The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation

Abstract

SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.

Keywords: PKN1; Salmonella; SspH1; Ube2D; bacterial effector; bacterial pathogenesis; ubiquitin; ubiquitin ligase (E3 enzyme); ubiquitin transfer; ubiquitin-conjugating enzyme (E2 enzyme); ubiquitylation (ubiquitination).

Figure 1.

Domain architecture and topology of SspH1. A, a type 3 secretion system (T3SS) targeting sequence is present at the N terminus of SspH1, followed by the substrate-binding LRR domain and catalytic ubiquitin ligase E3 domain. The positions of the catalytic cysteine, Cys-492, the NSD (cyan), and the CSD (gray) are depicted. B, the topology of SspH/IpaH E3 domains, based on the structure of SspH2 (PDB code 3G06), can be divided into an NSD (cyan) and a CSD (gray), which are connected by a short linker (magenta). C, the NSDs and CSDs from E3 domains of SspH2 (gray) and IpaH3 (red; PDB code 3CVR) were separately superposed. The individual subdomains align with backbone RMSDs of structurally conserved residues of <1 Å. D, alignment of the SspH2 and IpaH3 CSDs reveals a 20° rotation in the position of NSD helix 7 relative to the CSD in the two structures.

Figure 2.

NMR spectra show that the SspH1 E3 can be divided into two separately folded subdomains. A and B, SspH1 NSD (magenta spectrum) (A) and CSD (cyan spectrum) (B) yield dispersed ¹H,¹⁵N TROSY NMR spectra. C, spectrum of the SspH1 E3 domain (black spectrum). D, dispersed resonances of the E3 domain overlay well with those from the NSD and CSD. NMR spectra were collected on 200 μm ¹⁵N-labeled SspH1 NSD, CSD, or 100 μm E3 domain at 25 °C in 25 mm sodium phosphate, 150 mm sodium chloride, and 10% D₂O at pH 7.

Figure 3.

Ube2D3∼Ub binding is localized to the SspH1 CSD. ¹H,¹⁵N TROSY NMR spectral overlays of [¹⁵N]Ube2D3-O-Ub alone (black spectra) or in the presence (red spectra) of SspH1 constructs. A, 100 μm Ube2D3-O-Ub with or without 50 μm SspH1 E3 domain. B, 200 μm Ube2D3-O-Ub with or without 200 μm NSD. C, 200 μm Ube2D3-O-Ub with or without 200 μm CSD. D, Ube2D3–Ub residues whose resonances are perturbed upon binding to the SspH1 E3 domain or CSD are plotted on a surface structure representation of the Ube2D3∼Ub conjugate (PDB code 3UGB).

Figure 4.

The tip segment of the CSD thumb region, bounded by Tyr-642 and Gly-666, is required for binding Ube2D3∼Ub conjugate. A, homology model of SspH1 based on the structure of SspH2 showing the NSD (cyan), catalytic Cys-492 (yellow spheres), and the CSD (gray). Residues in the tip segment targeted for mutagenesis are shown as magenta spheres. B, single point mutants L655A, A661Q, and G666A disrupt the synthesis of unanchored poly-Ub chains by the SspH1 E3 domain. Assay conditions were as follows: 1 μm E1, 5 μm Ube2D3, 2 μm SspH1, 50 μm Ub, 5 mm MgCl₂, 5 mm ATP, pH 7, 37 °C. C, ¹H,¹⁵N TROSY NMR spectral overlay of 100 μm [¹⁵N]Ube2D3-O-Ub in the absence (black spectrum) and presence of 50 μm G666A SspH1 E3 domain (red spectrum). Only minor perturbations of Ube2D3-O-Ub resonances are observed, indicating significantly reduced binding.

Figure 5.

Ube2D3∼Ub binding to CSD thumb helices places the E2∼Ub thioester bond in a reactive conformation. A, homology model of SspH1 E3 domain showing the locations of Cys-492, Glu-639, Ser-653, and Ser-672 (red spheres) for incorporation of TEMPO spin labels. B, ¹H,¹⁵N TROSY spectral overlays of 150 μm [¹⁵N]Ube2D3-O-Ub in the presence of 75 μm SL-active (red spectra) and SL-quenched (black spectra) SspH1 E3 domains. C, resonance perturbations specifically induced by spin labels mapped onto a surface representation of Ube2D3-O-Ub. SL639 preferentially broadens peaks corresponding to Ube2D3 (green surface). SL672 preferentially broadens peaks in the Ub subunit (red surface). SL492 affects residues surrounding an entrance to the E2∼Ub active site (cyan surface). D, SL639 and SL672 form productive complexes with Ube2D3∼Ub, as shown by the ability to transfer Ub to the E3 active site of SspH1 C492K. Reaction conditions were as follows: 1 μm E1, 2 μm Ube2D3, 5 μm SspH1, 100 μm Ub, 5 mm MgCl₂, 5 mm ATP, pH 7, 37 °C. E, cartoon depicting the NSD, CSD, location of spin labels, and Ube2D∼Ub bound to the SspH1 thumb region.

