The goal of this procedure is to provide guidance for the production, crystallization and structural determination of human IKK1-alpha in order to understand the mechanistic basis of its signaling function and to establish platforms for rational drug design. This method should help researchers determine the structure of IKK1, which would provide answers to key queries in the field of immune signaling. This technique describes a streamlined path to obtaining crystals of IKK proteins, which are rather refractory to crystallization and to provide various critical information in overcoming bottlenecks in determining the structures.
Generally, individuals new to this method will struggle, because generating milligram quantities of soluble, well-behaved protein, requires its expression in insect cells, and crystallization can be done under only a highly narrow window of conditions. The molecular replacement procedure used in determining the structure of IKK1 can also be very tricky, especially due to poor deflection property of the crystals, not at the site of the asymmetric unit containing many IKK1 molecules in the absence of appropriate search models. Demonstrating the procedures will be Kyle Shumate and Sonjiala Hotchkiss, students from my laboratory.
On day one, plate Sf9 cells in two milliliters of Sf903 insect cell medium, in each well of a six-well plate and incubate at 27 degrees Celsius. Passage the cells when they reach a density of two to three times 10 to the six cells per milliliter in suspension by diluting it to fresh media, at a density of approximately six times 10 to the five. On day two, dilute eight microliters of Sf9 transfection reagent in 100 microliters of SF-903 or Grace's Insect Medium.
Then vortex the mixture briefly. In a separate tube, dilute one microgram of kit-purified recombinant bacmid in 100 microliters of the same medium. Combine the diluted DNA and transfection reagent.
After incubating at room temperature for 15 to 30 minutes, remove the medium from the well. Then add one milliliter of fresh medium. Next, add the diluted DNA transfection reagent mixture dropwise onto the cells, adjusting the drop size such that it does not dislodge adherent cells.
After six hours, add 1.5 milliliters of fresh medium on top of the cells. Incubate the cells at 27 degrees Celsius, checking the wells periodically every 12 hours for signs of viral infection. On day five, remove the medium containing cells, 60 to 72 hours post-infection.
After transferring the medium into tubes, centrifuge for five minutes at 500 times g and four degrees Celsius. When finished, save the supernatant, which is the P1 virus stack. On day one, resuspend the previously prepared His-IKK1 cell pellet in 40 milliliters of lysis buffer.
Place the cell suspension on ice and lyse the cells by sonication at 60%to 70%duty cycles and five to 10 pulses of 30-second duration at an interval greater than one minute. Clarify the lysate by centrifugation at greater than or equal to 28, 000 g for 45 minutes at four degrees Celsius. After preparing a Nickel-NTA Agarose resin, according to the text protocol, elute the His-tagged IKK1 alpha protein under gravity flow using 20 milliliters of elution buffer.
Collect one to 1.5 milliliter fractions. After combining the fractions containing the protein, digest the sample with TEV protease overnight at four degrees Celsius. On the morning of day two, incubate the protein with one millimolar of ATP, in the presence of magnesium chloride, beta-glycerophosphate, sodium fluoride and sodium orthovanadate for one hour at 27 degrees Celsius.
Following incubation, filter the protein solution through a 0.45 micron filter. Then load approximately six milliliters of the sample on to a 120 milliliter preparative size-exclusion column, attached to an automated liquid chromatography system. After equilibration, run the size-exclusion chromatography at a flow rate of one milliliter per minute and collect two-milliliter fractions.
During the run, monitor elution at 280 and 254 nanometers. After pulling the pure fractions, concentrate in a 30-kilodalton, molecular weight cut-off centrifugal concentrator, following the manufacturer's instructions. Then dispense 25-microliter aliquots of the concentrated protein and flash-freeze in liquid nitrogen.
Using a robot, pipette 80 to 100 microliters of a crystallization reagent into the reservoir of a 96-well plate. Using a crystallization robot, dispense and mix 0.2 to 0.25 microliters of IKK1 and its inhibitor complex with the same volume of reservoir solution. Immediately after setting the drops, seal each plate with optically clear films to avoid evaporation.
Incubate one plate at 18 degrees Celsius and the other plate at four degrees Celsius in a cold room. Use a stereo microscope with a polarizer to check for appearance of crystal in each drop, every day, for the first seven days and then at longer intervals. After preparing IKK1 and its inhibitor complex as previously described, transfer the well solutions to each well of a 24-well plate.
Place one to 1.5 microliters of well solution on a clean glass coverslip. Add an equal volume of IKK1 and its inhibitor complex to the glass coverslip and mix gently by pipetting up and down three to four times. After applying grease on the rings of each well of the plate, turn the glass coverslip over and place on the respective well with forceps.
Then seal the well by pressing down on the glass coverslip. After setting up all the drops in the 24-well plate, incubate in the cold room, in the dark. Occasionally check the drops under a microscope for appearance and growth of crystals.
Once crystal growth is complete, gently remove the glass coverslip containing the crystal and place it onto a solid surface with the crystal drop facing upwards. Gently add 10 microliters of cryo A solution on top of the drop and gently mix by pipetting so that the crystal is not touched. Using a microscope, slowly remove some liquid from the crystal and keep about five to eight microliters of the solution.
Gently add 2.5 to four microliters of cryo B solution onto the drop and gently mix by pipetting. Cover the glass coverslip with a small Petrie dish to avoid direct airflow over the crystal. After waiting five minutes, carefully pick a single crystal from the drop in an appropriate cryoloop mounted on a proper base.
Flash-freeze the crystal in liquid nitrogen. Then store the crystal containing cryoloop in a puck immersed in liquid nitrogen in a Dewar flask until ready for X-ray diffraction at the synchrotron. After extensive trials with several different IKK1 variants, crystals were obtained with one truncated construct, which displayed suitable X-ray properties only in the presence of the IKK inhibitor XII.
The combined effect of low-resolution X-ray data with weak intensities, a large number of IKK1 molecules in the asymmetric unit and conformational variation of the IKK1 monomeric-dimeric model, compared to known IKK2 models, made it difficult to obtain a molecular replacement solution, IKK1, and determine its structure. Different IKK2 structures indicated different inter-monomer orientation within its dimers so that the distance between the alpha carbons of P578 in the two kinase domains, in four different dimer models, varied between 39 and 61 angstroms. Obtaining a useful search model was possible because of the low-resolution cryoelectron microscopy map and a high-accuracy model of IKK1 domains that could be generated based on a high-resolution IKK2 structure.
The initial model indicated an orientation of kinase domain rotated 24 degrees relative to that of an IKK2 monomer and an N-terminal opening of 58 angstroms. Using one of the dimers with the 52-angstrom opening as a model, six dimers in the asymmetric unit were located. Once mastered, this technique can be executed in a couple of months, if it is performed properly.
While attempting this procedure, it's important to remember that obtainment of a soluble, active and well-behaved protein is the first critical step. After watching this video, you should have a good general understanding of all the steps that need to be followed in painstaking detail, to be successful in crystallizing and determining the structure of IKK1 protein. Learning this procedure, there's still structures of other IKK family proteins that can also be determined in order to answer additional questions about kinase-mitogen signaling.
