Dataset Information

Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal ?-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 tvMyb3(53-180) as the essential fragment for DNA recognition and report the crystal structure of tvMyb3(53-180) 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 ?-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, (1)H-(15)N 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 Arg(153) and the fragment K(170)KRK(173). We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein.

SUBMITTER: Wei SY

PROVIDER: S-EPMC3245928 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Wei Shu-Yi SY Lou Yuan-Chao YC Tsai Jia-Yin JY Ho Meng-Ru MR Chou Chun-Chi CC Rajasekaran M M Hsu Hong-Ming HM Tai Jung-Hsiang JH Hsiao Chwan-Deng CD Chen Chinpan C

Nucleic acids research 20110908 1

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 tvMyb3(53-180) as the essential fragment for DNA recognition and report the crystal structure of tvMyb3(53-180) 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 R ...[more]

PMID: 21908401

Similar Datasets

Project description:Ornithine decarboxylase (ODC), the lead enzyme in polyamine biosynthesis, was partially purified from Trichomonas vaginalis and its kinetic properties were studied. The enzyme appears to be of special significance in this anaerobic parasite, since the arginine dihydrolase pathway generates ATP as well as putrescine from arginine. ODC from T. vaginalis had a broad substrate specificity, decarboxylating ornithine (100%), lysine (1.0%) and arginine (0.1%). The enzyme had a pH optimum of 6.5, a temperature optimum of 37 degrees C and was pyridoxal 5'-phosphate-dependent. Attempts to separate ornithine- from lysine-decarboxylating activity by thermal-stability and pH-optima curves were not successful. Although Km values for ornithine and lysine were 109 and 91 microM respectively, and the Vmax values for these substrates were 1282 and 13 nmol/min per mg of protein respectively, the most important intracellular substrate is ornithine, since intracellular ornithine levels are 3.5 times those of lysine and extracellular putrescine levels are 7.5 times those of cadaverine. Ornithine was also an effective inhibitor of lysine-decarboxylating activity (Ki 150 microM), whereas lysine was relatively ineffective as inhibitor of ornithine-decarboxylating activity (Ki 14.5 mM). Crude ODC activity was localized (86%) in the 43,000 g supernatant and 3303-fold purification was obtained by (NH4)2SO4 salting and DEAE-Sephacel, agarose-gel and hydroxyapatite chromatography steps. The enzyme bound difluoro[3H]methylornithine ([3H]DFMO) with a ratio of drug bound to activity of 2500 fmol/unit, where 1 unit corresponds to 1 nmol of CO2 released from ornithine/min. The enzyme had a native M(r) of 210000 (gel filtration), with a subunit M(r) of 55,000 (by SDS/PAGE), suggesting that the trichomonad enzyme is a tetramer. From the subunit M(r) and binding ratio of DFMO, there is about 137 ng of ODC per mg of T. vaginalis protein (0.013%). The significant amount of ODC protein present supports the view that putrescine synthesis in T. vaginalis plays an important role in the metabolism of the parasite.

Project description:The secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host-parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and β-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used β-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two β-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two β-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure