Assembly, analysis and architecture of atypical ubiquitin chains

Ubiquitin (Ub) chains regulate many cellular processes, but several chain types including Lys6-linkages have remained unstudied. Here we analyse the bacterial effector E3 ligase NleL (Non-Lee-encoded effector ligase) from enterohaemorrhagic Escherichia coli (EHEC) O157:H7, which assembles Lys6- and Lys48-linked Ub polymers. Linkage-specific human deubiquitinases (DUBs) are used to show that NleL generates heterotypic Ub chains, and branched chains are efficiently hydrolysed by DUBs. USP DUBs cleave Lys6-linked polymers exclusively from the distal end, while OTUD3, a DUB with Lys6-preference, can cleave Lys6 polymers at any position within the chain. NleL is utilised to generate large quantities of Lys6-linked polyUb. Crystallographic and NMR spectroscopy analysis reveals that an asymmetric interface between Ile44 and Ile36 hydrophobic patches of neighbouring Ub moieties is propagated in longer Lys6-linked Ub chains. Interactions via the Ile36 patch can displace Leu8 from the Ile44 patch, leading to marked structural perturbations of Ub.