Figure 1.
NleE specifically targets TAB2/3 in the NF-κB pathway.
(A) Back-methylation analysis of NleE modification of cellular TAB2. Flag-TAB2 co-expressed with a vector or NleE in 293T cells was immunopurified and incubated with recombinant NleE in the presence of 3H-SAM. NleEΔ6, NleE mutant with deletion of 209IDSYMK214. (B–D) Mass spectrometry analysis of NleE modification of TAB2 (B), HOIL-1L (C) and Sharpin (D) during EPEC infection. Shown are the extracted ion chromatograms of triply charged NZF-domain peptides from TAB2, HOIL-1L-V195R or Sharpin-S346R purified from infected 293T cells. The unmethylated peptides are shown in blue trace and the methylated ones are in red with the methylated cysteine residue in red. The V195R and S346R mutations were introduced to facilitate mass spectrometry identification of the tryptic peptides. Cca, carbamidomethylated cysteine generated from iodoacetamide treatment during sample preparation; Cme, NleE-methylated cysteine.
Figure 2.
Crystal structure of NleE in complex with SAM.
(A) Stereoview of NleE structure in ribbon diagram with green α-helices, magenta β-strands and grey loops. SAM is in orange sticks. Secondary structure elements are labeled in sequential numbers (α1–10, β1–3). (B) Electrostatic surface potential of NleE structure. The color scale from red (−5 kT/e) to blue (5 kT/e) was calculated in PyMol. (C) A close view of the SAM binding core. SAM is meshed with σ-A weighted Fo-Fc simulated annealing omit map contoured at 3.0σ. SAM interaction residues (Arg107, Glu191 and Tyr212) are also in sticks. Polar interactions are represented by black dashed lines with a number denoting the distance in angstrom. The color scheme follows that in (A).
Figure 3.
Functional analyses of NleE SAM-binding mutants.
(A, B) Effects of SAM-binding site mutations on methylation of TAB2-NZF. GST-TAB2-NZF was incubated with NleE or indicated mutants in the absence (upper) or presence (lower) of exogenous SAM, followed by native-PAGE analysis (A). 3H-SAM was added into the reaction and the reaction was analyzed by SDS-PAGE electrophoresis in (B). FL indicates the full-length NleE. (C) Luciferase assays of the activity of NleE mutants in blocking the NF-κB signaling. 293T cells were transfected with TAB3 together with an indicated NleE variant. The Y axis is on the logarithmic scale. Error bar, standard deviation. (D) Mass spectrometry analysis of NleE-R107A mutant modification of TAB2 during EPEC infection. Shown are the extracted ion chromatograms of triply charged NZF-domain peptides from TAB2 purified from infected 293T cells. The unmethylated peptides are shown in blue trace and the methylated ones are in red with the methylated cysteine residue in red. Cca, carbamidomethylated cysteine generated from iodoacetamide treatment during sample preparation; Cme, NleE-methylated cysteine.
Figure 4.
Structural comparison of NleE-SAM complex with other SAM-dependent methyltransferases.
(A) Representative methyltransferase structures. The upper and lower panels show the ribbon diagram and the corresponding topology, respectively. α-helices, β-sheets and loops are in green, magenta and grey, respectively. SAM/SAH is shown as orange sticks in the ribbon diagram and orange rhombus in the topology diagram. The knot α-helixes in Class IV and V methyltransferases are in red and the linking loops are drawn as cyan lines. (B) The conformation of SAM in NleE (upper) and its atomic nomenclature (lower). (C) Structural alignment of SAM in NleE with that in other methyltransferases using the ribose ring as the reference. (D) The conformational difference between NleE-bound SAM and SAMs in other five classes of methyltransferases. SAM conformation is denoted by three dihedral angles indicated in the table. The atomic nomenclature of SAM follows that in (B).
Figure 5.
MD simulation shows Cys673 in TAB2-NZF being structurally favorable for methyltransfer from NleE.
(A) The dynamic motion (indicated by the RMSD values) (upper left), the change of interaction energy (upper right) and the S-C distance (lower left) of the largest four clusters in Cys673 or Cys687-restrainted 15-ns MD simulation (shown are snap shots of the last 10-ns simulation). Representative poses of the four clusters (black, red, blue and green curve) obtained from protein docking performed with restrained distance from Cys673/Cys687 to the methyl carbon in SAM (S-C distance) were used as starting templates for the 15-ns MD simulation shown. (B) Overall view of the docked NleE-SAM-NZF complex. NleE is shown as white surface model; the SAM is in orange sticks; TAB2-NZF is in blue sticks with the Zn represented by a red dot.
Figure 6.
NleE binds to an N-terminal region in TAB2/3 for efficient recognition.
(A) Domain illustration of TAB3 and summary of NleE interaction with TAB3 truncations. Interaction strength is scored by the number of + (also see the related data in Figure S4). CUE, CUE ubiquitin binding domain; TBD, TAK1-binding domain; CC, coiled-coil domain; NZF, Nlp4-like zinc finger domain. (B) Yeast two-hybrid assay of NleE interaction with TAB3 truncations. Yeast strain AH109 was transformed with plasmid combinations as illustrated (bait+prey). Plasmid combinations resulting in a positive interaction are highlighted in red. (C) Co-immunoprecipitation interaction assay of NleE and TAB3 truncations in transfected 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).
Figure 7.
NleE targets ZRANB3-NZF domain for cysteine methylation and disrupts its ubiquitin-chain binding.
(A) 3H-SAM labeling assay of NleE methylation of different GST-NZFs. (B) 3H-SAM labeling assay of NleE methylation of full-length Flag-TAB2/ZRANB3 purified from control or NleE-expressing 293T cells (Flag-ZRANB3NleE). (C–E) Assays of ubiquitin-chain binding of NleE-modified ZRANB3-NZF or full-length ZRANB3. Indicated ubiquitin chains were subjected to pulldown by purified GST-ZRANB3-NZF that was left untreated or had been incubated with recombinant NleE (C). Lysates of 293T cells transfected with Flag-ZRANB3 and NleE were subjected to pulldown by indicated ubiquitin chains (D and E). Shown are immunoblots of the pulldown (Ub chain pulldown) and total cell lysates (Input). FL indicates the full-length NleE. (F) Mass spectrometry analysis of NleE modification of ZRANB3 during EPEC infection. Shown are the extracted ion chromatograms of doubly charged NZF-domain peptides from ZRANB3 purified from infected 293T cells and digested by Glu-C. The unmethylated peptides are shown in blue trace and the methylated ones are in red with the methylated cysteine residue in red. Cca, carbamidomethylated cysteine generated from iodoacetamide treatment during sample preparation; Cme, NleE-methylated cysteine. (G) Assays of ubiquitin-chain binding of NleE-modified Flag-ZRANB3 during EPEC infection. Lysates of 293T cells transfected with Flag-ZRANB3 and infected with indicated EPEC strain were subjected to Lys63-linked ubiquitin-chain pulldown. Shown are immunoblots of the pulldown (K63 Ub chain pulldown) and total cell lysates (Input).