Abstract
Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three Myb-like transcriptional factors (tvMyb1, tvMyb2 and tvMyb3) that differentially bind to two closely spaced promoter sites, MRE-1/MRE-2r and MRE-2f. Here, we defined a fragment of tvMyb2 comprising residues 40-156 (tvMyb2&sub4;&sub0;₋&sub1;₅₆) as the minimum structural unit that retains near full binding affinity with the promoter DNAs. Like c-Myb in vertebrates, the DNA-free tvMyb2&sub4;&sub0;₋&sub1;₅₆ has a flexible and open conformation. Upon binding to the promoter DNA elements, tvMyb2&sub4;&sub0;₋&sub1;₅₆ undergoes significant conformational re-arrangement and structure stabilization. Crystal structures of tvMyb2&sub4;&sub0;₋&sub1;₅₆ in complex with promoter element-containing DNA oligomers showed that 5'-a/gACGAT-3' is the specific base sequence recognized by tvMyb2&sub4;&sub0;₋&sub1;₅₆, which does not fully conform to that of the Myb binding site sequence. Furthermore, Lys&sup4;&sup9;, which is upstream of the R2 motif (amino acids 52-102) also participates in specific DNA sequence recognition. Intriguingly, tvMyb2&sub4;&sub0;₋&sub1;₅₆ binds to the promoter elements in an orientation opposite to that proposed in the HADDOCK model of the tvMyb1&sub3;₅₋&sub1;&sub4;&sub1;/MRE-1-MRE-2r complex. These results shed new light on understanding the molecular mechanism of Myb-DNA recognition and provide a framework to study the molecular basis of transcriptional regulation of myriad Mybs in T. vaginalis.