Casein kinase-2–mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2

N- and C-terminal SIMs in IE2 interact with SUMO1 and SUMO2

Bioinformatics analysis using JASSA (24) predicted three SIMs in IE2: SIM1, 199–202; SIM2, 410–413; and SIM3, 501–504, as shown in Fig. 1A. Among these, SIM1 was previously identified as a bona fide SIM by pulldown assays in cells (15). However, the rest of the SIMs was not investigated before. The affinity of the SIM–SUMO interaction is typically weak, which is difficult to capture by pulldown experiments. Alternatively, NMR spectroscopy can detect interaction over a broad range of affinities, including weak interactions (25). We designed peptides corresponding to the putative SIMs and tested the binding of the SIMs to SUMO1 by NMR (Table 1). Perturbations due to the altered chemical environment upon ligand binding are reflected in a shift of the backbone amide resonances in the ¹⁵N-edited HSQC spectra. The IE2–SIM1 peptide was titrated into a sample of ¹⁵N-isotope–labeled SUMO1, and a series of ¹⁵N-edited HSQC experiments monitored its effect on ¹⁵N-SUMO1. An overlay of the HSQCs is given in Fig. 1B, and two expanded areas of the HSQC are plotted in Fig. 1C. A subset of SUMO1 peaks shifted consistently with increasing concentrations of IE2–SIM1. The chemical shift perturbations (CSP) against SUMO1 residues, given in Fig. 1D, indicated that the maximum perturbations occurred in the residues 35–55, which include the β-strand β2, α-helix α1, and the loop between them (Fig. 1E). The interface corresponds to the canonical interface observed in other SUMO/SIM complexes (17). The same titration experiments were repeated for IE2–SIM2 and IE2–SIM3 domains in separate experiments. IE2–SIM3, but not IE2–SIM2, bound to SUMO1 (Fig. S1A). The pattern of CSP in SUMO1 upon binding IE2–SIM3 is similar to IE2–SIM1, indicating that both the SIMs bind at the same interface.

We repeated the titration experiments with IE2–SIMs against ¹⁵N-labeled SUMO2 and monitored the effects using ¹⁵N-edited HSQC spectra of ¹⁵N-SUMO2. Similar to SUMO1, a significant subset of SUMO2 peaks shifted upon titration with IE2–SIM1 as shown in Fig. S1, B and C. The most perturbed region is between β2 and α1 of SUMO2, which is the known SUMO2/SIM interface (Fig. 1, F and G). Additionally, IE2–SIM3, but not IE2–SIM2, interacted with SUMO2 (Fig. S1D).

The CSPs observed during titration were fit against peptide/protein concentration to yield the dissociation constant (K_d) of the complex (Figs. S2 and S3 and Table 1). The K_d value of the N-terminal IE2–SIM1 was 6-fold lower than the C-terminal IE2–SIM3, indicating that the N-terminal SIM has a higher affinity for SUMO than the C-terminal SIM. Both IE2–SIM1 and IE2–SIM3 do not have paralog specificity and have similar affinities for SUMO1 and SUMO2. Taken together, although IE2–SIM1 and IE2–SIM3 interacted with both SUMO1 and SUMO2, the interaction between IE2–SIM1 and SUMO1/2 was stronger. Hence, the IE2–SIM1 was studied further.

Casein kinase 2 phosphorylates two serines adjacent to IE2–SIM1

IE2 phosphorylation modulates its transactivation activity (19). Previous phosphorylation studies have focused on specific sites or domains in IE2 (19–21). A comprehensive report of phosphorylation sites in the cellular condition is missing. Thus, we studied the sites of IE2 phosphorylation in HEK293T cells taking a proteomics approach (Fig. 2A, Fig. S4). Interestingly, in addition to various newly identified phosphorylation sites, two serines adjacent to IE2–SIM1, Ser-203 and Ser-205, were phosphorylated in cells (Fig. 2A). Ser-203 and Ser-205 are putative CK2 target phosphorylation sites. An in vitro phosphorylation assay of IE2–SIM1 by CK2 was carried out to confirm whether these sites are indeed modified by CK2 (Fig. 2B). IE2–SIM1 peptide does not include any serines apart from Ser-203 and Ser-205. It consists of a threonine, which is not the putative CK2 phosphorylation site. Phosphorylation of IE2–SIM1 was carried out using γ-ATP, resolved on SDS-PAGE, and detected by autoradiography. As shown in Fig. 2C, phosphorylated IE2–SIM1 was observed in the reaction with active CK2, whereas the heat-inactivated CK2 could not phosphorylate IE2–SIM1. IE2–ppSIM1 was further analyzed by MALDI-TOF to find out whether it was phosphorylated at a single serine or both the serines (Fig. 2D). Although mass (m/z) for IE2–SIM1 was 2098 Da, the same for phosphorylated IE2–SIM1 was 2258 Da. The difference of 160 Da corresponds to the addition of two phosphate groups, indicating that indeed CK2 phosphorylates IE2–SIM1 at Ser-203 and Ser-205.

