0.73 0.73
 Contrast, Δρ (1010 cm−2) 2.91 2.97
 Molecular mass Mr [from I(0)] 13200 ± 1000 11800 ± 1000
 Molecular mass Mr (expected) 13911 13358
Software employed
 Primary data reduction scatterBrain (v.1.0)
 Data processing PRIMUS (v.3.2) and GNOM (v.4.6)
Ab initio modelling DAMMIN (v.5.3)
 Validation and averaging DAMAVER (v.2.8.0)
 Computation of model intensities CRYSOL (v.2.8.3)
†The Rg for n-NmDsbD showed mild concentration dependence above a concentration of 1.0 mg ml−1, decreasing from ∼17.5 Å at 0.37 mg ml−1 to ∼16 Å at 5.88 mg ml−1. The 0.74 mg ml−1 data set was used for further analysis and modelling as it was deemed to be the highest concentration at which the effects of concentration were negligible. The change in Rg for c-NmDsbD showed no obvious systematic trend in the concentration range measured. A concentration of 1.12 mg ml−1 was chosen for further analysis and modelling.
[Figure 1]
Figure 1
Small-angle X-ray scattering from n-NmDsbD and c-NmDsbD. (a) Measured scattering data for n-NmDsbD (red) and c-NmDsbD (blue; scaled by a factor of 0.1 for clarity). Overlaid are the calculated scattering curves for n-EcDsbD (PDB entry 1l6p; black curve, χ2 = 2.38) and c-EcDsbD (PDB entry 1uc7; yellow curve, χ2 = 1.65). Inset: Guinier plots of the scattering data. (b) The pair-distance distribution functions for n-NmDsbD (red) and c-­NmDsbD (blue). (c) Probable domain shapes for n-NmDsbD (SASBDB entry SASDCH7; red envelope, resolution = 19 ± 2 Å) and c-NmDsbD (SASBDB entry SASDCJ7; blue envelope, resolution = 18 ± 2 Å) obtained from ab intio modelling against the scattering data. The crystal structures of n-­EcDsbD (PDB entry 1l6p) and c-EcDsbD (PDB entry 1uc7) are shown as cartoons. The grey envelopes represent the total volume encompassed by the 16 aligned models for n-NmDsbD (χ2 = 1.90 ± 0.01) and c-NmDsbD (χ2 = 1.60 ± 0.01).

2.3. Crystallization

Initial high-throughput crystallization experiments were performed at 293 K either in-house using a Mosquito crystallization robot (TTP Labtech) or a Crystal Gryphon Liquid Handling System (Art Robbins Instruments) or at the CSIRO Collaborative Crystallisation Centre (http://www.csiro.au/C3), Melbourne, Australia. For n-NmDsbDOx, c-NmDsbDOx, n-NmDsbDRed and c-NmDsbDRed, 96-well hanging-drop plates were prepared using 71, 44, 60 and 20–40 mg ml−1 protein solutions, respectively, and commercially available crystallization screens (Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2, Index and Salt RX from Hampton Research, PACT premier and JCSG-plus from Molecular Dimensions Ltd and Precipitant Synergy from Jena Bioscience) and an in-house malonate grid screen (pH 5–7 and 0.2–3.4 M malonate).

Initial crystallization experiments for the reduced proteins (n-NmDsbDRed and c-NmDsbDRed) were performed using a Crystal Gryphon Liquid Handling System (Art Robbins Instruments). Crystallization experiments for n-NmDsbDRed were set up with 60 mg ml−1 protein solution, whereas experiments for c-NmDsbDRed were set up with both 20 and 40 mg ml−1 protein solutions. 96-well sitting-drop plates (200 nl protein solution and 200 nl reservoir solution equilibrated against 70 µl reservoir solution) were prepared using commercially available crystallization screens (PEG/Ion 1 and 2, Salt RX, Index, Crystal Screen and Crystal Screen 2 from Hampton Research and PACT premier, JCSG-plus and Morpheus from Molecular Dimensions Ltd).

All crystal-optimization experiments were carried out at 293 K in 24-well hanging-drop VDXm plates using 18 mm siliconized cover slips (Hampton Research), a reservoir volume of 500 µl and a drop size of 2 µl (1 µl protein and 1 µl reservoir solution).

