Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease

Crystal Structure of the human TIP41 Orthologue, TIPRL, reveals a novel fold and a binding site for the PP2AC C-TERMINUS

Submitted by Luciane Willcox ([email protected]) on 2017-04-11T19:37:48Z No. of bitstreams: 1 Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.pdf: 2132858 bytes, checksum: f3c011ca732ddc9cb6872d8a9f79a48c (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-07-26T14:29:12Z (GMT) No. of bitstreams: 1 Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.pdf: 2132858 bytes, checksum: f3c011ca732ddc9cb6872d8a9f79a48c (MD5)Made available in DSpace on 2017-07-26T14:29:12Z (GMT). No. of bitstreams: 1 Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.pdf: 2132858 bytes, checksum: f3c011ca732ddc9cb6872d8a9f79a48c (MD5) Previous issue date: 2016Institute of Chemistry, University of Campinas, Campinas, SP, Brazil. / Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil. / Institute of Biology, University of Campinas, Campinas, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil. / Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil.Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Diamond Light Source, Chilton, UK.Diamond Light Source, Chilton, UK.Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition