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The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P63. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P63 crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P63 crystal form had the best diffraction quality, yielding data extending to 2.3 Å resolution.
The Gram-negative bacterium Neisseria meningitidis is a major causative agent of bacterial meningitis, an infection hallmarked by inflammation of the protective membranes of the brain and spinal cord (Stephens et al., 2007 ). Six serogroups of N. meningitidis (A, B, C, X, Y and W135) are responsible for most life-threatening meningococcal meningitis disease and account for worldwide morbidity and at least 10 000 deaths annually. Although several vaccines are currently available for N. meningitidis, none are effective for serogroup B, which causes more than 50% of infections in industrialized countries (Girard et al., 2006 ).
The N. meningitidis outer membrane protein (OMP) PorB is a pore-forming β-barrel protein that is essential for the survival of the organism. PorB contributes to neisserial infection through multiple unusual mechanisms (Massari et al., 2003 ). Firstly, PorB spontaneously transfers from N. meningitidis and inserts into the host mitochondrial outer membrane. In the mitochondrion, PorB may interact with the mitochondrial voltage-dependent anion channel (Massari et al., 2000 ; Müller et al., 2002 ), which is likely to influence the mitochondrial membrane potential and cellular apoptosis (Müller et al., 1999 ; Massari et al., 2000 ). Furthermore, ATP and GTP may regulate the selectivity and gating of PorB in the mitochondrial membrane (Rudel et al., 1996 ), which may contribute to host accommodation of this channel.
The location of PorB in the bacterial outer membrane allows PorB to be recognized by the immune system (Singleton et al., 2005 ; Massari et al., 2006 ). This makes N. meningitidis PorB an attractive target for vaccine development. As a result, both purified PorB and outer membrane vesicles containing PorB are currently being used to develop new vaccines against N. meningitidis (Girard et al., 2006 ; Nøkleby et al., 2007 ). Purified recombinant PorB induces an immune response in mouse models, which suggests that a protein-based vaccine could be efficacious (Wright et al., 2002 ).
Biochemical and spectroscopic characterization of PorB have demonstrated that PorB is composed of 30–40% β-strands and is likely to form a 16-stranded trimeric porin (van der Ley et al., 1991 ; Minetti et al., 1997 ; Derrick et al., 1999 ). A topology model suggests that the pore is regulated by long inter-strand loops that gate transmembrane solute translocation (van der Ley et al., 1991 ; Derrick et al., 1999 ). This architecture for regulation of gating is distinct from the large folded C-terminal plug domain that hallmarks OMP transporters (Moeck & Coulton, 1998 ; Wiener, 2005 ). Here, we report the crystallization and X-ray diffraction analysis of PorB from N. meningitidis.
The gene encoding PorB was amplified from a clinical isolate of N. meningitidis (Kilic et al., 2006 ; a generous gift from Dr YiWei Tang, Department of Infectious Disease, Vanderbilt University Medical Center) using the previously described sense (5′-GGG GTA GAT CTG CAG GTT ACC TTG TAC GGT ACA ATT AAA GCA GGC GT) and antisense (5′-GGG GGG GTG ACC CTC GAG TTA GAA TTT GTG ACG CAG ACC AAC) primers (Feavers et al., 1992 ). The PCR-amplified fragment was digested with BglII/XhoI and then cloned into the BamHI/XhoI sites of the pET21b expression vector without any affinity tags. Recombinant PorB expression was performed using modified Escherichia coli BL21 (DE3) cells (Locher & Rosenbusch, 1997 ). Cells were grown in LB medium at 303 K with 100 µg ml−1 ampicillin until an OD600 of 0.5 was reached. PorB expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM. The cells were incubated with shaking for 4 h at 310 K and then harvested by centrifugation at 5000g for 20 min at 277 K. Using this protocol, PorB was expressed into inclusion bodies (Qi et al., 1994 ). Cells expressing seleno-l-methionine-incorporated PorB were grown in minimal medium supplemented with 25 mg l−1 seleno-l-methionine (SeMet) by inhibition of the methionine-biosynthesis pathway (van Duyne et al., 1993 ).
