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 sites enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexes with Ets proteins on the B cell-specific mb-1 promoter through interactions between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bind the promoter, but not the closely related Ets protein SAP1a. Here we show that, while several different mutations increase binding of SAP1a to an optimized 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 SAP1a is not sufficient for recruitment by Pax-5, but instead involves protein-protein interactions mediated by the acidic side chain. Recruitment of Ets proteins by Pax-5 requires Gln22 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 sites. 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. Together, our results define specific amino acid requirements for Pax-Ets ternary complex assembly and show that the mechanism is conserved between evolutionarily related proteins of diverse animal species. Moreover, the data suggest that interactions between Pax and Ets proteins are an important mechanism that regulates fundamental biological processes in worms and humans.
Fitzsimmons
D
D
Integrated Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Lutz
R
R
Wheat
W
W
Chamberlin
H M
HM
Hagman
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001402:B-Lymphocytes / Q000378:metabolism; D051925:CD79 Antigens; D017173:Caenorhabditis elegans / Q000235:genetics; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D051757:PAX5 Transcription Factor; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011947:Receptors, Antigen, B-Cell / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D015703:Antigens, CD; D051925:CD79 Antigens; D004268:DNA-Binding Proteins; D015801:Helminth Proteins; D046911:Macromolecular Substances; D051757:PAX5 Transcription Factor; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011947:Receptors, Antigen, B-Cell; D014157:Transcription Factors; D004247:DNA