Cysteine methylation disrupts ubiquitin-chain sensing in NF-κB activation

NF-kappa B is crucial for innate immune defence against microbial infection(1,2). Inhibition of NF-kappa B signalling has been observed with various bacterial infections(3,4). The NF-kappa B pathway critically requires multiple ubiquitin-chain signals of different natures(5,6). The question of whether ubiquitin-chain signalling and its specificity in NF-kappa B activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-kappa B signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-kappa B signalling. NleE harboured an unprecedented S-adenosyl-L-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger ( NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF ( truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-kappa B activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-kappa B signalling.