Phosphorylation increases the affinity between IE2–SIM1 and SUMO1/2

Phosphorylation of SIMs may regulate the SUMO–SIM interaction (26). NMR titrations were repeated using a synthetic peptide of IE2–SIM1, where the two serines Ser-203 and Ser-205 are phosphorylated (IE2-ppSIM1, Table 1), to examine the effect of Ser-203/205 phosphorylation on its interaction with SUMO1/2. The pattern of CSPs observed in SUMO1 upon titration with IE2–ppSIM1 is similar to that observed during interaction with unphosphorylated IE2–SIM1 (Fig. S5A), indicating that the interface of binding is identical. However, several peaks went into an intermediate exchange during the titration, suggesting that either the affinity between SUMO1 and the IE2–SIM1 has increased upon phosphorylation and/or phosphorylation of IE2–SIM1 gave rise to dynamic exchange processes at the binding interface. Fitting of NMR chemical shifts against ligand/protein concentration yielded the dissociation constant to be 7.1 (±0.4) μm, which is 8-fold lower than unphosphorylated IE2–SIM1 (Fig. S5B and Table 1). When IE2–ppSIM1 was titrated to SUMO2, the interface of interaction was similar to IE2–SIM1 (Fig. S6A). The K_d of interaction with SUMO2 was 7.2 (±1.6) μm, confirming a significantly tighter binding upon phosphorylation (Fig. S6B). In summary, phosphorylation increased the interaction between IE2–SIM1 and SUMO1/2.

It was important to determine the structure of the complex between SUMO and IE2–ppSIM1 to understand that the molecular mechanism of phosphorylation induced a tighter SUMO/IE2–SIM1 interaction. A ¹³C,¹⁵N-filtered (F1) and ¹³C,¹⁵N-edited (F2) NOESY HSQC were acquired on a sample of ¹³C,¹⁵N-SUMO1/IE2–ppSIM1 at the stoichiometric ratio of 1:1.5 (SUMO1/IE2-ppSIM1) (Fig. 3A). This experiment exclusively detects the intermolecular NOEs between SUMO1 and IE2-ppSIM1. ¹H-¹H TOCSY and ¹H-¹H NOESY experiments on free IE2–ppSIM1 provided the proton chemical shifts of IE2-ppSIM1. The chemical shifts of SUMO1 in the complex were assigned by comparing the ¹⁵N-¹H–edited HSQC and the ¹³C-¹H–edited HSQC of ¹³C,¹⁵N-SUMO1/IE2–ppSIM1 complex with the assignments of free SUMO1. Using the intermolecular NOEs as distance restraints, the structure of the SUMO1/IE2–ppSIM1 complex was solved by HADDOCK (Table 2 provides the structural statistics) (27). Fig. S7A shows the 20 lowest energy structures, which superposed well with an r.m.s.d. of 0.5 Å. In the structure, the hydrophobic residues Ile-200 and Ile-202 packed into the hydrophobic patch between the β2-strand and α1-helix (Fig. 3B). Two hydrogen bonds between Asp-204 in IE2 and Lys-46 in SUMO1 also stabilized the interface. The two salt bridges between the side-chain phosphate oxygen atoms of the phosphorylated serines pSer-203/pSer-205 in IE2–ppSIM1 and Lys-39 of the β2-strand in SUMO1 are responsible for the higher affinity between IE2–ppSIM1 and SUMO1 (Fig. 3C).

The structure of the SUMO2/IE2–ppSIM1 complex was studied to determine whether a similar mechanism operates between phosphorylated IE2–SIM1 and SUMO2. We prepared a sample of ¹³C,¹⁵N-labeled SUMO2/IE2–ppSIM1 at the stoichiometric ratio of 1:1.5 (SUMO2/IE2-ppSIM1). ¹³C,¹⁵N-Filtered (F1) and ¹³C,¹⁵N-edited (F2) NOESY HSQC of this sample detected several intermolecular NOEs between SUMO2 and IE2–ppSIM1 (Fig. S7B), which were used to determine the SUMO2/IE2–ppSIM1 structure. The 20 lowest energy structures superimposed with a low r.m.s.d. of 0.4 Å (Fig. S7C). Similar to SUMO1, the IE2–Ile-200 and Ile-202 side chains are buried in the hydrophobic patch between β2 and α1 of SUMO2 (Fig. 3D). The oxygen atoms of phosphate groups of pSer-203 and pSer-205 form salt bridges with Lys-33, Lys-35 in β2 and His-17 in β1 (Fig. 3E). The additional salt bridges between the phosphoserines in IE2–ppSIM1 and SUMO explain the higher affinity of the IE2–SUMO noncovalent interaction upon phosphorylation of IE2.

