The Pellino proteins are one of the several families of E3 ubiquitin ligases that direct ubiquitination events immediately following Toll and interleukin-1 receptor (TIR) activation. Polyubiquitination of known Pellino substrates, such as the interleukin-1 associated kinase (IRAK1), is a necessary step in mediating downstream signaling events that are responsible for eliciting a proper immune response. To elucidate Pellino's role in TIR signaling, we are investigating the molecular basis of Pellino substrate specificity. We previously determined the X-ray crystal structure of the human Pellino2 substrate recognition domain and found that it contains a non-canonical example of a well-characterized phosphothreonine (pT)-binding domain, the forkhead-associated (FHA) domain. In an attempt to determine the specific substrate-binding motif of Pellino2, we identified an IRAK1 truncation variant (aa 1-197, IRAK1-197) that interacts with Pellino2 in a phosphorylation dependent manner. Substitution of each threonine in IRAK1-197 with alanine identified T141 as the critical phosphorylated threonine on IRAK1-197 that Pellino2 specifically recognizes. A synthetic phosphopeptide corresponding to the sequence centered on T141 of IRAK1 (pT141 peptide) binds to Pellino2 with a Kd value in the 1 uM range; this data was assessed in a fluorescence polarization binding assay, and independently verified using isothermal titration calorimetry. Binding analyses of other mammalian Pellino isoforms (Pellino 1, 3A, and 3B) to the pT141 peptide reveals differences in binding affinities and specificities. These differences are hard to reconcile due to the high degree of sequence identity among the Pellino isoforms, and cannot be readily explained by the Pellino2 FHA domain crystal structure. Thus, to further explore the molecular basis of these differences, we are working towards determining the X-ray crystal structures of the other Pellino isoforms.