Crystals of n-NmDsbDOx only grew in 20%(w/v) PEG 6000, 0.01 M zinc chloride, 0.1 M MES pH 6 (PACT premier). This condition was optimized by varying different components and diffraction-quality single hexagonal crystals (Figs. 2[link]a and 2[link]c) grew overnight using protein at 66 mg ml−1 in 18–20%(w/v) PEG 6000, 0.1 M MES pH 6.4, 0.02 M ZnCl2.

[Figure 2]
Figure 2
Crystals and diffraction patterns for n-NmDsbD (this domain shares 30% sequence identity with n-EcDsbD; PDB entry 1l6p). (a) n-NmDsbDOx crystals with approximate dimensions of 0.30 × 0.25 × 0.40 mm. (b) n-NmDsbDRed crystals with approximate dimensions of 0.1 × 0.05 × 0.1 mm. Diffraction images are shown for (c) n-NmDsbDOx and (d) n-NmDsbDRed crystals.

Initial screening for n-NmDsbDRed produced microcrystals in 2.5 M ammonium sulfate, 0.1 M Tris pH 8.5 (condition 1; from Salt RX), 0.1 M HEPES pH 7.5, 1.4 M sodium citrate tribasic dehydrate (condition 2; from Index), 5% Tacsimate pH 7.0, 0.1 M HEPES pH 7.0, 10%(w/v) PEG MME 5000 (condition 3; from Index), 0.1 M ammonium citrate tribasic pH 7.0, 12%(w/v) PEG 3350 (condition 4; from Index), 20%(w/v) PEG 3350, 0.2 M potassium nitrate (condition 5; from JCSG) and 0.1 M bis-tris propane pH 7.5, 20%(w/v) PEG 3350, 0.2 M sodium nitrate (condition 6; from PACT premier).

Optimization screens were prepared using protein at 30 and 60 mg ml−1, and the best initial hits and small hexagonal diffraction-quality crystals (Figs. 2[link]b and 2[link]d) grew amongst salt crystals within 2 d in 2.5 M ammonium sulfate, 0.1 M Tris pH 9.1 with a protein concentration of 60 mg ml−1.

Initial screening of c-NmDsbDOx yielded crystals in only one condition consisting of 5%(w/v) PEG 400, 2 M sodium citrate/citric acid pH 7.5 (Precipitant Synergy), and diffraction-quality crystals were obtained using protein at 42–38 mg ml−1 in 5%(w/v) PEG 400, 1.7–2.2 M citrate/citric acid pH 7.0–7.8. These thin spear-shaped crystals grew under a skin after between 4 and 8 d (Figs. 3[link]a and 3[link]c).

[Figure 3]
Figure 3
Crystals and diffraction patterns for c-NmDsbD (this domain shares 38% sequence identity with c-EcDsbD; PDB entry 2fwe). (a) c-NmDsbDOx crystals with approximate dimensions of 0.10 × 0.05 × 0.50 mm. (b) c-NmDsbDRed crystal cluster. Diffraction images are shown for (c) c-NmDsbDOx and (d) c-­NmDsbDRed crystals.

Initial screening of c-NmDsbDRed yielded crystals in the following conditions: 0.2 M zinc acetate dehydrate, 0.1 M sodium cacodylate trihydrate pH 6.5, 18%(w/v) PEG 8000 (condition 1), 2.4 M sodium malonate pH 7 (condition 2), 0.1 M Tris–HCl pH 8.5, 2 M ammonium sulfate (condition 3; from Crystal Screen), 2 M ammonium sulfate (condition 4; from Crystal Screen) and 2.1 M DL-malic acid (condition 5; from JCSG-plus). Diffraction-quality crystals of c-NmDsbDRed were obtained using protein at 30 mg ml−1 in 0.2 M zinc acetate dihydrate, 0.1 M sodium cacodylate tri­hydrate pH 7.5, 18%(w/v) PEG 8000. Clustered crystals grew in 3 d, or overnight with microseeding, and were subsequently manipulated to isolate single crystals for diffraction data collection (Figs. 3[link]b and 3[link]d).

All final crystallization conditions for the crystals used for data collection are described in Table 3[link].

