The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-ΚB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif49 GITR52 , was critical for TAB2 and TAB3 binding. NleE mutants lacking49 GITR52 were unable to methylate TAB3, and wild type NleE but not NleE49AAAA52 where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the49 GITR52 motif. Ectopic expression of an N-terminal fragment of NleE (NleE34-52 ) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the49 GITR52 motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response.