Cloning, expression and purification of TbpA
The N. meningitidis TbpA sequence from strain K454 (B15:P1.7,16) was subcloned into pET20b (Novagen) containing an N-terminal 10X-His tag. TbpA mutants were created using site-directed mutagenesis using QuikChange (Stratagene). For structural studies, mutation of M889 to Tyr improved expression levels. TbpA was expressed in BL21(DE3) cells at 20°C without induction in terrific broth (TB) and carbenicillin (carb). Expression for the mutants followed the same protocol.
For purification, cells were resuspended in lysis buffer (50 mM Tris-HCl, pH7.5, 200 mM NaCl, 1 mM MgCl2, 10 µg /ml DNaseI, 100 µg/ml AEBSF) and lysed by two passages through an Emulsiflex C3 (Avestin) homogenizer at 4°C. The lysate was centrifuged at 12,000xg for 10 minutes remove unlysed cells and the supernatant was incubated with 2% Triton X-100 for 30 minutes at room temperature. The mixture was centrifuged at 160,000xg for 90 minutes at 4°C. The membrane pellets were resuspended in 50 mM Tris-HCl, pH7.5, 200 mM NaCl, 20 mM imidazole and solubilized by constant stirring using 5% Elugent for 16 hours at 4°C. Solubilized membranes were centrifuged at 265,000xg for 60 minutes at 4°C and the supernatant filtered and applied to a 15-ml Ni-NTA column. TbpA was eluted using 250 mM imidazole. Peak fractions were concentrated and applied to an S-300HR Sephacryl size exclusion column (GE Healthcare) using 20 mM Tris-HCl, pH7.5, 200 mM NaCl, 0.8% C8E4 and 0.02% NaN3. Peak fractions were verified using SDS-PAGE and Western blot analysis using an anti-His monoclonal antibody (Sigma).
Cloning, expression and purification of TbpB
The TbpB sequence (starting at residue L22) from N. meningitidis K454 was codon optimized and synthesized by GenScriptand subsequently subcloned into a pET28b vector (Novagen). TbpB was expressed in T7-Express cells (NEB) at 37°C with IPTG induction at an OD600 of 0.75 – 1.0 with continued expression for four hours. Mutants were expressed using the same protocol.
For purification, cells were harvested and resuspended in 5ml PBS per gram of cell paste and supplemented with 10µg/ml AEBSF and 100µg/ml DNaseI. Cells were lysed by French press and then centrifuged for 45 minutes at 38,400xg. The supernatant was applied to a Ni-NTA column and washed with 10 column volumes of PBS. A final wash was performed with PBS containing 20mM imidazole before elution with PBS / 250 mM imidazole. Eluted protein was then dialyzed against PBS overnight at 4°C. For constructs where the His tag was removed, TEV-HIS protease was added, the sample was dialyzed and then passed through a second Ni-NTA column to remove uncleaved protein and protease. Finally, samples were purified by size exclusion chromatography in PBS / 0.02% NaN3.
Crystallization and data collection
For crystallization of the TbpA-hTf complex, apo-human transferrin (Sigma) was mixed with TbpA at a 2:1 ratio and incubated on ice for 1 hour. The complex was isolated using Sephacryl S300HR chromatography equilibrated with 20 mM Tris-HCl, pH7.5, 200 mM NaCl, 10 mM Na-citrate, 1 mM EDTA, 0.8% C8
(Anatrace) and 0.02% NaN3
. Fractions corresponding to the TbpA-hTF complex were verified using SDS-PAGE, pooled and concentrated to 10 mg/ml. Heptane-l,2,3-triol was added to 3% final concentration, incubated on ice for 30 minutes and then the protein sample was filtered prior to crystallization. Sparse matrix screening was performed using a TTP Labtech Mosquito crystallization robot using hanging drop vapor diffusion and plates incubated at 21°C. The best crystals were grown in 24 well Linbro plates (Hampton Research) from 20% Peg3350 and 200 mM BaBr2
. Data were collected at the SER-CAT beamline of the Advanced Photon Source of Argonne National Laboratory and data processed using HKL200034
. The space group was P21
with one mol/ASU and final cell parameters a=91.014, b=129.362, c=198.589, α=90.00, β=90.00, γ=90.00.
