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Many microbial pathogens deliver effector proteins, via type III secretion system, into infected host cells. Elucidating the function of these effectors is essential for our understanding of pathogenesis. Here I describe biochemical and structural characterization of an effector protein (NleL) from E. coli O157:H7, a widespread pathogen causing severe food borne diseases. NleL functionally and structurally mimics eukaryotic HECT E3 ligases and catalyzes formation of unanchored polyubiquitin chains using K6 and K48 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 E3sstructures 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. In the structure of NleL/UbcH7 complex, UbcH7 binds at the end of the N-lobe that is closer to the C-lobe as observed in the structure of SopA/UbcH7 complex. The conserved phenylalanine of E2s (Phe63 of UbcH7) is involved heavily in the binding. The C-lobe of NleL in the complex rotates as much as ~168? towards UbcH7 compared with the structures of the isolated NleLs and the catalytic cysteines of E2 and E3 are within 7? of each other. In the structure of SopA/UbcH7 complex, the C-lobe bends towards the putative substrate binding domain (β-helix domain). The orientation of the C-lobes in the NleL and SopA complexes differs by ~180?, demonstrating that large movements of the C-lobes are possible, and the two complexes could represent two different stages of the transfer of ubiquitin from E2 to the substrate. These results provide critical knowledge towards understanding the molecular mechanism by which pathogens utilize the host ubiquitination system during infection.