Biochemical and Structural Studies of a HECT-like Ubiquitin Ligase from Escherichia coli O157:H7

David Yin-Wei Lin,Jianbo Diao,Daoguo Zhou,Jue Chen

doi:10.1074/jbc.m110.167643

David Yin-Wei Lin, Jianbo Diao + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.167643

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Abstract

Many microbial pathogens deliver effector proteins via the type III secretion system into infected host cells. Elucidating the function of these effectors is essential for our understanding of pathogenesis. Here, we describe biochemical and structural characterization of an effector protein (NleL) from Escherichia coli O157:H7, a widespread pathogen causing severe foodborne diseases. We show that NleL functionally and structurally mimics eukaryotic HECT E3 ligases and catalyzes formation of unanchored polyubiquitin chains using Lys6 and Lys48 linkage. The catalytic cysteine residue forms a thioester intermediate with ubiquitin. The structure of NleL contains two domains, a β-helix domain formed by pentapeptide repeats and a bilobed catalytic domain reminiscent of the N- and C-lobe architecture of HECT E3s. Six structures of NleL observed in two crystal forms revealed a large range of different positions of the C-lobe relative to the N-lobe, indicating that the helix linking the two lobes is extremely flexible. Comparing the structure of NleL with that of the Salmonella homolog SopA showed that the orientation of the C-lobes differ by as much as 108°, suggesting that large movements of the C-lobe may be required to facilitate the transfer of ubiquitin from E2 to the substrate. These results provide critical knowledge toward understanding the molecular mechanism by which pathogens utilize the host ubiquitination system during infection.

Highlights

In eukaryotes, ubiquitination plays important roles in many cellular functions, including protein degradation, DNA repair, cell-cycle progression, trafficking, and endocytosis [1]
The E1 enzyme activates a free ubiquitin (Ub)2 by forming a thioester bond between its active site cysteine and the carboxyl terminus of Ub using the energy from ATP hydrolysis; the activated Ub is passed to the active cysteine on the E2 conjugating enzyme; E3 mediates the transfer of the
RING E3s catalyze ubiquitination by serving as a molecular scaffold to bring the Ub-charged E2 and the substrate into close proximity, whereas HECT E3s directly participate in the chemistry by forming a thioester bond with the Ub molecule brought by E2 and direct it to the substrate protein

Summary

The abbreviations used are

Ubiquitin; polyUb, polyubiquitin; MES, 4-morpholineethanesulfonic acid; NEL, novel E3 ligase. Structural and mutagenesis studies [5] suggest that the conformational flexibility of the HECT domain is important for transferring Ub from E2 to the substrate. The ubiquitination pathway is absent in bacteria, some pathogenic bacteria deliver effector proteins into eukaryotic host cells to function as E3 ligases (9 –13). We reported that SopA from Salmonella is an E3 ligase structurally similar to eukaryotic HECT E3s [22, 23]. We biochemically and structurally characterized NleL (non-Lee-encoded effector ligase), a homologue of SopA from Escherichia coli O157:H7. The relative orientations between the two lobes revealed in the structures of NleL and SopA are markedly different, suggesting that like eukaryotic HECT E3s, the flexibility of the C-lobe is essential in Ub transfer

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION