NleG Type 3 effectors from enterohaemorrhagic Escherichia coli are U-Box E3 ubiquitin ligases

Abstract

NleG homologues constitute the largest family of Type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9' family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56+/-2 microM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.

Figure 1. Multiple sequence alignment of NleG effectors.

The amino acid sequence of NleG2-3 (Sakai ID: ECs2156), NleG (ECs1824), NleG9′ (ECs1828), NleG2-2 (Ecs1994), NleG2-4′ (ECs2229), NleG5-2 (ECs2154), NleG6-1 (ECs1995), NleG6-2 (ECs2155), NleG6-3′ (ECs3488), NleG7′ (ECs2226), NleG8-2 (ECs3486), NleG2-1′ (ECs1811), and NleG5-1 (ECs1996) were aligned using ClustalX . The secondary structure of the NleG2-3[90–191] fragment, as determined by NMR, is shown above the alignment. Secondary elements corresponding to the RING/U-box motif are shaded darkly, while other secondary elements are shaded lightly. Mutation sites are designated with shaded triangle above the alignment when made in NleG2-3 and under the alignment when NleG5-1 was used as template. The positions of the N-terminal amino acid of NleG C-terminal fragments used in this study are denoted by an asterisk. Residues which were line-broadened upon addition of UBE2D2 are denoted by dark grey spheres above the alignment.

Figure 2. Solution structure of NleG2-3[90–191] (A) 2D ¹⁵N-HSQC spectrum of NleG2-3[90–191] at 25°C and pH 7.0.

Selected peaks corresponding to reduced and oxidised conformations are labeled as r (green) and o (blue), respectively. (B) Chemical shift difference (Δ_ppm) between NleG2-3[90–191] reduced and oxidised conformations plotted against the residue number. Δ_ppm was calculated using the formula Δ_ppm = [(Δδ² _HN + (Δδ_N/5)²]^1/2, where δ_N and δ_HN are the chemical shift differences for ¹HN and ¹⁵N between the two forms, respectively. The positions of Cys141 and Cys177 forming the disulfide bond are denoted. (C) Ensemble of 20 refined structures of NleG2-3[90–191] in the reduced (left) and oxidised (right) conformations, with the positions of side chains of residues with different conformations between the two forms highlighted. Positions of secondary structure elements on the reduced conformation are denoted.

Figure 3. Overall ribbon diagram of NleG2-3[90–191].

Secondary elements corresponding to the RING/U-box motif are coloured cyan, while other elements of the structure are coloured green. (A) Overall structure of reduced NleG2-3[90–191] with the N- and C-terminus and secondary structure elements labelled. (B) Superimposition of reduced NleG2-3[90–191] (same as (A)) with RING finger 38 protein (PDB 1X4J, red); the N- and C-termini of both molecules labelled.

Figure 4. In vitro activity of NleG proteins.

(A) Immunoblot analysis using anti-ubiquitin antibodies of reactions performed in the presence of ATP, ubiquitin, E1, UBE2D2 and full length or C-terminal fragments of His₆-NleG isoforms. (B) Immunoblot analysis with anti-ubiquitin antibodies (top panel) and SDS PAGE electrophoresis (bottom panel) of reactions performed with ATP and in the presence or absence of ubiquitin, E1, UBE2D2 E2 and NleG5-1[1–203]. (C) Immunoblot analysis with anti-ubiquitin antibodies (top panel) and anti-His₆ antibodies (bottom panel) of reactions performed with E1, wild type ubiquitin (WT) or ubiquitin derivative (K”O”) in the presence or absence of His₆-NleG5-1[1–203] and UBE2D2 E2 enzyme. All reactions were incubated at 30°C for the times indicated.

Figure 5. Interaction of NleG2-3 and NleG5-1 with human E2 enzymes.