IE2–SIM1 enhanced SUMOylation of IE2

SIMs often regulate the SUMOylation of a protein in-cis (28). The identified SUMOylation sites in IE2 are Lys-175 and Lys-180, which are adjacent to IE2–SIM1. In vitro SUMOylation assays were carried out to assess the effect of IE2–SIM1 on the SUMOylation of IE2. A FITC was attached to the N-terminal region of IE2 (aa 172–210), which we termed the IE2 N-terminal domain (IE2–NTD, Fig. 4A). IE2–NTD includes both the SUMOylation sites Lys-175 and Lys-180 as well as the IE2–SIM1. Sumoylation reactions were carried out using IE2–NTD as the substrate. Robust and rapid poly-SUMOylation of IE2–NTD with SUMO1 could be observed in the reaction (Fig. 4B). The reaction was repeated with a SIM-mutated IE2–NTD (IE2–NTDm), where the central hydrophobic residues CIVI were mutated to AAAA. IE2–NTDm showed a significantly reduced rate of SUMOylation (Fig. 4C). The efficiency of SUMOylation was estimated by the decay rate of unmodified or apo IE2–NTD signal against time. Faster decay reflected a higher rate of SUMOylation. Apo IE2–NTD signal decayed rapidly compared with IE2–NTDm, which indicated efficient SUMOylation of IE2–NTD due to the presence of IE2–SIM1 (Table 3 and Fig. 4D). When SUMOylation of IE2–NTD was repeated with SUMO2, the rate of SUMOylation was again higher in IE2–NTD than IE2–NTDm (Fig. 4E, Fig. S8, A and B, and Table 3), indicating that the IE2–SIM1 promotes IE2 SUMOylation.

The poly-SUMOylation of IE2–NTD would increase its size and anisotropy. Hence, the SUMOylation of the substrate IE2–NTD can be monitored in real time by measuring the change in fluorescence anisotropy of IE2–NTD (Fig. 4F). As a control, a reaction without ATP was carried out, where the absence of SUMOylation did not change the anisotropy (Fig. S8C). In the SUMOylation reaction using SUMO1 and IE2–NTD/IE2–NTDm as the substrate, the rate of increase in fluorescence anisotropy was higher for IE2–NTD than IE2–NTDm, confirming that the IE2–SIM1 increased SUMOylation of IE2–NTD. When the reaction was repeated with SUMO2, the rate of SUMOylation was higher than SUMO1 (Table 3). This could be because SUMO2 has a consensus SUMOylation motif at Lys-11, which enhances the rate of poly-SUMOylation. Nevertheless, IE2–SIM1 increased the rate of SUMOylation (Fig. 4G and Table 3). Together, the IE2–SIM1 enhanced the SUMOylation of IE2 significantly.

Mechanism of SIM enhanced IE2 SUMOylation

In principle, IE2–SIM1 could increase the SUMOylation of IE2 either by reducing K_m (increasing affinity) between the substrate IE2 and the enzyme UBC9∼SUMO conjugate or by increasing the activity (K_cat) of the enzyme UBC9∼SUMO. To determine the exact effect of IE2–SIM1, we carried out a kinetic study of the IE2 SUMOylation. IE2–NTD is poly-SUMOylated rapidly, which made quantification of the SUMOylated species difficult. We used the mutant of SUMO2 (K11R-SUMO2) that is deficient in poly-SUMOylation, which allowed us to effectively monitor the mono-SUMOylated IE2 (Fig. 5A and Fig. S9). Kinetic analysis of IE2 SUMOylation revealed that IE2–SIM1 reduced the K_m of IE2 by 2.5-fold (Fig. 5B) but did not alter the V_max (K_cat) significantly (Fig. 5C). Therefore, IE2 SIM1 increases the specificity constant (K_cat/K_m) by decreasing the K_m value of the SUMOylation reaction (Table 4). The presence of IE2–SIM1 significantly increased the SUMOylation of IE2 in cellular conditions (Fig. 5, D and E).

