Protein expression and purification
DNA coding for amino acid residues 166−382 of the SAE2 subunit was amplified through polymerase chain reaction, and inserted into the vector pET-28a for expression of the E1 Cys domain protein with a His6
-tag on the C-terminus. For all mutants, site-directed mutagenesis were carried out with the QuikChange PCR kit (Stratagene), using designed primers to create the desired amino acid residue changes. The sequences of all new plasmids were confirmed by DNA sequencing before protein expression and purification. All the recombinant proteins were expressed and purified using a Ni-NTA column as described previously (Lin et al., 2002
). For NMR studies, proteins were labeled with 15
C or 2
H as described previously (Liu et al., 1999
). The E. coli strain for expressing full-length E1 was a generous gift from Dr. Christopher Lima (Sloan-Kettering Institute, NY).
Enzyme activity assays
All SUMO conjugation assays were conducted at 37°C in the presence of ATP and its regeneration system (50 mM Tris, pH 7.5, 5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate, 3.5 Units/ml creatine kinase, and 0.6 Units/ml inorganic pyrophosphatase). For E1•SUMO thioester formation, 20 μM SUMO and 5 μM E1 were incubated for 10 minutes. For conjugation to substrate RanGAP1, 15 μM SUMO and 15 μM RanGAP1418−587 were incubated together with 0.5 μM E1 and 0.5 μM Ubc9 for 3 minutes. The reactions were terminated by adding SDS-gel loading buffer, and the mixtures were resolved via SDS-polyacryamide gel electrophoresis.
In order to examine the transfer of SUMO from E1 to E2, SUMO transfer assays were performed as follows. E1•SUMO thioester complex was formed first in a mixture containing ATP regeneration system (see above), 1 μM Alexa680 (Invitrogen) fluorescent dye-labeled SUMO, and 1 μM E1 protein. After 10 minutes of incubation at 37 °C, the reaction was halted by adding EDTA to a final concentration of 30 mM to remove Mg2+. Due to fast transfer rates, all the solutions for the following steps were pre-cooled on ice, and the assay was carried out in the cold room. Various concentrations of Ubc9 were added to the E1•SUMO/EDTA mixture, and samples were taken at different time points after the start of the assay and stopped by mixing with non-reducing 4M urea/SDS gel-loading buffer; the samples were fractionated by SDS-PAGE. The gel was briefly rinsed with water before the fluorescent image of the gel was taken with a Li-Cor infrared fluorescence scanner in combination with the software Odyssey. The software ImageQuant 5.2 was used for quantification.
For the steady-state kinetic experiment, preliminary assays were carried out to determine the appropriate E1 concentration range in which the rate of RanGAP1-SUMO conjugation is linearly dependent on the E1 concentration. In addition, the optimal assay duration was also determined for the examination of initial rates of the product formation. In the final kinetic assays, 10 μM RanGAP1 was mixed with 5μM SUMO1, 100 nM E1, an ATP regeneration system, and serial concentrations of E2 proteins (25, 40 and 62.5 nM for wild-type Ubc9; 100, 160 and 250 nM for Q130A mutant; 800, 1250 and 2000 nM for A131D mutant; 160, 250, 400 nM for E132A mutant; and 500, 800, 1250 nM for Y134A mutant). The reactions were carried out for 10 minutes at 37 °C before it was stopped by the addition of an equal volume of SDS-gel loading buffer containing DTT. The samples were resolved by SDS-polyacrylamide gel electrophoresis, and the bands were detected by Western blot with mouse monoclonal antibody against SUMO-1 (Abgent) and IRDye 680-labeled secondary antibody (Li-Cor Biosciences), in combination with the Odyssey infrared imaging system. SUMO protein alone was also loaded onto the gel in a series of known amount in parallel to the assay samples to ensure that the quantification of the protein bands is in the linear range of the detection. Each data point was repeated three times, and the amount of assay product formed in each reaction was calculated as the percentage of the band intensity of RanGAP1-SUMO over that of free SUMO and RanGAP1-SUMO combined. For the Lineweaver-Burk plot, the reciprocal of the amount of RanGAP1-SUMO formed per minute was plotted against the reciprocal of Ubc9 concentration, and the Km and Vmax values were extracted from linear fittings of each set of data.