For crystallization of the TbpA-hTf C-lobe complex, hTf C-lobe31
was mixed in a 2:1 ratio with TbpA and the complex isolated by Sephacryl S300HR chromatography using 20 mM Tris-HCl, pH7.5, 200 mM NaCl, 0.1% LDAO and 0.02% NaN3
. Final crystal conditions consisted of 21% PEG 1000, 100mM sodium acetate buffer (pH 4.8), 200mM NaCl, 0.1% LDAO and 3% heptane-1,2,3-triol. Data were collected and processed as described for the TbpA-hTf complex. The space group was P21 with one mol/ASU with final cell parameters a=58.055, b=107.592, c=130.721, α=90.00, β=94.48, γ=90.00.
TbpB was crystallized from a 10mg/ml solution with 2.0 M NaCl and 2.0 M ammonium sulfate. Data were collected at the GM/CA CAT beamline of the Advanced Photon Source of Argonne National Laboratory and data were processed using HKL200034
. The space group was P21 with two molecules per ASU with final cell parameters a=75.288, b=82.761, c=111.882, α=90.00, β=105.95, γ=90.00.
For diferric hTf crystallization, 100 mg of holo hTf (Sigma) was solubilized and further purified by Sephacryl S300HR chromatography using 20 mM Tris-HCl, pH7.5 and 200 mM NaCl. The protein was then concentrated to ~50mg/mL crystallized using 100mM HEPES, pH 7.5, 1.6 M ammonium sulfate, and 2% PEG 1000, with red-tinted crystals appearing only after several months and being extremely sensitive to even slight temperature changes. Drops containing the crystals were quickly hydrated with 3.4 M ammonium sulfate immediately prior to being flash cooled in liquid nitrogen and stored for data collection. Data were collected at the SER-CAT beamline of the Advanced Photon Source of Argonne National Laboratory and data were processed using HKL200034
. The space group is C2 with six molecules per ASU and final cell parameters a=254.53, b=173.00, c=150.15, α=90.00, β=123.26, γ=90.00.
For hTf C-lobe crystallization, (holo)C-lobe32
was mixed with excess TbpB N-lobe and the complex isolated by size exclusion chromatography as above in in 25mM Tris pH8.0, 200mM NaCl. The complex was concentrated to ~10mg/ml and broad screening performed using a Mosquito crystallization robot. Several crystallization conditions were observed, however none were red in color as might be expected for iron bound crystals and most contained citrate, which is a known iron chelator. Data were collected and analyzed as for TbpB. The space group was I422 with one mol of hTf C-lobe/ASU with final cell parameters a=95.847, b=95.847, c=204.140, α=90.00, β=90.00, γ=90.00. NO TbpB N-lobe was present in the crystals.
For TbpA-hTf, we were unable to collect useful heavy atom derivatives for experimental phasing and selenomethionine-substituted TbpA protein yields were not sufficient for crystallization. We eventually used molecular replacement in PHASER-CCP435
to solve the TbpA-hTF complex structure. Here, we first searched for each of the two domains (N-lobe and C-lobe) of hTF using the deposited coordinates (PDB code 2HAV), which produced good solutions with Z-scores above 8. However, while the electron density for the hTF molecule was reasonable, the electron density for TbpA was poor and could not be used for model building. Our attempts at using known TonB-dependent transporter structures as search models (barrel and plug, together and individually) were unsuccessful (low Z-scores and LLG scores). We then aligned the TbpA sequence to our structure-based sequence alignment reported in our recent review4
and found that TbpA contained many conserved regions characteristic of TonB-dependent transporters. Using the alignment between TbpA and it closest relative, FhuA (10% identity, ClustalW), and trimming the extracelluar loops, 500 models within an RMSD of 5 Å were produced using Modeller (Accelrys). Each of these models was then used for molecular replacement within PHASER-CCP435
, with two of them producing Z-scores above 8. The solution with the highest LLG (containing both hTF and the TbpA model) was refined in PHENIX36
producing an initial R/Rfree of 0.43/0.48. Further model building was performed using COOT37
and subsequent refinement done in PHENIX36
. During the final states of refinement, extra density was observed which was mapped to residues N413 and N611 of hTF, both of which are reported as possible N-linked glycosylation sites. Therefore, N-linked glycans were built for these two residues. The final structure was solved to 2.60 Å with R/Rfree values of 0.22/0.28. The TbpA-hTf C-lobe structure was solved by molecular replacement using the coordinates from the TbpA-hTF (full-length) structure reported here. Two search models were formed, one for TbpA and one for hTf C-lobe. PHASER-CCP435
was used for molecular replacement and subsequent refinement performed using PHENIX36
. The structure was solved to 3.1 Å resolution wth final R/Rfree values of 0.24/0.29.