Three mechanisms of IE2–SIM1 enhanced SUMOylation are possible (Fig. 6, A–C). The binding of IE2–SIM1 to the SUMO conjugated at the active site of Ubc9 can increase the rate of SUMOylation (Fig. 6A). Alternatively, SUMO has a noncovalent interaction with UBC9 at its “backside binding” area, and this interaction could also enhance IE2 SUMOylation (Fig. 6B). Moreover, UBC9 is SUMOylated covalently at Lys-14, which could also impact IE2–SUMOylation (Fig. 6C). NMR titration experiments studied the hypothesis in Fig. 6A. ¹⁵N labeled WT–UBC9 was titrated with IE2–NTD, and the observed CSPs in UBC9 are plotted in Fig. 6D. The IE2–NTD was truncated from the N-terminal end such that Lys-180 was the sole acceptor lysine present in IE2–NTD. The high CSPs at the α′–α2 and α2–α3 loops indicated the binding site of acceptor Lys-180 and UBC9. The other significant CSPs were observed in the α1–β1 region. The pattern of CSPs was consistent with NMR titrations of UBC9 with SUMO-acceptor sites of p53 and c-Jun (29). Then, C93K–UBC9∼¹⁵N-SUMO1 conjugates were purified and titrated with IE2–NTD. The pattern of CSPs on SUMO1 matched with the CSPs observed in isolated SUMO1/IE2–SIM1 complex (Fig. 6E), indicating that the IE2–SIM1 within the IE2–NTD binds to the same interface on the SUMO1 conjugated to UBC9. IE2–NTD does not bind to the UBC9 active site when the active site is mutated to lysine (C93K–UBC9), and hence the binding to the C93K–UBC9∼¹⁵N-SUMO1 conjugate could not be studied (Fig. S10A). Nevertheless, combining the NMR data of the IE2–SIM1/SUMO interaction, the IE2–NTD/UBC9∼SUMO1 interaction, and the UBC9∼SUMO1 structure (PDB code 1Z5S), a model of UBC9∼SUMO1/IE2–NTD complex was determined by Xplor-NIH (Fig. 6F and Fig. S11A and Table S1). The structure shows how the IE2–SIM1 binds to SUMO1, whereas the acceptor Lys-180 attacks the active site (Fig. 6I). Interestingly, the essential negatively-charged residue of the ψKX(D/E) motif, which is Glu-182 in this context, forms a salt bridge with the positively charged Lys-101 in the β4α2 loop of UBC9. Lys-101 is essential for the recognition of substrates like p53, PML, and IκB (30). The molecular details provided by the structural model explain the underlying mechanism of substrate recognition by Lys-101 in UBC9.

A structural model corresponding to Fig. 6B was built using the NMR CSP data, the structure of UBC9∼SUMO1, and the structure of UBC9/SUMO (PDB), where SUMO binds to the “backside” β-sheet of UBC9 (Fig. 6G and Figs. S11B and S12). Again, the Glu-178 forms a salt bridge with Lys-101. The structural model of Fig. 6C was determined using the covalently linked SUMO1–UBC9 structure (PDB code 2VRR, Fig. 6H, and Figs. S11C and S12). In this case, the Glu-178 prefers to form a salt bridge with Lys-74, which is present on the β-strand β4. Lys-74 is vital to identify substrates like RANGAP1 (31). Lys-175 is further away from the IE2–SIM1 than Lys-180 and had more flexibility to access the active site of UBC9 when the IE2–SIM1 binds to either conjugated, covalently linked, or noncovalently linked SUMO. Overall, structural modeling suggested that all three possibilities of SIM-enhanced SUMOylation are possible in IE2–NTD.

SUMOylation assays were performed with appropriate substitutions of UBC9 to delineate the effects of each mechanism. The noncovalent interaction between UBC9 and SUMO involves histidine 20 in UBC9, and the H20D mutation abolishes the interaction (32). Alternatively, The K14R–UBC9 abolishes the covalent SUMO conjugation. The IE2–NTD SUMOylation was studied by initiating a SUMOylation reaction using E1, SUMO1, and WT–UBC9 or H20D–UBC9 or K14R–UBC9. Gel-shift assays or real-time fluorescence anisotropy assay monitored the SUMOylation of IE2–NTD. When the covalent- or noncovalent-mediated interactions between UBC9 and SUMO were abolished individually, the rate of IE2–NTD SUMOylation did not change significantly (Fig. 6, H and I). A possible cause of the result is that the mechanisms involving the covalent or noncovalent UBC9–SUMO interaction do not contribute much to IE2 SUMOylation. However, the three mechanisms could be redundant, so that when one is interrupted, the others compensate.

