Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal β-hairpin conformation

Abstract

Trichomonas vaginalis Myb3 transcription factor (tvMyb3) recognizes the MRE-1 promoter sequence and regulates ap65-1 gene, which encodes a hydrogenosomal malic enzyme that may play a role in the cytoadherence of the parasite. Here, we identified tvMyb353–180 as the essential fragment for DNA recognition and report the crystal structure of tvMyb353–180 bound to MRE-1 DNA. The N-terminal fragment adopts the classical conformation of an Myb DNA-binding domain, with the third helices of R2 and R3 motifs intercalating in the major groove of DNA. The C-terminal extension forms a β-hairpin followed by a flexible tail, which is stabilized by several interactions with the R3 motif and is not observed in other Myb proteins. Interestingly, this unique C-terminal fragment does not stably connect with DNA in the complex structure but is involved in DNA binding, as demonstrated by NMR chemical shift perturbation, 1H-15N heteronuclear-nuclear Overhauser effect and intermolecular paramagnetic relaxation enhancement. Site-directed mutagenesis also revealed that this C-terminal fragment is crucial for DNA binding, especially the residue Arg153 and the fragment K170KRK173. We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein.

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

“…The structural organization of Myb3 is comprised of a highly ordered R1R2R3 domain, followed by a small hairpin and a polybasic sequence in a direction C-terminal to the structured domain ( Fig. 2A) (41). Clustered mutations were first introduced into the polybasic sequence to study its role in Myb3's nuclear translocation.…”

“…This notion was tested in Myb3, which belongs to the R1R2R3 subfamily and the DBD of which contain a unique small ␤-hairpin C-terminal to the bundled helical structure (see Fig. S4 in the supplemental material) (41). In our previous study (18), subcellular localization of HA-Myb3 in transfected T. vaginalis was detectable in only a small fraction of cells, while the tagged protein was not detected in cells depleted of iron (data not shown).…”

“…In vertebrates, there are three Myb proteins in the R1R2R3 subfamily, exemplified by human c-Myb, B-Myb, and A-Myb (32). Recently, the structures of the DBDs in Myb1, Myb2, and Myb3 were each resolved (21,24,41). Like human c-Myb (27), the R2R3 domains of Myb1 and Myb2 display optimal DNA-binding activities and are comprised of six ␣-helices arranged in two bundles (21,24), while the optimal DNAbinding activity of Myb3 requires an additional unique hairpin structure in the flexible tail C-terminal to R2R3 (41) (see Fig.…”

Regulation of Nuclear Translocation of the Myb1 Transcription Factor by TvCyclophilin 1 in the Protozoan Parasite Trichomonas vaginalis

“…These Myb proteins share the conserved R2R3 DNA-binding domains (DBDs) 3 of the eukaryotic Myb protein family (19), but each has a variable N or C terminus that is much shorter than the variable C-terminal regulatory regions of vertebrate c-Myb, A-Myb, and B-Myb (20). Similar to c-Myb, the DBDs in Myb1, Myb2, and Myb3 are composed of six helices arranged in two bundles with two antiparallel ␤-sheets unique to Myb3 forming a small hairpin following the helices (21)(22)(23). Intriguingly, the DBDs of Myb2 and Myb3 were also shown to mediate their own nuclear translocation (24,25).…”

The cell survival pathways of the primordial RNA-DNA complex remain conserved in the extant genomes and may function as proto-oncogenes

“…Of the three tvMybs, the tvMyb3 protein shows 83% aa sequence identity with human cMyb. Myb proteins are widely expressed in various avian and mammalian hosts and in plants [48][49][50][51]. The ciliates Sterkiella (also known as Oxytricha) and the ciliaphora Euplotes both carry myb genes in their genomes [52].…”