Purification of N. meningitidis PorB was modified from a previously described protocol (Qi et al., 1994 ; Tanabe & Iverson, 2009 ). Cell pellets were resuspended in breaking buffer (10 mM Tris–HCl, 5 mM EDTA pH 8.0) supplemented with 1 µg ml−1 DNaseI and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and were lysed by sonication on ice (60 Sonic Dismembrator, Fisher Scientific). The insoluble material was isolated by centrifugation at 30 000g for 30 min in an SS-34 rotor (Sorvall). The pellet was resuspended in TSE buffer (20 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 mM EDTA) supplemented with 2% Triton X-100. The suspension was stirred for 1 h and insoluble material was sedimented by centrifugation at 30 000g for 20 min. The pellet was washed three times with TSE buffer supplemented with 2% Triton X-100 and then with TSE buffer alone in order to remove the detergent.
Approximately 1 g of inclusion bodies (from 400 ml LB culture) was resuspended in 1 ml denaturing buffer (50 mM Tris–HCl pH 7.5, 200 mM NaCl, 7.2 M urea) and sonicated for 1 min in a bath sonicator; it was then quickly diluted with 2 ml detergent buffer [50 mM Tris–HCl pH 7.5, 200 mM NaCl, 10% lauryldimethylamine N-oxide (LDAO)] and sonicated for 1 min in a bath sonicator. After 10 min incubation, the insoluble material was sedimented by centrifugation at 20 000g for 20 min. The clarified soluble material was passed through a 0.22 µm filter and loaded onto a Hi-Load Superdex 200 16/60 size-exclusion column equilibrated with TSE buffer supplemented with 0.15% LDAO. The elution time was consistent with a trimer (Fig. 1 a). For SeMet PorB, all buffers were supplemented with 5 mM 2-mercaptoethanol.
Prior to crystallization, the purified protein was concentrated to 15 mg ml−1 in crystallization setup (CS) buffer (20 mM Tris–HCl pH 7.5, 200 mM NaCl) supplemented with one of several types of zwitterionic detergents [0.1% LDAO, 0.01% tetradecyl-N,N′-dimethylglycine (TDDG), 0.02% Fos-choline 14, 0.002% Fos-choline 16, 0.2% LAPAO (3-laurylamido-N,N′-dimethylpropyl aminoxide), 0.3% Anzergent 3-12 or 0.03% Anzergent 3-14]. The sample purity was assessed by SDS–PAGE of protein samples suspended in SDS sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol and 0.004% bromophenol blue) without heat treatment (Fig. 1 b). The identity of PorB was independently confirmed by protein excision, in-gel digestion with trypsin protease and subsequent MALDI–TOF/TOF and ESI–LC/MS/MS mass spectrometry at the Vanderbilt University Proteomics core facility, giving a combined coverage of 89.5% of the predicted protein sequence. The β-sheet percentage of PorB at 0.7 mg ml−1 was confirmed by circular-dichroism (CD) spectroscopy (Fig. 1 c) at 298 K using a Jasco J-810 CD spectropolarimeter. The solution was in a 1 mm path-length cell and the spectra were recorded with 20 nm min−1 scanning time. The protein concentration was estimated using the BCA protein assay (Pierce).
Initial sparse-matrix crystallization screening of 15 mg ml−1 PorB sampled 1440 chemically distinct crystallization conditions using a Mosquito crystallization robot (TTP LabTech). Both sitting and hanging drops comprised of 200 nl protein solution plus 200 nl reservoir solution were equilibrated against 100 µl reservoir solution at either 277 or 291 K. Following the identification of preliminary crystallization leads, crystals were optimized manually using the hanging-drop vapor-diffusion method with 1 µl protein solution plus 1 µl reservoir solution incubated over 1 ml reservoir solution.