Table 3
Crystallization

  n-NmDsbDOx n-NmDsbDRed c-NmDsbDOx c-NmDsbDRed
Method Hanging drop Hanging drop Hanging drop Hanging drop
Plate type VDXm plates VDXm plates VDXm plates VDXm plates
Temperature (K) 291 291 291 291
Protein concentration (mg ml−1) 66 61 40 30
Buffer composition of protein solution 0.025 M HEPES, 0.05 M NaCl pH 6.7 0.025 M HEPES, 0.05 M NaCl pH 6.7 0.025 M HEPES, 0.05 M NaCl pH 6.7 0.025 M HEPES, 0.05 M NaCl pH 6.7
Composition of reservoir solution 18–20%(w/v) PEG 6000, 0.1 M MES pH 6.4, 0.020 M ZnCl2 2.5 M ammonium sulfate, 0.1 M Tris pH 9.1 5%(w/v) PEG 400, 1.7–2.2 M citrate/citric acid pH 7.0–7.8 0.2 M zinc acetate dehydrate, 0.1 M sodium cacodylate trihydrate pH 7.5, 18%(w/v) PEG 8000
Volume and ratio of drop 2 µl, 1:1 2 µl, 1:1 2 µl, 1:1 2 µl, 1:1
Volume of reservoir (µl) 500 500 500 500

2.4. X-ray diffraction data measurements

For diffraction data measurements, n-NmDsbDOx, c-NmDsbDOx and n-NmDsbDRed crystals were cryoprotected by soaking them for 2 min in mother liquor supplemented with 20% glycerol, PEG 400 and glycerol, respectively. c-NmDsbDRed crystals were cryocooled in reservoir solution. X-ray diffraction data were collected at 100 K on the microfocus beamline (MX2) at the Australian Synchrotron using an ADSC Quantum 315r detector. Diffraction data were collected over a total angular rotation of 180° for both the n-NmDsbDOx and n-NmDsbDRed crystals, with an oscillation angle of 1° and an exposure time of 1 s. A total of 180° of diffraction images were also collected for the c-NmDsbDOx crystal, with an oscillation angle of 0.5° and an exposure time of 1 s. For the c-NmDsbDRed crystal the oscillation range per image was 1° over a total angular rotation of 200°, with an exposure time of 0.5 s. Diffraction data were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997[Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307-326.]) for the n-NmDsbDOx and c-NmDsbDOx data sets, and using iMosflm (Battye et al., 2011[Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. (2011). Acta Cryst. D67, 271-281.]) and AIMLESS from the CCP4 suite (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]) for the n-NmDsbDRed and c-NmDsbDRed data sets.

3. Results and discussion

The two periplasmic domains of the thiol-disulfide reductase DsbD from N. meningitidis, n-NmDsbD and c-NmDsbD, were recombinantly expressed in E. coli and purified to homogeneity using three chromatographic steps (affinity, reverse affinity and size-exclusion chromatography) for crystallization trials.

We first analyzed n-NmDsbD and c-NmDsbD using SAXS not only to determine the quality of the recombinant proteins in solution but also to obtain low-resolution information on the shape and conformation of these domains (Fig. 1[link]). The SAXS data displayed a low experimental noise level, with a linear Guinier plot consistent with a homogeneous, monodisperse solution of both NmDsbD domains. The molecular weights of n-NmDsbD and c-NmDsbD estimated from extrapolation to I(0) were 13.2 and 11.9 kDa, respectively. Ab initio modelling was employed to determine the low-resolution structure of both domains. 16 low-resolution models were generated and subsequently aligned, averaged and filtered to produce the probable shape of the protein (Fig. 1[link]c). The agreement between the crystal structures of the n-EcDsbD and c-EcDsbD domains and the red and blue envelopes provides convincing evidence that the n-NmDsbD and c-NmDsbD domains adopt similar three-dimensional structures in solution to their E. coli counterparts.

