Conservation and divergence of the G-interfaces of Drosophila melanogaster septins.

Septins possess a conserved guanine nucleotide-binding (G) domain that participates in the stabilization of organized hetero-oligomeric complexes which assemble into filaments, rings and network-like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. Here, we report the crystal structure of the heterodimer formed between the G-domains of Sep1 and Sep2, the first from an insect to be described to date. A G-interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G-interface formed between the two copies of Pnut which occupy the centre of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt-bridge network. Whereas wild-type Pnut purifies as a monomer, the reintroduction of the salt-bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly.

SAXSMoW 3.0: New advances in the determination of the molecular weight of proteins in dilute solutions from SAXS intensity data on a relative scale.

SAXSMoW (SAXS Molecular Weight) is an online platform widely used over the past few years for determination of molecular weights of proteins in dilute solutions. The scattering intensity retrieved from small-angle X-ray scattering (SAXS) raw data is the sole input to SAXSMoW for determination of molecular weights of proteins in liquid. The current updated SAXSMoW version 3.0 determines the linear dependence of the true protein volume on their apparent protein volume, based on SAXS curves calculated for 67,000 protein structures selected from the Protein Data Bank. SAXSMoW 3.0 was tested against 43 experimental SAXS scattering curves from proteins with known molecular weights. Our results demonstrate that most of the molecular weights determined for the nonglycosylated and also for the glycosylated proteins are in good agreement with their expected molecular weights. Additionally, the average discrepancies between the calculated molecular weights and their nominal values for glycosylated proteins are similar to those for nonglycosylated ones.

Seryl-tRNA synthetase specificity for tRNASec in Bacterial Sec biosynthesis.

tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.

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.

In vitro and in vivo characterization of the multiple isoforms of Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferases.

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.

The unique tRNASec and its role in selenocysteine biosynthesis.

Selenium (Se) is an essential trace element for several organisms and is mostly present in proteins as L-selenocysteine (Sec or U). Sec is synthesized on its L-seryl-tRNASec to produce Sec-tRNASec molecules by a dedicated selenocysteine synthesis machinery and incorporated into selenoproteins at specified in-frame UGA codons. UGA-Sec insertion is signaled by an mRNA stem-loop structure called the SElenoCysteine Insertion Sequence (SECIS). tRNASec transcription regulation and folding have been described showing its importance to Sec biosynthesis. Here, we discuss structural aspects of Sec-tRNASec and its role in Sec biosynthesis as well as Sec incorporation into selenoproteins. Defects in the Sec biosynthesis or incorporation pathway have been correlated with pathological conditions.