Crystal form 1 (C222; Fig. 2 a) was grown by mixing 1.0 µl protein solution (CS buffer plus 0.1% LDAO, with protein at 15 mg ml−1) with an equal volume of reservoir solution containing 50 mM Tris–HCl pH 8.5, 13%(w/v) PEG 1500, 0.03%(w/v) Anzergent 3-10 and 3%(w/v) DMSO. Crystals grew to approximately 0.2 × 0.2 × 0.1 mm at 291 K and formed in between 3 and 5 d. Prior to data collection, the crystals were quickly dragged through crystallization solution supplemented with 30%(w/v) PEG 1500 and 15%(w/v) glycerol for cryoprotection and were flash-cooled in liquid nitrogen.
Crystal form 2 (R32; Fig. 2 b) and crystal form 3 (P63; Fig. 2 c) were grown by mixing 1.0 µl protein solution [CS buffer plus 0.01%(w/v) TDDG, with protein at 15 mg ml−1] with an equal volume of reservoir solution containing 100 mM MES buffer pH 6.0–6.5, 50 mM CsCl, 28–32%(w/v) Jeffamine M-600. The R32 crystals preferentially formed from native PorB and grew to maximum dimensions of approximately 0.10 × 0.10 × 0.02 mm in 2–3 weeks at 291 K. Prior to data collection, the crystals were quickly dragged through a crystallization solution with the Jeffamine M-600 concentration increased to 35–40%(w/v) for cryoprotection, followed by flash-cooling in liquid nitrogen. The P63 crystal form used the same crystallization conditions as the R32 crystal form but preferentially grew from SeMet-incorporated protein. The crystals grew to maximum dimensions of approximately 0.05 × 0.05 × 0.1 mm and formed in 2–3 months.
Crystal quality was assessed by diffraction on Stanford Synchrotron Radiation Laboratory (SSRL) beamlines 9-2 and 11-1, Advanced Light Source (ALS) beamlines 8.2.2 and 8.3.1, Cornell High Energy Synchrotron Source (CHESS) beamlines A1 and F2 and the Advanced Photon Source (APS) Industrial Macromolecular Crystallography Association (IMCA-CAT; ID-17), Southeast Regional Collaborative Access Team (SER-CAT; ID-22) and Life Sciences Collaborative Access Team (LS-CAT; ID-21-D/F/G) beamlines. Iterative rounds of detergent and additive screening improved the diffraction quality of the crystals as assessed by diffraction-based feedback using synchrotron radiation. Even using this procedure, the best crystals of crystal form 1 (C222) diffracted to a maximum resolution of 5 Å after careful optimization of the crystallization conditions. As a result, this crystal form was abandoned for structure determination.
Following optimization, crystal form 2 (R32) diffracted isotropically to 2.9 Å resolution (Fig. 3 a) and a complete native data set was collected at 100 K on SSRL beamline 11-1 using a MAR 325 CCD detector. The exposure time was 15 s for each frame and used an oscillation width of 1° per image.
Data for both native and SeMet-incorporated PorB in crystal form 3 (P63) were collected using a wavelength of 0.978 Å at the APS LS-CAT ID21-G on a MAR 225 CCD detector. A data set consisting of 120 frames was collected with a rotation angle of 90° and an exposure time of 5 s per frame. All data were processed and scaled using the HKL-2000 program package (Otwinowski & Minor, 1997 ).
Crystals of the OMP PorB were grown in three different crystal forms (Fig. 2 ). Initial C222 crystals grew from PorB solubilized in 0.1% LDAO and used PEG 1000–2000 as the precipitant. These crystals initially diffracted to 20 Å resolution. To optimize diffraction quality, Anzergent and DMSO were included as additives and the precipitant was altered to PEG 1500. However, these alterations only improved the diffraction quality of these crystals to 5 Å resolution.