Despite the only difference between n-NmDsbDOx and n-NmDsbDRed or c-NmDsbDOx and c-NmDsbDRed being the presence or absence of a disulfide bond, the domains crystallized in different conditions in their two redox forms. Single crystals of n-NmDsbDOx grew in 18–20%(w/v) PEG 6000, 100 mM MES pH 6.4, 20 mM ZnCl2. n-NmDsbDRed crystals were obtained in 2.5 M ammonium sulfate, 0.1 M Tris pH 9.1. c-NmDsbDOx crystallized in 5%(w/v) PEG 400, 1.7–2.2 M citrate/citric acid pH 7.0–7.8 and crystals of c-NmDsbDRed grew in 0.2 M zinc acetate dehydrate, 0.1 M sodium cacodylate trihydrate pH 7.5, 18%(w/v) PEG 8000 (Figs. 2[link] and 3[link]). The n-NmDsbDOx, n-NmDsbDred, c-NmDsbDOx and c-NmDsbDRed crystals diffracted to resolutions of 2.3, 1.6, 2.3 and 1.7 Å and belonged to space groups P213, P321, P41 and P1211, respectively. Assuming that the n-NmDsbDOx, n-NmDsbDRed, c-NmDsbDOx and c-NmDsbDRed crystals contain six, two, two and one molecules per asymmetric unit, respectively, their respective Matthews coefficients (VM) are 3.19, 3.29, 2.29 and 2.09 Å3 Da−1 and their corresponding solvent contents are 61.50, 62.65, 46.37 and 41.17%, respectively (Matthews, 1968[Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497.]). Diffraction data statistics are shown in Table 4[link]. Further studies are in progress to elucidate the structure and mode of action of these catalytic domains in NmDsbD.

Table 4
Data collection and processing

Values in parentheses are for the highest resolution shell.

  n-NmDsbDOx n-NmDsbDRed c-NmDsbDOx c-NmDsbDRed
Diffraction source Australian Synchrotron Australian Synchrotron Australian Synchrotron Australian Synchrotron
Wavelength (Å) 0.9537 0.9537 0.9537 0.9537
Temperature (K) 100 100 100 100
Detector ADSC Quantum 315r ADSC Quantum 315r ADSC Quantum 315r ADSC Quantum 315r
Rotation range per image (°) 1 1 0.5 1
Total rotation range (°) 180 180 180 200
Exposure time per image (s) 1 1 1 0.5
Space group P213 P321 P41 P1211
a, b, c (Å) 147.64, 147.64, 147.64 116.98, 116.98, 45.79 35.80, 35.80, 188.74 43.25, 28.06, 45.80
α, β, γ (°) 90, 90, 90 90, 90, 120 90, 90, 90 90, 101, 90
Predicted solvent content (%) 61.50 62.65 46.37 41.17
Resolution range (Å) 50.00–2.60 (2.69–2.60) 45.79–1.60 (1.69–1.60) 50.00–2.30 (2.38–2.30) 42.46–1.70 (1.79–1.70)
Total No. of reflections 432319 504136 131158 46022
No. of unique reflections 32934 47439 10483 12150
Completeness (%) 99.9 (100) 100 (100) 99.8 (100) 99.8 (99.9)
Redundancy (multiplicity) 13.1 (12.6) 10.6 (10.5) 12.5 (12.7) 3.8 (3.9)
I/σ(I)〉 14.0 (4.6) 11.4 (1.8) 20.4 (6.3) 7.5 (2.2)
Rr.i.m. 0.09 (0.365) 0.08 (1.06) 0.07 (0.281) 0.067 (0.357)
Rp.i.m. 0.051 (0.213) 0.046 (0.594) 0.039 (0.155) 0.059 (0.306)
†Calculated by multiplying Rmerge = [\textstyle \sum_{hkl}\sum_{i}|I_{i}(hkl)- \langle I(hkl)\rangle|/][\textstyle \sum_{hkl}\sum_{i}I_{i}(hkl)], where I(hkl) is the intensity of individual reflections, by the factor [N/(N − 1)]1/2, where N is the data multiplicity.

Acknowledgements

This research was undertaken on the MX and SAXS-WAXS beamlines at the Australian Synchrotron, Victoria, Australia. We acknowledge the use of The University of Queensland Remote Operated Crystallization X-ray Facility (UQROCX), Brisbane, Australia, the CSIRO Collaborative Crystallisation Centre (http://www.csiro.au/C3), Melbourne, Australia and the La Trobe Comprehensive Proteomics Platform, Melbourne, Australia.

Funding information

This work was supported by an Australian Research Council (ARC) project grant (DP150102287), a British Society for Antimicrobial Chemotherapy (BSAC) project grant and a National Health and Medical Research Council (NHMRC) Project Grant (APP1099151). BH is supported by an ARC Future Fellowship (FT130100580). RS is supported by a Research Training Program (RTP) Scholarship. CMK was supported by the National Health and Medical Research Council (APP546003).

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