The TbpB structure was solved by molecular replacement using PDB code 3HOL. An initial model was created using the Swiss Model server39
that was subsequently divided into four different search domains. PHASER-CCP435
was used for molecular replacement and subsequent refinement performed using PHENIX36
. The structure was solved to 2.40 Å resolution with final R/Rfree values of 0.25/0.30.
The diferric hTf crystal structure was solved by molecular replacement using Phaser-CCP444
. Search models for the N-lobe and C-lobe were created separately with the program Chainsaw (CCP4) using the existing diferric porcine Tf coordinates (PDB code 1H76). Six copies of each lobe (6 molecules of hTf total) were found in the ASU and the iron sites were easily observed in the difference density. These iron sites were further verified in an anomalous difference electron density map. Refinement was performed using PHENIX45
and the structure was solved to 2.1 Å resolution with final R/Rfree values of 0.19/0.23.
The non-glycosylated hTF-Clobe structure was solved by molecular replacement using PDB code 2HAU. An initial search model was formed by truncating the N-lobe domain. PHASER-CCP435
was used for molecular replacement and subsequent refinement performed using PHENIX36
. The structure was solved to 1.7 Å resolution with final R/Rfree values of 0.17/0.19. For all structures, figures were made with PyMOL (Schrodinger) or Chimera40
and annotated and finalized with Adobe Illustrator.
Whole cells (2µL, 0.01g/ml) and cell lysates (unmodified for TbpB samples, or incubated for 3 hours with 2mM EDTA and 1% DDM at room temperature for TbpA samples) were spotted onto nitrocellulose membrane and allowed to dry at room temperature. The membranes were then blocked with PBST 2% BSA for 15 minutes, washed and probed with HRP conjugated hTf (1:1000) (Jackson ImmunoResearch) for 15 minutes. The membrane was then washed and imaged using the colorimetric substrate 3,3’-diaminobenzidine (Sigma) where the appearance of a red dot indicated specific binding of the hTF-HRP conjugate. The results from the mutants were compared to wild type Tbp to determine their effect on hTF binding.
Whole cells (100µL at 10 mg/ml or 1 mg/ml in PBS) of wild-type TbpA, empty vector control (pET20b), and TbpA mutants were added to a NUNC polystyrene 96-well plate (Fisher Scientific) and incubated at 37°C overnight. Wells were washed 2X with PBST and then blocked with PBST 2% BSA for 30 minutes and probed with hTf-HRP (1:1000) for 15 minutes. Wells were washed 2X in PBST, 2X in PBS, and then developed using 100 µL 3,3’,5,5’ - tetramethylbenzidine substrate (TMB, Sigma) for 5 minutes and terminated using Stop solution (Sigma). Absorbances of each well were determined using a BioRad iMark plate reader at 450nm and data normalized and compared to wild-type TbpA. Each experiment was performed in triplicate and data reported with standard errors.
Antibody blocking assays
Using the TbpA-hTf crystal structure reported here, we designed four different peptides based on four loops from TbpA (loops 3, 7,11, and plug loop) to be used as antigens for polyclonal antibody development (Precision Antibody). A fifth polyclonal antibody was developed using purified full length TbpA (1X PBS 7.4, 0.1% DDM). An ELISA was designed to probe whether or not these antibodies could block hTF binding. Here, TbpA-HIS (20ng) was incubated for 20 minutes in a final volume of 100 µL either alone or in the presence of each antibody (1:20) individually in PBS containing 0.05% Cymal-6 (Anatrace). In addition, we tested the antibodies that targeted TbpA loops in combinations to determine if an additive affect could be observed. Each sample was then transferred to a 96-well Ni-NTA Agarose HiSorb plate (Qiagen) and incubated for 30 minutes and washed 2X with PBST + 0.05% Cymal-6. Assays were performed as described in the previous section. In a second set of ELISAs, TbpA-HIS was first bound to the Ni-NTA Agarose HiSorb plate prior to incubation with antibodies. Results were analyzed and initial graphs made using Microsoft Excel. The graphs were then imported, annotated and finalized with Adobe Illustrator.
