A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases - PubMed (original) (raw)

A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases

Cindy M Quezada et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

SspH2 is an E3 ubiquitin ligase. Ubiquitin ligase activity of SspH2. Purified recombinant GST-SspH2, GST-SspH2C580A, GST-SspH1, or GST-SspH1C492A (as indicated) were incubated with FLAG-tagged ubiquitin in the presence of ATP, E1, and recombinant UbcH5b. Aliquots of the reaction were analyzed by 6–12% SDS-PAGE and immunoblotted with anti-FLAG antibody to detect ubiquitin conjugates. The indicated components of the reaction were omitted as controls.

Fig. 2.

SspH2 consists of an LRR domain and an NEL domain. (A) The overall structure of SspH2 is shown as a ribbon diagram, with the LRR domain in orange and the NEL domain in red. The catalytic cysteine residue is shown in a space-filling rendering (blue). N, NH2 terminus; C, COOH terminus. (B) Sequence and secondary structure of the crystallized region of SspH2 are shown. Secondary structure is numbered separately for the 2 domains. (C) Comparison of YopM (PDB ID 1JL5) and SspH2 LRR domains. Proteins were aligned to maximize the overlap at the N terminus, revealing the divergence between the folds as the chain progresses. (D) Partially transparent molecular surface of the SspH2 LRR domain with the nonpolar residues lining the concave surface (blue) (residues in patch F270, W288, F290, C325, W328, Y365, W368, Y370, I388, M408, and Y430). Orientation similar to panel C.

Fig. 3.

Structure of the NEL domain of SspH2. (A) Comparison of the SspH2 NEL domain with 2 bacterial E3 ligases: SopA, a HECT family of cysteine-dependent E3 ubiquitin ligases from Salmonella (PDB ID 2QYU), and AvrPtoB, a RING finger/U-box protein (PDB ID 2FD4). The catalytic cysteine residues are shown in a space-filling format (blue). (B) Molecular surface representation of the crystallized construct of SspH2 with the LRR and NEL domains in orange and red, respectively. The catalytic cysteine is show in blue. Hydrophobic residues in 2 clusters are show in green (LRR domain) and yellow (NEL domain, residues F570, F586, F587, V594, V597, M619, F620, L622, V633, and W645). (C) Residues near the catalytic cysteine of SspH2. Hydrogen bonds are shown as yellow spheres between atoms, and atoms of nitrogen and oxygen are shown in blue and red, respectively.

Fig. 4.

The amino terminal domain of SspH2 regulates NEL domain ligase activity. (A) E3 ligase activity of the amino and carboxy terminal domains. Purified recombinant SspH2, SspH2C580A, SspH226–487, and SspH2488–788 were incubated with FLAG-tagged ubiquitin in the presence of ATP, E1, and recombinant UbcH5b. Aliquots of the reaction were analyzed by 6–12% SDS-PAGE and immunoblotted with anti-FLAG antibody to detect ubiquitin conjugates. As controls the indicated components of the reaction were omitted. (B) Comparison of the ubiquitin ligase activities of SspH2 and SspH2488–788. Different amounts (as indicated) of purified recombinant SspH2 or SspH2488–788 were tested for their ubiquitin ligase activity as indicated above. After in vitro ubiquitination reactions, free ubiquitin chains (supernatant) and E3–ubiquitin conjugates (pellet) were separated and analyzed by SDS-PAGE and immunoblotted with anti-FLAG antibody. Bottom: Coomassie-stained gel representing the concentration of GST-tagged E3 used in the above titration. (C) Time course comparison of the ubiquitin conjugation reaction of SspH2 and SspH2488–788. Purified recombinant GST-SspH2 or GST-SspH2488–788 (250 nM) were incubated with FLAG-tagged ubiquitin in the presence of ATP, E1, and recombinant UbcH5b. Aliquots of the reactions were removed at the indicated times and analyzed by 8% SDS-PAGE and immunoblotted with anti-FLAG antibody. (D) Removal of the LRR domain increases the E3 ligase activity of SlrP and SspH1. Purified recombinant GST-SspH1, GST-SspH1400–700, GST-SlrP, GST-SspH1458–765, GST-SspH2, or GST-SspH2488–788 (250 nM) were analyzed as above. (E) Disruption of the interaction of the N-terminal domain with the NEL domain relieves autoinhibition of the E3 ligase activity of full-length SspH2. Recombinant GST-SspH2, GST-SspH2C580A, GST-SspH2488–788, GST-SspH2479/481/483A, and GST-SspH2479/481/483A, C580A were incubated with FLAG-tagged ubiquitin in the presence of ATP, E1, and recombinant UbcH5b. Aliquots of the reactions were removed at the indicated times and analyzed by 8% SDS-PAGE and immunoblotted with anti-FLAG antibody.

Fig. 5.

The amino terminal domain of SspH2 mediates localization within host cells. FLAG epitope–tagged SspH2, SspH21–487, or SspH2488–788 were transiently expressed in cultured HeLa (A) or polarized MDCK (B) cells and their localization examined by immunofluorescence confocal microscopy using an antibody directed to the tag (green), rhodamine phallodian to stain polymerized actin (red) and where indicated POPO1 to stain DNA (blue).

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A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases - PubMed (original) (raw)