Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly

Combinatorial association of DNA-binding proteins on composite binding site s enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexe s with Ets proteins on the B cell-specific mb-1 promoter through interactio ns between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bi nd the promoter, but not the closely related Ets protein SAP1a. Here we sho w that, while several different mutations increase binding of SAP1a to an o ptimized Ets binding site, only conversion of Val68 to an acidic amino acid facilitates ternary complex assembly with Pax-5 on the mb-1 promoter. This suggests that enhanced DNA binding by SAP1 a is not sufficient for recruit ment by Pax-5, but instead involves protein-protein interactions mediated b y the acidic side chain. Recruitment of Ets proteins by Pax-5 requires GIn2 2 within the N-terminal beta -hairpin motif of its paired domain. The beta -hairpin also participates in recognition of a subset of Pax-5-binding site s. Thus, Pax-5 incorporates protein-protein interaction and DNA recognition functions in a single motif. The Caenorhabditis elegans Pax protein EGL-38 also binds specifically to the mb-1 promoter and recruits murine Ets-1 or the C.elegans Ets protein T08H4.3, but not the related LIN-1 protein. Toget her, our results define specific amino acid requirements for Pax-Ets ternar y complex assembly and show that the mechanism is conserved between evoluti onarily related proteins of diverse animal species. Moreover, the data sugg est that interactions between Pax and Ets proteins are an important mechani sm that regulates fundamental biological processes in worms and humans.