This protocol facilitates the successful structure determination of the IKK binding domain of NEMO, in its unbound form. And details of its production and crystallization. The protocol, takes advantage of this stabilization of the native confirmation of the IKK binding domain fragment of NEMO, through coiled-coil adapters, which facilitate crystallization and structure determination.
The structural biology of NEMO, as a target, provides an important advantage in the development of NEMO inhibitors for the treatment of inflammatory and autoimmune diseases and cancer. This protocol may be extended to the structure determination of NEMO complexes, with peptide or small molecule inhibitors for drug discovery or optimization. The protein refolding and concentration, during the protein production stage, are fundamental to obtaining a pure NEMO dimer.
Limiting aggregation and ensuring solubility under crystallization conditions. Demonstrating the procedures will be Tamar and Amy, graduate students in our laboratory. Begin by adding 20 milliliters of terrific broth solution.
And 20 microliters of a 100 milligram per milliliter stock solution of ampicillin. To a 125 milliliter Erlenmeyer flask. Followed by a few microliters of cell glycerol stock, from 80 degrees Celsius storage, of BL21DE3 competent cells.
Transformed with vector. After shaking the starter culture overnight, at 37 degrees Celsius and 220 rotations per minute, dilute the cells to an OD600 of 0.1 and 250 milliliters of terrific broth. And add ampicillin to a final concentration of 100 micrograms per milliliter.
When the OD600 of the culture reaches 0.8 to 1, add IPTG to the culture, to a 500 micromolar concentration. And grow the cells for four hours, at 37 degrees Celsius, until the OD600 reaches six to 10. At the end of the incubation, sediment the cells by centrifugation.
And re-suspend the pellet in 40 milliliters of lysis buffer. Split the re-suspended cells into two 20 to 25 milliliter aliquots. And use a French press to apply approximately 25, 000 pounds per square inch of pressure to the cells, two to three times per aliquot, in a cold room.
Next, add urea to the cell lysates, to a final concentration of eight molar. And incubate the cell solution on a rocking platform for two to 16 hours, at room temperature. The next morning, transfer the lysates to ultra-centrifuge tubes, to at least three quarters full, per tube.
And ulrta centrifuge the lysates, for 45 minutes. Decant the supernatants into a 50 milliliter conical tube. And load the urea incubated supernatant onto an IMAC column, at three milliliters per minute.
When all of the supernatant has run through the column, wash the column for 10 column volumes, with binding buffer, at three milliliters per minute. And perform gradient elution of the NEMO-EEAA protein from 10 to 500 millimolar imidazole, over a 12 column volume gradient. Collecting one milliliter aliquots of the eluate, into a fraction collection plate.
After SDS page analysis, pull the fractions containing the pure target protein and measure the protein concentration by Bradford assay, according to standard protocol. Select the eluded fractions, containing protein. To cleave the HIS6 tag and to remove excess imidazole from the sample, add TEV protease, at a one to 10 weight ratio, of TEV cleaved protein, to the target protein sample.
And dilate the sample overnight in four liters of 20 millimolar Tris. 150 millimolar sodium chloride and two millimolar DTT solution. The next morning, load the TEV cleaved NEMO-EEAA sample, onto a second IMAC column, at one milliliter per minute.
Collecting the flow through in one milliliter fractions, in a 96 bowl fraction collection plate. Wash the column with five volumes of 20 millimolar Tris, 150 millimolar sodium chloride and 10 millimolar imidazole solution, at one milliliter per minute. After the last wash, elute the TEV and uncleaved HIS6 NEMO-EEAA, with three column volumes of 20 millimolar Tris, 150 millimolar sodium chloride, 500 millimolar imidazole and two millimolar DTT solution, into a 50 milliliter flask.
Pull the flow through fractions, containing cleaved NEMO-EEAA construct and concentrate the sample with a stirred cell concentrator, to five milliliters. After overnight dialysis as demonstrated, load five milliliters of the sample onto 16 millimeter by 60 centimeter size exclusion chromatography columns. Repeating as necessary, according to the sample volume.