Paramagnetic Spin-labeling and NMR Experiments
Ubc9 was first treated with excess tris(2-carboxylethyl)phosphine (TCEP) for approximately two hours at room temperature to ensure that all Cys residues were reduced, followed by dialysis to remove excess TCEP. A 15-fold excess of MTSL dissolved in acetylnitrile was added to the Ubc9 solution, and the reaction was carried out at 4°C overnight. Excess MTSL was removed by extensive dialysis. The complex was prepared with a molar ratio of 1:1.1 Cys domain:Ubc9 and had pH of 7.2.
N-enriched Cys domain was used to acquire the following spectra for resonance assignments: HSQC, TROSY, TROSY-HNCA, TROSY-HN(CO)CACB and TROSY-HNCACB. All spectra were acquired at 25°C on a 600 MHz Bruker Avance instrument equipped with a cryo-probe. For each complex of spin-labeled mutant Ubc9 and 15
N-labeled SAE, two HSQC experiments were acquired; one with MTSL in the oxidized state (paramagnetic state) and one with MTSL reduced by addition of 5-fold excess concentrated ascorbic acid to the NMR tube, which only increased the volume of NMR sample by 1%. The latter 15
N-HSQC was acquired two hours after the addition of ascorbic acid. All spectra were processed and analyzed with the programs nmrPipe, nmrDraw (Delaglio et al., 1995
), and nmrView. The intensity ratios of oxidized versus reduced spectra were obtained from the peak heights, and further normalized to the largest ratio value before distance constraints were calculated.
Intermolecular distance constraints between the spin label on Ubc9 and amide protons of the SAE2 domain were calculated from paramagnetic line broadening effects using a protein rotational correlation time of 13 ns, based on the molecular weight of the complex (Battiste and Wagner, 2000
). Because the complex is of low affinity, the rotational correlation time could be influenced significantly by the unbound states. However, theoretical simulation shows that when the overall correlation time changes from 6.5 ns to 26 ns, the calculated distances change about 3 Å in comparison to the distance calculated with correlation time of 13 ns. This range is covered by the 6 Å upper and lower bounds. The distance restraints were defined as follows. Residues with oxidized:reduced peak intensity ratios of 0 were set with upper limits of 20
and the lower limits were set to 1.8 Å. Residues with oxidized:reduced peak intensity ratios between 0 and 0.65 have both lower and upper limits defined by calculations. For residues with peak intensity ratios greater than 0.75, their lower limits were calculated, but upper limits were set at an arbitrary 62 Å to ensure that they are sufficiently large and that the exact value does not influence the docking results. The distance constraints used in the structural calculation are between the sulfur atom of Ubc9's cysteine and the protons of SAE2. Therefore, in order to compensate for the difference in the distances between the sulfur group and the unpaired electron as well as the flexibility of the proxyl group, 6 Å bounds were used for the constraints. A total of 107 distance constraints was used in the structural calculation.
The structure of the Cys domain-Ubc9 complex was calculated using the program HADDOCK (Dominguez et al., 2003
). The X-ray structures of the Cys domain and Ubc9 were used as the starting structures. An ensemble of five Cys domain structures with diverse loop region structures, as generated by the program Modeller, was used as an input file. The two loop regions of SAE2 (residues 212−238 and 291−304) were defined as “fully flexible”. Other residues of the Cys domain and Ubc9 that showed significant chemical shift perturbation as well as significant solvent exposure (>50% for Cys domain and >30% for Ubc9) were defined as “semi-flexible”. All other residues on both proteins were fixed in the calculation. The chemical shift changes were added as ambiguous constraints in the final stages of structural calculation, which did not significantly alter the structure of the complex, but slightly improved the convergence of the structure. A total of 1000 structures were initially generated. The top 250 structures were subjected to simulated annealing calculations, and the best 20 structures were analyzed and shown here.