Mutation analysis of the Pip interaction domain reveals critical residues for protein-protein interactions

The PU.1 interaction partner (Pip) is a mem ber of the interferon regulator y factor family that regulates gene expression through heterodimerization w ith the ETS transcription factor PU.1. Binding of Pip atone to DNA is weak, and usually it is recruited by phosphorylated PU,I to form a strong ternar y complex with specific DNA sequences, An approach combining sequence homol ogy analysis, secondary structure predictions, and a precise mutational str ategy has been used to determine critical residues within the Pip heterodim erization domain that contribute to ternary complex formation. We have deli mited the Pip interaction domain to residues 245-422 by using deletion anal ysis. Site-directed mutagenesis of conserved polar amino acids within two p redicted alpha-helices contained in this region, and which are highly conse rved in the IRF family, confirmed the importance of these residues for Pip- PU.1 interaction with DNA as well as for trans-activation activity. Our res ults suggest the existence of a functional epitope essential for heterodime rization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners.