Molecular Mechanism Of The Ubiquitin-Conjugating Enzyme Ubc13 Deamidation By Shigella Effector OspI

Pathogens deliver large amounts of effectors into the host cell to target the key molecules to promote their invasion and proliferation during infection. Ashigella effector OspI secreted by its specific type Ⅲ secretion system interacts with the host ubiquitin-conjugating enzyme Ubcl3 and deamidates the glutamine residue at position 100 to a glutamic acid residue, which disrupts the ubiquitin ligase TRAF6 induced K63-linked polyubiquitination and inhibits the activation of the NF-κB signaling pathway.In order to study the molecular mechanism of the Ubcl3 deamidation by OspI, we purified OspI and Ubcl3 to get the complex crystal, and finally obtained the complex structure at 2.3 A resolution. In the complex, OspI uses papain-like conformation, while Ubcl3 adopts the classic UBC fold of ubiquitin-conjugating enzymes. There exists extensive interactions between Ubcl3 and OspI, al helix of Ubcl3 binds the negatively charged region in OspI, L1 and L2 loops of Ubcl3 interact with the hydrophobic patch of OspI. Biochemical experiments reveal that mutations of the residues involved in the interaction between Ubcl3 and OspI have an effect on the OspI-binding to Ubcl3 and inhibit NF-κB activation.Upon binding Ubcl3, the catalytic pocket of OspI which is covered by Asn61 in wild-type alone structure is opened and the Gln100 of Ubcl3 is placed into the catalytic center. The notable conformation change in OspI triggered by Ubcl3 binding is involved in the catalysis.In the catalytic pocket of Ospl, the hydrogen interaction between Asp160 and His145 promotes the thiolate-imidazolium ion pair formation between the catalytic residue Cys62 and His145, then Cys62 is activated. The Gln100 in Ubcl3 is attacked by Cys62 and deamidated to the glutamic acid residue.As a deaminase, OspI selectively chooses Ubcl3 as substrate, the residues in al helix and L2 loop of Ubc13 determine the specificity for OspI recognition. The host deubiquitinating enzyme OTUB1 and some host ubiquitin ligases also bind to the al helix, L1 and L2 loops in Ubcl3, which indicates that host proteins and OspI recognize Ubcl3 through the same surface.This study demonstrates molecular mechanism of Ubc13 deamidation by OspI and a convergence of E2 recogintion by host and bacterial proteins.