Phosphorylation of IE2–SIM1 further enhances IE2 SUMOylation

The SUMOylation assays were repeated with IE2–NTD and phosphorylated IE2–NTD (IE2–ppNTD) to find out whether the phosphorylation of IE2–SIM1 enhances the SUMOylation IE2. FITC-tagged IE2–NTD was incubated with CK2 for phosphorylation. The CK2 was subsequently inactivated, and the IE2–NTD was checked by mass spectrometry to ensure that the serines Ser-203 and Ser-205 were phosphorylated. The phosphorylated IE2–NTD (IE2–ppNTD) was later used as a substrate in the SUMOylation assay (Fig. 7A). The rate of SUMOylation was higher in IE2–ppNTD than in IE2–NTD (Fig. 7B). At 60 min, 50% of total IE2–ppNTD was SUMOylated, whereas IE2–NTD was only SUMOylated up to 30% (Fig. 7C).

The altered rate of SUMOylation upon SIM phosphorylation was also monitored in real time by the fluorescence anisotropy assay using SUMO1 and IE2–NTD/IE2–ppNTD as the substrate (Fig. 7D). The rate of increase in anisotropy was measured to be 1.2 × 10⁻⁴/min for WT IE2–NTD, which almost doubled to 2.1 × 10⁻⁴/min for IE2–ppNTD, indicating that phosphorylation of IE2–SIM1 enhances the SUMOylation of IE2–NTD.

SUMOylation of IE2 was measured in HEK293T cells to assess whether the effect of phosphorylation on SUMOylation of IE2 is persistent in full-length IE2 in cellular conditions. HA-tagged IE2 or a double phospho-inactive mutant IE2 pm S203A/S205A was co-transfected with SUMO1, lysed 24/48 h post-transfection, and probed with HA antibody (Fig. 7, E and F). The amount of IE2 pm∼SUMO1 was significantly lower than IE2∼SUMO1, indicating that the phosphorylation of serines near SIM1 enhances SUMOylation of IE2. The enzyme kinetics experiments were repeated with IE2–NTD and IE2–ppNTD. The experiments were carried out at 16 °C to slow down the reaction and effectively measure the kinetics (Fig. 7G and Fig. S13). The kinetic analysis given in Table 4 indicates that the phosphorylation reduces the K_m value of the reaction by 3.5-fold but does not change the V_max significantly (Fig. 7, H and I). Together, phosphorylation of two serines near IE2–SIM1 by CK2 increases the SUMOylation of IE2.

Phosphorylation of IE2–SIM1 increases transactivation by IE2

IE2 functions as a transactivator for various viral promotors and auto-repressors for its promotor. IE2 SUMOylation and IE2 SIM1 are essential for its function. Ser-203 and Ser-205 phosphorylation enhanced both the IE2–SIM1/SUMO interaction and IE2–SUMOylation. The role of phosphorylation on the IE2-mediated transactivation was examined by a luciferase assay, where the luciferase gene is expressed under the IE2-responsive promoter (pUL54-Luc). Consequently, the level of luciferase expression was proportional to the IE2 transactivation activity. A phosphorylation-inactive S203A, S205A double mutant (pmIE2) and a SIM mutant (CIVI to AAAA, smIE2) of IE2 were designed. PUL54-Luc was transfected into HEK293T along with HA-IE2 (WT or mutants) and SUMO1 (Fig. S14). Transactivation activity of pmIE2 is reduced by ∼25% compared with WT–IE2, indicating that phosphorylation of Ser-203 and Ser-205 is vital for transactivation (Fig. 8A). Interestingly, the activity of pmIE2 is comparable with smIE2, indicating that SIM phosphorylation is necessary for the SUMO-dependent transactivation activity of IE2.

The effect of phosphorylation was also checked with co-transfection of only IE2 and pUL54-Luc, but not SUMO. Interestingly, the activity of WT–IE2 with endogenous SUMO reduced by 50% compared with overexpressed SUMO (Fig. 8B). IE2 SUMOylation increases upon overexpression of SUMO (Fig. S15). The increase of both SUMOylation and transactivation activity of IE2 upon overexpression of SUMO underscores the importance of IE2 SUMOylation for its transactivation activity. Alternatively, increased SUMOylation of IE2-associated transcription factors may also increase the IE2-mediated transactivation (Fig. 8D). Nevertheless, the activity of pmIE2 dropped by 40% compared with WT–IE2, emphasizing the significance of SIM phosphorylation when the amount of SUMOylated transcription factors or SUMO is not abundant (Fig. 8B). Additionally, the importance of Ser-203/205 phosphorylation on the auto-repression activity of IE2 was studied using similar luciferase assays where Luc was expressed under MIEP. Expression of luciferase decreases on overexpression of IE2 indicating MIEP repression by IE2. Interestingly, MIEP repression is relieved in pmIE2, indicating the importance of SIM phosphorylation for the auto-repression activity (Fig. 8C). IE2 SUMOylation is important for MIEP repression, and SIM phosphorylation enhances IE2 SUMOylation. Our data suggest that SIM phosphorylation augments SUMOylation-dependent activity of IE2 as an auto-repressor.