The R32 and P63 crystals were both grown from TDDG-solubilized PorB with identical crystallization conditions. Both of these crystal forms exhibited reasonable diffraction quality after optimization (Fig. 3 , Table 1 ). Interestingly, PorB crystals in the hexagonal P63 space group (Fig. 2 c) were initially identified in crystallization trials of SeMet protein. SeMet PorB only forms crystals in P63. In comparison, the native protein preferentially crystallizes in the R32 space group, but a small percentage (<5%) of native protein formed crystals in space group P63. Despite growing from identical crystallization conditions, the P63 crystals exhibited a distinct morphology compared with the R32 crystal form (Figs. 2 b and 2 c). Data collected at the LS-CAT ID21-G merged to 2.3 Å resolution (Fig. 3 b; Table 1 ).
Specific volume calculations (Matthews, 1968 ) based on the unit-cell parameters and the molecular weight suggested that there could be either one or two PorB molecules per asymmetric unit in the C222 crystals, with a Matthews coefficient (V M) of 6.1 Å3 Da−1 and a solvent content of 79% for one molecule in the asymmetric unit and a V M of 3.0 Å3 Da−1 and a solvent content of 59% for two molecules in the asymmetric unit. The R32 and P63 crystal forms both contained one molecule in each asymmetric unit. For one molecule in the asymmetric unit, the R32 crystals have a V M value of 3.3 Å3 Da−1 and a solvent content of 62%, while the P63 crystal form has a V M of 2.9 Å3 Da−1 and a solvent content of 58%.
N. meningitidis PorB contains only two methionine residues and does not have significant sequence similarity to any porin of known structure. As a result, phasing of the N. meningitidis PorB primarily used the method of multiple isomorphous replacement with anomalous scattering after heavy-atom incorporation. Since the SeMet PorB had a dramatically increased likelihood of forming in the P63 crystal form, which had the highest diffraction limit, heavy-atom cocrystallizations and soaks were performed on SeMet protein. Three derivatives, Lu3+, Er3+ and WO4 2−, were prepared by soaking SeMet PorB crystals with 1 mM lutetium acetate for 3 h, 1 mM sodium tungstate for 3 h or 5 mM erbium acetate for 3 d. Data were collected using the wavelengths and beamlines listed in Table 2 . The location of the position of Lu3+ was identified using the SHELXD (Sheldrick, 2008 ) subroutine in the program SHARP (de La Fortelle & Bricogne, 1997 ). Sites for the other two heavy atoms and the SeMet locations were identified using difference Fourier maps phased with the Lu3+ derivative. The details of the structure determination and analysis will be published elsewhere.
This work was supported by a grant from the Ellison Medication Foundation (AG-NS-0325) and NIH grants GM081816 and GM079419. MT was supported by The Uehara Memorial Foundation. Portions of this research were carried out at beamlines 9-2 and 11-1 at Stanford Synchrotron Radiation Light Source (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program and the National Institute of General Medical Sciences. Use of Industrial Macromolecular Crystallography Association (IMCA-CAT) ID-17, Southeast Regional Collaborative Access Team (SER-CAT) ID-22 and the Life Sciences Collaborative Access Team (LS-CAT) ID21-G beamlines at Advanced Photon Source (APS) was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-06CH11357. Beamline 8.3.1 at Advanced Light Source (ALS) is supported by the Director, Office of Science, Office of Basic Energy Sciences of the US Department of Energy under Contract No. DE-AC02-05CH11231. The Cornell High Energy Synchrotron Source (CHESS) A1 and F2 stations are supported by the NSF and NIH/NIGMS via NSF award DMR-0225180 and the MacCHESS resource is supported by NIH/NCRR award RR-01646. We thank Dr YiWei Tang for the clinical isolate of N. meningitidis, Dr David Friedman for mass-spectroscopic analysis, Dr Jessica Vey, Timothy Panosian, and Thomas Tomasiak for experimental assistance with the data collection and Dr Jessica Vey, Timothy Panosian and Tarjani Thaker for critical reading of the manuscript.