Structural Insights on the Mycobacterium
tuberculosis Proteasomal ATPase Mpa
Tao Wang, Hua Li, Gang Lin, Chunyan Tang, Dongyang Li, Carl Nathan, K. Heran
Darwin, and Huilin Li
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of Mpa-ID, Mpa_N97∆, ∆, Mpa’’’, and Mpa-ID’’’
For production of Mpa-ID (amino acids 97-245) primers Nde-mpa-97-ccf2
(GGAATTCCATATGCCGCCCAGTGGCTACGGCGTC) and Not-throm-mpa-245
were used to amplify nucleotides 288 through 735 while adding a thrombin cleavage site and
hexa-histidine tag to make the plasmid pET24b+mpa97-245. For cloning the amino-terminal 97
amino acid truncation plasmid, primer Nde-mpa-97-ccf2 was used with Not-throm-mpa-20r
the production of the triple mutant Mpa’’’ we used the Stratagene QuikChange Multi Site-
Directed Mutagenesis Kit. The following three oligonucleotides with the desired mutations
(underlined) were simultaneously used on the template pET24b(+)mpa2 (Darwin et al., 2005):
mpaW187A (CGAGGAACGCGTTGTTGCGCTGGCTGATCCCCTGATCGC). The cloned
PCR product was sequenced between the ATG-containing NdeI site and the mpa SacII site at
~950 bp. The NdeI-SacII site, containing all three mutations, was subcloned into the parental
plasmid pET24b(+)mpa2, making pHD138. To make the triple mutated Mpa-ID, we used the
same primers as above for making Mpa-ID but used pHD138 as the template. The final plasmid
was named pHD139.
For all cloning, Pfu Ultra (Stratagene) was used for the polymerase chain reactions and the
amplified products were cloned into pET24b(+) (Novagen) using enzymes from New England
Biolabs. The inserts were sequenced by GENEWIZ (South Plainfield, NJ). Plasmids were
transformed into DH5 α (Gibco, BRL) and ER2566 (Chong and Garcia, 1994) for expression.
Bacteria producing Mpa-ID were grown in Luria-Bertani medium at 37°C to OD600 value
of 0.6. Gene expression was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)
overnight at 20°C. The bacteria were harvest by low speed centrifugation (6000 × g, 4°C) and
resuspended in buffer containing 25 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 300 mM NaCl, 1 mM
β-mercaptoethanol (β-ME). Cells were broken by three passes through a microfluidizer. The
lysed bacteria were centrifuged at 30,000 ×g at 4 °C for 1 hour, and the pellet containing intact
cells and debris was discarded. The supernatant was loaded onto a 5 ml Qiagen Ni2+ affinity
column with 20 mM imidazole in the loading buffer, and washed to UV280 stable. To further
remove contaminations, the column was washed by 50 ml high salt buffer containing 25 mM
Tris-HCl pH 8.0, 1 M NaCl, 2 mM MgCl2, 1 mM beta-mercaptoethanol, 20 mM imidazole. The
Mpa-ID was eluted with buffer containing 300 mM imidazole. The eluate was buffer exchanged
to 1× PBS and treated by thrombin for His tag removal. A second Ni2+ affinity chromatography
was performed and the flow-through was collected and further purified by the Resource Q ion
exchange column and the Superdex-200 gel filtration column. The purified Mpa-ID was dialyzed
in buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 1 mM β-ME), and concentrated with a Vivaspin
tube (WMCO 10 kDa) to a final concentration of 10.0 mg/ml. Purification of Mpa-N97∆, Mpa
mutant and Mpa-ID mutant followed the same procedure with similar conditions. Native Gel
(4~15% gradient Ready Gel) was purchased from Bio-Rad, run with the standard Tris-HCl Gel
method, and stained by Coomassie blue.
Electron Microscopy of Mpa-20SOG
20 µl 20S proteasomes at a concentration of 10 mg/ml in 10 mM Tris (pH8.0), 100 mM NaCl, 1
mM β-mercaptoethanol was mixed with 20 µl Mpa or Mpa_N97∆ at 20 mg/ml in 10 mM Tris
(pH8.0), 100 mM NaCl, 5 mM MgCl2, 1 mM β-mercaptoethanol, with a molar ratio of ~ 1:4, on
ice for 20 minutes, in the presence of 1 mM ATPγS. For negative staining EM, a 5-µl drop of the
diluted mixture was applied to a glow-discharged 300-mesh copper grid covered with a thin layer
of carbon film. After removing the excess solution by blotting with filter paper, the sample grid
was stained with two 5-µl drops of 2% (w/v) uranyl acetate aqueous solution. Excess stain
solution was blotted and the grids were left to air dry. Images were recorded on Kodak SO-163
negative film in low-dose condition (10 e/Å2) in a JEOL JEM-2010F electron microscope
operating at 200 kV with magnification of 50,000X and the under-focus value of 1~1.5-µm. Film
was developed in Kodak D-19 solution and digitized using a Nikon Supercool scanner 8000ED
at a step size of 12.7 µm, corresponding to a pixel size of 2.54 Å. The software package EMAN
(Ludtke et al., 1999) was used to process the particle images of the Mpa-20SOG complexes.