Figure 6.

HDX-MS for SspH1 E3 and E3-Ub domains. A, fractional deuteration of SspH1 E3 peptides at 3 s (red), 1 min (blue), and 30 h (black) after mixing with buffered D₂O. A fractional deuteration value of 1 represents complete substitution of exchangeable protons for deuterons. Values are plotted relative to the middle residue of each peptide analyzed. The secondary structure of SspH1 E3 domain, based on the structure of SspH2, is shown above the HDX plot. B, difference in HDX exchange values between SspH1 E3 and E3-Ub at 3 s (red), 1 m (blue), and 30 h (black) after mixing with buffered D₂O. Values of 0 indicate similar observed exchange rates, whereas values < 0 indicate slower exchange in the E3-Ub conjugate. C, heat map of HDX-MS data shown in A collected at 3 s after mixing plotted on a homology model of SspH1 based on the SspH2 structure. The fractional deuteration of each peptide is plotted on a continuous color gradient from white (no exchange) to red (complete exchange). The active-site cysteine is shown as yellow spheres. D, difference HDX data (shown in B) comparing identical peptides in the SspH1 E3 domain and the E3-Ub conjugate at the 3-s time point. Red, regions that are more protected in the free SspH1 E3 domain; blue, regions more protected in E3-Ub.

Figure 7.

Mutations designed to restrict NSD and CSD conformations impair SspH1 ubiquitylation activity. A, Consurf analysis, comparing the amino sequences of 164 different SspH/IpaH E3 domains, showing the location of conserved residues in the SspH/IpaH family of E3s plotted onto a homology model of the SspH1 E3 domain. Highly conserved residues are shown in dark magenta. The positions of active-site Cys-492 and other residues discussed in this work are shown as spheres. B, ubiquitylation assays with G515P and H498K SspH1 E3 mutants show decreased ability of SspH1 to ubiquitylate the PKN1 substrate. Assays were performed using 1 μm wheat E1, 5 μm Ube2D3, 2 μm SspH1, 5 μm PKN1 1–201 (HRab), 100 μm Ub, 5 mm MgCl₂, pH 7, at 37 °C. Reactions were initiated by the addition of 5 mm ATP. C, single-turnover assays following the breakdown of Ube2D3∼Ub conjugate to free Ube2D3 and Ub in the absence and presence of SspH1 C492A LRR-E3 (left), WT LRR-E3 (middle), and G515P LRR-E3 (right). Assays were performed using 20 μm Ube2D3∼Ub and 8 μm SspH1 at pH 7 and 37 °C. Aliquots were quenched in nonreducing SDS sample buffer and analyzed by nonreducing SDS-PAGE. D, ubiquitylation assays for WT and H498K SspH1, as described in B, analyzed by nonreducing SDS-PAGE. The results show the accumulation of high steady-state levels of the SspH1 H498K∼Ub intermediate in the reaction mixture that persist after the WT reaction is complete.

Figure 8.

Proposed mechanism and domain motions for SspH/IpaH-catalyzed Ub transfer. A, SspH1 must bind both substrate (PKN1 HRb domain) and an activated E2∼Ub conjugate (Ube2D3∼Ub). The dynamics of the SspH1-binding site may ensure selectivity for the E2∼Ub conjugate. B, both subunits of the Ube2D3∼Ub conjugate grasp onto the thumb region of the CSD, adopting a semi-closed conformation. With the NSD in the proximal conformation, the E3 active site is positioned to rapidly react with Ube2D∼Ub. The trans-thiolation reaction could also be facilitated by the flexibility of the E2∼Ub–binding region. C, after Ub is transferred to the E3 active site, Ube2D3 dissociates from the complex. D, the E3 domain undergoes a conformational change to the distal conformation, with the NSD pivoting toward the substrate bound to the LRR domain. This motion is aided by the flexibility of the linker (magenta) connecting the NSD and CSD. This motion may be coupled to a large conformational change in the relative position of the LRR and E3 domains. The result is that Ub is brought into proximity of the substrate, where it is transferred from the E3 active site to a substrate lysine.