Protease accessiblity of TbpA and TbpA mutants
To confirm that TbpA and the TbpA mutants were being properly presented at the surface of the bacteria, we treated whole cells with trypsin (5 ug/mL final concentration) for 15 minutes at room temperature and the reaction was stopped by the addition of AEBSF (0.2 mg/ml final concentration). The cells were then centrifuged and supernatant removed. The pellets were then resuspended in LDS loading buffer, boiled for 10 minutes, centrifuged for 10 minutes, and then separated on a NuPAGE Novex 4–12% Bis-Tris gel. The samples were then transferred to a PVDF membrane using an iBlot system (Invitrogen) and Western blot analysis performed using a polyclonal anti-TbpA antibody (1:1000) and a monoclonal anti-HIS HRP conjugated antibody (Sigma) (1:5000). Here, each membrane was blocked with 2% BSA in 1X PBST for 15 minutes and then probed with either the anti-TbpA or anti-HIS HRP conjugated antibody for 30 minutes. The anti-TbpA membrane was then washed 2X with 2% BSA in 1X PBST and then probed with anti-Mouse HRP conjugated secondary antibodies for an additional 30 minutes. Both membranes were then washed 2X with 1X PBST, 2X with 1X PBS, and then imaged using the colorimetric substrate 3,3’-diaminobenzidine (Sigma). The results from the mutants were compared to wild type TbpA to determine which constructs were being presented on the surface of the cells.
Transferrin competition assays
To determine if the affinity of hTF to either TbpA or TbpB is affected by the conformation or coordination state of the N-lobe, we performed an ELISA competition assay using apo-hTF, holo-hTF, hTF-FeN (iron bound in N-lobe only), and hTF-FeC (iron bound in C-lobe only), which were expressed in BHK cells and purified as described23
. Here, HIS-TbpA (20 ng) or HIS-TbpB (20 ng) was incubated for 15 minutes in a final volume of 100 uL in a 96-well Ni-NTA HiSorb plate (Qiagen) in 1X PBS (0.05% cymal-6 was added to all buffers for HIS-TbpA). Wells were washed 2X with PBST, blocked with 2% BSA in PBST for 15 minutes, and then incubated with each of the transferrin constructs (apo, holo, FeN, FeC) (100 ng each) for 15 minutes. Wells were washed again 2X with PBST, and then probed for 20 minutes with HRP conjugated hTF (1:1000) (Jackson ImmunoResearch) in 100 µL final volume. Wells were washed again 2X with PBST and then 2X with PBS and imaged using 100 µL 3,3’,5,5’ - tetramethylbenzidine substrate (Sigma) for ~5 minutes and terminated using Stop solution (Sigma). Data were collected and analyzed as described above.
Small angle X-ray scattering analysis
The TbpB-hTF complex was dialyzed overnight at 4°C into TBS, pH 7.4 (25 mM Tris, 137mM NaCl, 3mM KCl) and then filtered using a 0.2µm spin filter. Data were collected at concentrations of 1, 2.5, and 5 mg/ml at Stanford Synchrotron Radiation Lightsource beamline BL4-2. Data reduction and analysis were performed using the beamline software SAStool. The program AutoGNOM of the ATSAS suite41
was used to generate P(r) curves and to the determine maximum dimension (Dmax) and radius of gyration (Rg) from the scattering intensity curve (I(q) versus q) in an automatic, unbiased manner, and rounds of manual fitting in GNOM42
were used to verify these values. Ab initio molecular envelopes were computed by the programs GASBOR24
. Ten iterations of GASBOR were averaged using DAMAVER43
. Docking of the TbpB and diferric hTF crystal structures into the molecular envelope was performed manually, guided by both previous docking studies20
and mutagenesis results. Figures were made with PyMOL and annotated and finalized with Adobe Illustrator.
Modeling the TbpA-TbpB-hTF triple complex
The in silico TbpA-TbpB-hTF triple complex was assembled based on our crystal structures (TbpA-(apo)hTF, diferric hTf, TbpB) and SAXS analysis (TbpB-(holo)hTF) reported here. The crystal structure (TbpA-hTF) was aligned with our TbpB-hTF model using the C1-subdomain of hTF as a reference, yielding a triple complex containing a 1:1:1 ratio of TbpA, TbpB, and hTF. Figures were made with PyMOL and/or Maya (Autodesk) and annotated and finalized with Adobe Illustrator.