Plasmid and peptides

HCMV IE2 peptides were synthesized from Lifetein. pET28-SUMO1(C-His), pET28-SUMO2(C-His), pET11-AOS1/UBA2, and pET28-ΔN364 SENP2 were gifted by Dr. Christopher Lima, Sloan Kettering Institute, New York. pET28-UBC9 was obtained from Addgene (25213). This construct was used as a template for site-directed mutagenesis to obtain H20D UBC9, K14R UBC9, and C93K UBC9. SUMO1-pQE80L and SUMO2-pET15b were obtained from TIFR, Mumbai, India. Mammalian expression vectors, pDEST-SG5 HA-IE2, pUL54-Luc, and pSG5 Flag-SUMO1, were kind gifts from Dr. Jin-Hyun Ahn, Sungkyunkwan University School of Medicine. The pDEST-SG5 HA-IE2 was used as a template for site-directed mutagenesis to generate CIVI199/200/201/202AAAA HA-IE2 and S203A/S205A HA-IE2.

Protein purification

All the proteins were expressed and purified from BL21 (DE3) cells in ¹⁵NH₄Cl-M9 medium for labeled proteins and in LB for unlabeled proteins. Labeled proteins were purified in phosphate buffer for NMR, and in Tris buffer for in vitro assays. ¹⁵N-Labeled SUMO1/2 were cultured at 37 °C to 0.8 OD₆₀₀ and induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 4–5 h. Cells were lysed by sonication in lysis buffer containing 50 mm Na₂HPO₄, pH 8.0, 20 mm imidazole, and 300 mm NaCl. Lysate was clarified by centrifugation, and supernatant was incubated with pre-equilibrated Ni-NTA beads for 1 h. After incubation, flow-through was collected, and beads were washed with at least 5 column volumes of lysis buffer. Protein was eluted by increasing the concentration of imidazole in lysis buffer. Fractions containing SUMO1/2 were concentrated and were further processed through a gel-filtration column (Superdex 75 16/600) in PBS buffer.

For in vitro assays, E1 (UBA2/AOS1) and E2 (UBC9) were purified with Ni-NTA affinity purification followed by gel filtration as discussed above, but in Tris buffer. Lysis/wash buffer composition was 50 mm Tris, pH 8, 350 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm β-mercaptoethanol, 20 mm imidazole. Elution buffer contained 25 mm Tris, pH 8, 150 mm NaCl, 1 mm β-mercaptoethanol, 250 mm imidazole, and gel filtration buffer contained 20 mm Tris, pH 8, 50 mm NaCl, 1 mm β-mercaptoethanol.

For SUMOylation assays, the mature forms of SUMO1 and SUMO2 were obtained by processing CHis-SUMO1 or CHis-SUMO2 with SENP2. After Ni-NTA purification, fractions containing CHis-SUMO1/2 were pooled and incubated with purified His-ΔN 364 SENP2 (1:1000 molar ratio) at room temperature until complete digestion of the C-terminal extension. SENP2 and unprocessed SUMO were removed by passing the reaction mixture through Ni-NTA beads, and flow-through containing mature SUMO was collected, concentrated, and further purified with Superdex 75. ΔN 364 SENP2-pET 28b was expressed in BL21 (DE3) and purified through Ni-NTA affinity purification as mentioned above. The buffer used for SENP2 purification is similar to the buffer used for E1 purification. After Ni-NTA, partially purified SENP2 was concentrated and stored.

In vitro biochemical assays

For SUMOylation of IE2–NTD or IE2–ppNTD or IE2–NTDm, 5 μm peptide and 5 μm SUMO1/2 were incubated with 1 μm E1 and 2.5 μm E2. The reaction was started by adding ATP. SUMOylation buffer contains 20 mm HEPES, pH 7.5, 50 mm NaCl, 5 mm MgCl₂, 0.1% Tween 20. The reaction was analyzed either on 12% SDS-PAGE or by a change in anisotropy. Gels were imaged for FITC fluorophore (λ_ex − 495 nm and λ_em − 519 nm). Although anisotropy was measured using MOS450 fluorimeter (λ_ex − 470 nm and λ_em − 520–560 nm).