(A) Western blot analysis of reactions using anti-ubiquitin antibodies performed in presence of ATP, ubiquitin, E1, the indicated human E2 enzymes and either NleG5-1[1–213] (top panel) or NleG2-3[55–191] (bottom panel). Reactions were performed at 30°C for 2 hours. The asterisk indicates the E2 enzymes supporting formation of multiple ubiquitinated protein species. The E2 nomenclature is in accordance with that used by the Human Genome Organization (http://www.genenames.org/genefamily/ube2.php). (B) Immunoblot analysis using anti-ubiquitin antibodies of reactions performed in the presence of ATP, ubiquitin, E1, UBE2D2 and NleG2-3 (left) or NleG5-1 (right) variants. The NleG2-3 variants include the wild type (WT) fragments NleG2-3[55–191] and NleG2-3[90–191] and C141A and C177A mutants of NleG2-3[90–191]. The NleG5-1 variants included wild type NleG5-1[1–213] and its C124A, C142A, C147A, C164A and C200A mutants. The samples were incubated at 30°C for the times indicated at the bottom of the panel. (C) Determination of dissociation constants of UBE2D2 with NleG5-1[1–213] wild type (WT) or its C200A variant. The change in fluorescence polarisation of fluorescein-labeled UBE2D2 is plotted as a function of NleG5-1 concentration with error bars indicating one standard deviation.

Figure 6. Detailed analysis of NleG interactions with UBE2D2.

(A) Overlay of the ¹N-¹H HSQC spectrum of ¹⁵N-labeled NleG2-3[90–191] recorded at 25°C in the absence (red) and in the presence (lilac) of unlabeled UBE2D2 at a 1:1 protein ratio. The resonances of NleG2-3 residues significantly affected by UBE2D2 are labeled. (B) Ribbon diagram of the structure of reduced NleG2-3[90–191] (green) with residues whose NH resonances were severely line-broadened in ¹N-¹H HSQC spectra upon addition of UBE2D2 coloured purple. (C) Surface representation of (B). (D). Superimposition of AvrPtoB (PDB 2FD4, contact residues and backbone in bleu) and reduced NleG2-3[90–191] (predicted contact residues, purple and backbone, green), with both structures shown by ribbon diagrams, with selected secondary structure elements of NleG2-3[90–191] and selected residues from both structures labelled. (E) Immunoblot analysis using anti-ubiquitin antibodies of reactions performed in the presence of ATP, ubiquitin, E1, UBE2D2 and NleG2-3[90–191] variants. The NleG2-3 variants include the wild type (WT) fragments NleG2-3[55–191] and I121A, I121IK, L123K, L152A, L152K, P160A, P160K, E164K and D145K mutants.

Figure 7. Characterisation of UBE2D2 surface interacting with NleG2-3 and NleG5-1.

(A) Alignment of the E3 ligase domains of reduced NleG2-3[90–191] (green) with the CHIP U-box/UBE2D2 complex (PDB 2OXQ) (yellow for the CHIP U-box, cyan for the E2 enzyme), as shown using ribbon diagrams. The transparent surface of reduced NleG2-3[90–191] is also shown. As with Figure 6, residues in NleG2-3[90–191] whose NH resonances were severely line-broadened in ¹N-¹H HSQC spectrum upon addition of UBE2D2 are coloured purple. Selected residues in UBE2D2 are shown by a stick representation and labelled, and selected secondary structure elements on the NleG2-3[90–191] ribbon are also labelled. (B) Immunoblot analysis using anti-ubiquitin antibodies of reactions performed in the presence of ATP, ubiquitin, E1, NleG5-1[1–213] (top and middle panels) or NleG2-3[90–191] (lower panel) and UBE2D2 wild type (WT) or its R5A, F62A and K63A variants. Samples were incubated at 30°C for the indicated time. (C) Determination of dissociation constants of NleG5-1[1–213] interactions with UBE2D2 wild type (WT) or its R5A, F62A and K63A variants. The change in fluorescence polarisation of fluorescein-labelled UBE2D2 and its variants is plotted as a function of NleG5-1[1–213] concentration with error bars indicating standard deviation.