At one milliliter per minute, and at two millimolar Tris, 100 millimolar sodium chloride and two millimolar DDT solution. After pulling the fractions, corresponding to the dimeric NEMO-EEAA, concentrate the sample in the stirred cell concentrator, with a molecular weight cut off membrane of three kilodalton, to a final concentration of 113 micromolar. Then, aliquot the protein for storage at four degrees Celsius, for up to one month.
For sparse matrix screening, add 60 microliters of sparse matrix solution into each of the 96 wells of a two drop chamber crystallization plate. For sit and drop vapor diffusion. And add the protein solution to a robotic drop setter.
Next, use a robotic drop setter, to dispense 100 nanoliters of protein solution at 1.65 milligrams per milliliter, in a one to one ratio with reservoir solution in drop one, to a final volume of 200 nanoliters. And add 66 nanoliters of protein solution with 134 nanoliters of reservoir solution, to a final volume of 200 nanoliters in drop two. Then, immediately seal the plate with three inch wide sealing tape.
Then, store the trays in a crystallization imager storage, at 20 degrees Celsius. Checking the images collected automatically, for the presence of crystals, starting two days after storing the plates. For seed stock generation, transfer the entire drop containing the crystal of interest, into 50 microliters of crystallization condition solution, in the provided vial, from a seed generation kit.
And vortex the seed stock, with 20 seconds of pulsing and 10 seconds of rest, for three minutes. Then, serially dilute the seed stock, in one to 10 increments, down to one to 10, 000. And store the dilutions at four degrees Celsius, until further use.
One to two days before shipment to synchrotron, cut the tape from the top of the well, with the crystal of interest. And add 0.5 microliters of crystallization solution containing 12%one, two propane DiO cryoprotectant, directly to the well. Gently dislodge the crystal with a cryo loop.
Loop the crystal from the well, and store the crystal containing cryo loop in a puck immersed in liquid nitrogen, until shipment for X-ray diffraction. After receiving the X-ray diffraction data, process the scaled intensities in the STAR and ISO server. Using an X-ray crystallography indexing cut off mean, of 1.2 for the diffraction limit surface for the data.
And load onto Phoenix. To determine the structure, use the X-ray structure of GCN4 as a search model for molecular replacement, using MRage in Phoenix. The 4DMD structure, will be defined as an ensemble.
And the MRage solution will successfully build to the structure portion, corresponding to the end terminal coiled-coil adapter of NEMO-EEAA. to the search model, for both chains in the dimer. The over expressed protein appears at a band, at approximately 14 kilodalton molecular weight marker, before the first IMAC column purification.
And displays a monomer band and a dimer band, at 14 and 28 kilodaltons, respectively, after the first elution. TEV cleavage is practically complete, following the elution, through the second IMAC column. Almost entirely as a dimer, at the expected molecular weight.
Size exclusion chromatography, displays a single peak, eluding between 60 to 65 milliliters, who are responding to the dimer. Fine screening produces crystals that can be utilized to produce a seed stock for the production of NEMO-EEAA crystals, for data collection. Here, representative NEMO-EEAA crystals diffraction profiles, are shown.
Structural analysis of the NEMO-EEAA protein, reveals a homodimeric irregular parallel coiled-coil, approximately 175 angstroms in length. The regular coiled-coil region, encompasses the ideal coiled-coil adapter sequence, at the end terminus. And the first two heptads of the NEMO proper sequence.
A regular coiled-coil, is also present at the C terminus. Starting at NEMO residue 97 and encompassing the C terminal ideal coiled-coil adapter. The IKK beta bound structure, displays a more open coiled-coil confirmation, to accommodate the ligand.
With a larger interhelical spacing by one to 2.2 angstroms in this region. The structure of a ligand in NEMO, offers a new target for the design of inhibitors through computational methods. And for the visual screening of binding pockets with small molecule libraries.
We now have a path for crystallization of the engineered NEMO construct in complex with small molecule ligands. To aid in developing and optimizing a new class of inhibitors.