IE2–ppNTD was obtained by phosphorylating IE2–NTD with purified CK2. Human CK2 was obtained from New England Biolabs (P6010S). IE2–NTD was phosphorylated in buffer provided by the manufacturer, which contains 50 mm Tris, pH 7.5, 10 mm MgCl₂, 0.1 mm EDTA, 2 mm DTT, and 0.1% Brij35. In a 50-μl reaction, 50 units of CK2 was sufficient to phosphorylate 50 μm IE2 peptide in 2 h at 30 °C. CK2 was heat-inactivated at 65 °C for 15 min after IE2 phosphorylation. IE2–NTD phosphorylation was analyzed using autoradiography or by MALDI-TOF MS. Phosphorylated IE2–NTD (IE2–ppNTD) was used as a substrate for SUMOylation assays.

C93K UBC9-SUMO1 conjugates were made either with ¹⁵N-C93K UBC9 or ¹⁵N-SUMO1. Conjugation reaction was performed in buffer containing 20 mm CAPS, pH 9.5, 50 mm NaCl, 5 mm MgCl₂, 3 mm DTT in the presence of 10 μm E1, 300 μm C93K UBC9, 500 μm SUMO1, and 3 mm ATP. The reaction was performed overnight at 37 °C. Conjugate formation was analyzed on an SDS gel before further purification by Mono Q and size-exclusion chromatography. Conjugation reaction mixture was diluted by 25 mm Tris, pH 8, to reduce salt concentration. The diluted reaction mixture was loaded onto the Mono Q column, and proteins were eluted with increasing concentrations of NaCl (up to 1 m). Fractions containing C93K UBC9-SUMO1 conjugate were pooled and further purified by SD75 in PBS buffer.

Single turnover reactions were performed for Michaelis-Menten kinetics experiments. UBC9∼K11R SUMO2 conjugates were formed in a 50-μl reaction with 1 μm E1, 10 μm E2, and 10 μm K11R SUMO2 in SUMOylation buffer. The reaction was started by adding E1 and was incubated at 37 °C for 10 min. The reaction was stopped by adding 1450 μl of quenching buffer. Quenching buffer contained 20 mm HEPES, pH 7.5, 50 mm NaCl, 5 mm EDTA, 0.1% Tween 20. IE2–NTD SUMOylation was performed by adding an equal volume of quenched reaction to different concentrations of serially diluted IE2–NTD peptides (so that conjugates and peptides are further diluted by half to give the desired concentration). Aliquots were taken at desired time points, and the reaction was stopped by SDS loading dye. The reaction was resolved on 12% SDS-PAGE in nonreducing conditions and was then transferred onto polyvinylidene difluoride membrane and blotted with SUMO2 antibody. All the biochemical assays were performed in triplicate, and the error bars represent the S.E.

NMR experiments

The NMR spectra of SUMO1 and SUMO2 were recorded at 298 K on 800 MHz Bruker Avance III HD spectrometer with a cryoprobe head, processed with NMRpipe (38) and analyzed with Sparky (39). All the NMR experiments with FHA-Chk2 were performed at 293 K. The SUMO1/2 samples were prepared in PBS buffer, with 5 mm DTT at pH 7.4 and 10% D₂O. ¹H-¹H TOCSY and ¹H-¹H NOESY were acquired and used to assign IE2–ppSIM. For NMR titration experiments, ∼3 mm peptides were titrated into ∼0.3 mm ¹⁵N-SUMO1 or ¹⁵N-SUMO2. The titration data were fit in 1:1 protein/ligand model using the equation, CSP_obs = CSP_max {([P]_t + [L]_t + K_d) − ([P]_t + [L]_t + K_d)² − 4[P]_t[L]_t]^1/2}/2[P]_t, where [P]_t and [L]_t are total concentrations of protein and ligand at any titration point. The SUMO/IE2–ppSIM1 NMR samples were prepared in PBS buffer, with 5 mm DTT at pH 7.4 and 10% D₂O. ¹³C,¹⁵N-filtered (F1) and ¹³C,¹⁵N-edited (F2) NOESY HSQC were collected on a ¹³C,¹⁵N-SUMO1/IE2–ppSIM1 (1:1.5) complex sample with a mixing time of 200 ms to measure intermolecular NOEs between SUMO1 and IE2-ppSIM1. A similar experiment was performed to obtain intermolecular NOEs in the SUMO2/IE2–ppSIM1 complex.