3D Crystallization of Mpa-ID and Diffraction Data Collection
The protein was prone to aggregation, so the detergent cyclohexylpropanoyl-N-
hydroxyethylglucamide (C-HEGA-9) was essential for crystallization (See Experimental
The hanging drop vapor diffusion method was used for crystallization of Mpa-ID.
Briefly, a 2-µl droplet of Mpa-ID at 10.0 mg/ml in 10 mM Tris, pH 8.0, 10 mM NaCl, 1 mM β-
ME was mixed with 2 µl of reservoir solution consisting of 0.1 M MES, pH 6.0, 50 mM MgCl2,
50 mM NaCl, 20% PEG-2000, 108 mM C-HEGA-9. Two types of crystals, one plate-like and
the other cubic, were obtained within one week of incubation at 21°C. Both plate-like and cubic
crystals were fragile and temperature sensitive. For cryo-freezing of the crystals, the crystal
solution was gradually replaced by a same solution but containing ethylene glycol and dimethyl
sulfoxide (DMSO) as cryo-protectants in a multi-step treatment. The final concentrations of the
cryo-protectants were 5% (v/v) ethylene glycol and 20% (v/v) DMSO. The crystals were picked
up and flash frozen in liquid nitrogen.
The halide soaking method was performed initially but the anomalous signal was too
weak to yield useful data. Subsequently, we screened an array of heavy atom derivatives by
native gel electrophoresis of the purified Mpa-ID, and found that mercury acetate
(Hg(CH3CO2)2) induced a clear upward shift of the protein on the native gel, so
Hg(CH3CO2)2was chosen for derivatizing the crystals. An artificial reservoir solution containing
2 mM Hg(CH3CO2)2 was gently pippetted onto the crystal drop and the mixture was incubated
for 2 h for heavy atom soaking. A back-soaking step was performed in solution containing no
heavy atoms in order to remove the non-specifically bound heavy atoms. The cryoprotectants
used for flash freezing heavy atom derivatized crystals were 10% (v/v) glycerol and 10% (v/v)
Crystal screening and dataset collection were carried out at X25 and X29 beam lines at
NSLS, Brookhaven National Laboratory. Both plate-like and cubic crystals diffracted well but
were radiation sensitive and the cryo-protectants adversely affected the quality of the crystals.
Most crystals were either twinned or highly anisotropic in diffraction quality, probably due to the
large unit cell, which reaches ~ 200 Å in one dimension. Furthermore, the unit cell parameters
varied among crystals even from the same crystallization batch. Therefore, hundreds of crystals
were screened to collect useful datasets. Although the unit cell parameters varied slightly, all
native and heavy atom derivative crystals had the same space group P21. The anomalous dataset
from the derivatized crystals were collected at 1.0066 Å, a wavelength corresponding to the peak
of the scanning spectrum.
Crystallographic Data Reduction and Structure Determination
All datasets were indexed, integrated and scaled using HKL2000 (Otwinowski, 1997). For
experimental phase solution, the native-II dataset (2.6 Å) and the mercury-derivative dataset
(3.0Å) were used for single isomorphous replacement (SIR) and/or single anomalous diffraction
(SAD) runs with HKL2MAP-Shelx program (Pape and Schneider, 2004; Uson and Sheldrick,
1999) (Table 1). Seventeen heavy atom positions were determined. The phases were
subsequently improved with the 6-fold non-crystallographic symmetry (NCS) using
Solve/Resolve in the PHENIX suite (Terwilliger, 2004). The initial model was improved by
manual docking in COOT (Emsley and Cowtan, 2004). One partial hexamer model was used for
molecular replacement using PHASER-PHENIX suite with the native-I dataset (2.0 Å). The
higher resolution dataset significantly improved the electron density map. This dataset contained
partial pseudo-merohedral twinning. Detwining only marginally improved the electron density
due to the near rectangular unit cell of the crystal (β=90.31°). Further sequence assignment and
model building were done in COOT. Two hexamers of Mpa-ID were refined to 2 Å by using
CNS1.21 (Brunger, 2007; CCP4, 1994) and Refmac5.5 (CCP4, 1994) with application of
appropriate NCS restraints (Table 2).
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(A) A gallery of the negatively stained EM raw images of Mpa_N97∆-20SOG complex particles.
(B) Mpa-20SOG complex particles.
(C) Eight selected 2D class averages of the complex particles formed by the native Mpa with
20SOG, demonstrating the flexible binding of Mpa on one end of open gate core particles. The
particles are aligned with Mpa rings on the top of the 20SOG core particles.
(A) Stereo view of the 2Fo-Fc electron density map (1σ) of the short loop (Leu-168 – Arg173)
connecting the β6 and β7 strands. At the resolution of 2.0 Å, the amino acid side chains are well
(B) The 2Fo-Fc electron density (1σ) at the long loop region (Leu-188 and Gly-217) connecting
β8 and β9 strands. This loop is fully traced only in one of the six subunits; in other subunits, it is