Biochemical and structural studies of a HECT-like ubiquitin ligase from Escherichia coli O157:H7.

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 Lys(6) and Lys(48) 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.