Electron microscopic analysis
The triple complex (TbpA-TbpB-(holo)hTF) was prepared from separately purified components by first forming a complex between TbpB and (holo)hTf which was purified by size exclusion chromatography in 1X PBS. Cymal-6 was added to a final concentration of 0.05% and purified TbpA (in 1X PBS, 0.05% Cymal 6) was added to the mixture using an excess of the TbpB-(holo)hTf complex. The triple complex (which retains a 1:1:1 stoichiometry) was isolated by sized exclusion chromatography in Buffer A (1X PBS in 0.05% Cymal 6) and used immediately for EM experiments. The complex was diluted with buffer A to an optimal concentration for EM (determined empirically to be ~1 µg/mL). Drops (4µL each) were applied to carbon coated, glow-discharged EM grids (EMS, USA). After 1 min, the grid was blotted, washed twice with Buffer A, once with distilled water, and then stained with 2% uranyl acetate. Grids were observed with a CM120 La-B6 electron microscope (FEI), operating at 120kV. Micrographs were recorded on SO163 film (Kodak) at a nominal magnification of 45,000, and digitized on a Nikon Coolscan 9000 at a rate corresponding to 1.55 Å/pixel. The large majority of particles distributed evenly on the grid and were essentially uniform in size (~ 90–110 Å in diameter), indicative of a homogeneous population.
The particles were variable in substructure, suggesting that the molecules deposit on the grid in a variety of orientations. Accordingly, a data set of 4240 particles was subjected to a "reference-free" classification, using SPIDER58, EMAN59, and Bsoft60. Images were picked uaing a box 256×256 pixel box, and binned 4 times (to 6.2Å/pixel) to increase the signal-to-noise ratio and the speed of calculation. Initial reference-free classification and averaging were performed using EMAN; then further classification was done in SPIDER, using principal component analysis (PCA) with 3 cycles of iteration. We chose to obtain 56 final class averages, based on a cluster distribution obtained from PCA.
The coordinates of the modeled triple complex were converted to a density map and low-pass filtered to 15 Å. The sampling rate of the density map was set to be same as the EM images and 2D projecions were calculated at angular increments of 30° (Supplementary Fig. 14b
). Comparisons and matching between the EM class averages and the model reprojections were done in terms of cross-correlation coefficients. A few ambiguous cases were reassigned by visual assessment. Figures were made with Chimera and annotated and finalized with Adobe Illustrator.
Molecular dynamics simulations
For simulations of TbpA bound to (apo)hTf, a membrane-water system containing the protein complex was first built using VMD44
. The complex was placed in a DMPE bilayer as used previously45
, with the barrel of TbpA aligned with the membrane’s hydrophobic core, and then fully solvated. Disulfide bonds for three pairs in TbpA and 19 pairs in hTf were added based on S-S proximity. Ca2+
ions were added to a concentration of 100 mM, resulting in an initial size of 264,000 atoms. The system was equilibrated in stages for 13.5 ns, including 10 ns of fully unrestrained dynamics. The simulations were run using NAMD 2.733
in the NPT ensemble at a temperature of 310K and a pressure of 1 atm; after the first 3.5 ns of equilibration, the area of the membrane was fixed. Other simulation parameters were set identically to those used previously45
. For steered MD (SMD) simulations, the Cα atom of the TbpA N-terminal plug domain residue was pulled in the -z direction, away from the membrane, at a constant velocity of 5 Å/ns33,46
. To counterbalance the pulling forces, six residues at the extracellular periphery of the barrel domain were restrained in the z direction. An adaptive procedure was used to limit the maximum required system size during SMD simulations47
. When the extension of the unfolded region of the plug domain brought it near the periodic boundary, the simulation was stopped, the unfolded region of the plug domain distant from the barrel and membrane was cleaved, and the simulation restarted after a short equilibration of the water with the new N-terminal residue being pulled. With this procedure, utilized three times, approximately 150 Å of pulling was accomplished while keeping the system sizes below 300,000 atoms.
Sequence analysis and alignments
Sequence analysis and alignments were performed and analyzed with the programs STRAP48
. Figures were annotated and finalized with Adobe Illustrator.