Structure determination and modeling

Unambiguous restraints between the SUMO1 and IE2–ppSIM1 were determined from the intermolecular NOEs observed in the ¹³C,¹⁵N-filtered (F1) and ¹³C,¹⁵N-edited (F2) NOESY HSQC. Dihedral angles of IE2–ppSIM1 were determined from ¹H-¹H TOCSY and NOESY experiments. The dihedral angles were used to determine an extended structure of IE2-ppSIM1. The solution structure was calculated in HADDOCK (27) using the structure of SUMO1 (PDB code 4WJO) and the extended structure of IE2-ppSIM1. Rigid body energy minimization generated 1000 initial complex structures, and the best 200 by total energy were selected for torsion angle dynamics and subsequent Cartesian dynamics in an explicit water solvent. Default scaling for energy terms was applied. The interface of SUMO1 was kept semi-flexible during simulated annealing and the water refinement steps. Following the standard benchmarked protocol, cluster analysis of the 200 water-refined structures yielded a single clear ensemble. The SUMO2/IE2–ppSIM1 complex was similarly docked, except that intermolecular restraints between SUMO2 and IE2–ppSIM1 were used, and the starting structure of SUMO2 was taken from a crystal structure of free SUMO2 (PDB code 1WM3). The structures of IE2–SIM1/SUMO1 and IE2–SIM1/SUMO2 were deposited in the PDB with accession codes 6K5T and 6K5R.

The model of UBC9∼SUMO1/IE2–NTD was calculated in XPLOR-NIH. The intramolecular and intermolecular distance restraints were calculated from the structure of UBC9∼SUMO1 (PDB code 1Z5S). IE2–NTD was kept flexible throughout the structure calculation. The other restraints used were the SUMO1/IE2–ppSIM1 NOE restraints and UBC9/IE2–NTD CSPs measured in this work. During structure calculation, the distance of acceptor Lys-180 and the active site was restrained to 1.8 Å. A hundred structures were calculated, and the 10 lowest energy structures were used for analysis. The model of SUMO1/UBC9∼SUMO1/IE2–NTD, where an additional SUMO1 molecule is noncovalently bound to UBC9, was calculated similarly, except the SUMO1/UBC9 intermolecular restraints were estimated from the SUMO1/UBC9 structure (PDB: 2UYZ) and the SUMO1/IE2–SIM1 restraints were set between the noncovalent bound SUMO1 and IE2–SIM1. The model of SUMO1–UBC9∼SUMO1/IE2–NTD complex, where the SUMO1 is covalently bound to UBC9, was calculated similarly, except that SUMO1–UBC9 restraints were obtained from the SUMO1–UBC9 structure (PDB code 2VRR), and the SUMO1/IE2–SIM1 restraints were set between the covalently-bound SUMO1 and IE2–SIM1. The refinement statistics are given in Table S1.

Cell culture and transfection

HEK293T cells were maintained in Dulbecco's modified Eagle's medium with 10% serum. For any experiment, cells were seeded into 12-well tissue culture plates. Cells were transfected at 60–80% confluency with 500 ng of FLAG-SUMO1 and 500 ng of HA-IE2 (WT or mutant as mentioned) using Lipofectamine 3000 reagent. Cells were harvested 48 h post-transfection and lysed with 2× SDS loading dye. Lysates were run on a 12% SDS gel and were probed with HA antibody (CST-3724) after blotting.

For transactivation assays, HEK293T cells were seeded into a 12-well plate and were cultured to 70–80% confluency. Cells were transfected with 100 ng of pUL54-Luc, 5 ng of pTK-Renilla (transfection control), and 400 ng of WT–IE2 with or without 400 ng of FLAG-SUMO1. In the case of mutants (S203A/S205A-IE2 and C199A/I200A/V201A/I202A-IE2) transfected plasmid amount was increased to acquire similar expression as WT–IE2. Luciferase concentration was measured 36–40 h post-transfection by Dual-Glo–luciferase kit (Promega).

HA-IE2 was immunoprecipitated from HEK293T for PTM analysis by mass spectrometry. One 100-mm dish was transfected with 10 μg of HA-IE2 plasmid with Lipofectamine 3000. IP was performed 36h post-transfection. HA-tagged Sepharose beads (CST) were used for pulldown. The protocol provided by the manufacturer was followed for the IP. After IP, beads were directly loaded onto reducing SDS-PAGE, and immunoprecipitated proteins were resolved. The band, matching the size of the protein of interest, was excised and analyzed by mass spectrometry.