Structural analysis of the Trypanosoma brucei EIF4E6/EIF4G5 complex reveals details of the interaction between unusual eIF4F subunits.

Recognition of the mRNA 5' end is a critical step needed for translation initiation. This step is performed by the cap binding protein eIF4E, which joins the larger eIF4G subunit to form the eIF4F complex. Trypanosomatids have a minimum of five different eIF4F-like complexes formed through specific but not well-defined interactions between four different eIF4E and five eIF4G homologues. The EIF4E6/EIF4G5 complex has been linked with the stage-specific translation of mRNAs encoding the major Trypanosoma brucei virulence factors. Here, to better define the molecular basis for the TbEIF4E6/TbEIF4G5 interaction, we describe the identification of the peptide interacting with TbEIF4E6 in the region comprising residues 79-166 of TbEIF4G5. The TbEIF4E6-TbEIF4G5_79-116 complex reconstituted with recombinant proteins is highly stable even in the absence of cap-4. The crystal structure of the complex was subsequently solved, revealing extensive interacting surfaces. Comparative analyses highlight the conservation of the overall structural arrangement of different eIF4E/eIF4G complexes. However, highly different interacting surfaces are formed with distinct binding contacts occurring both in the canonical and noncanonical elements within eIF4G and the respective eIF4E counterpart. These specific pairs of complementary interacting surfaces are likely responsible for the selective association needed for the formation of distinct eIF4F complexes in trypanosomatids.

Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system.

Trypanosoma brucei belongs to a group of protozoans presenting fragmented large subunit rRNA. Its LSU rRNA equivalent to the 25S/28S rRNA of other eukaryotes is split into six fragments, requiring additional processing for removal of the extra spacer sequences. We have used a genetic complementation strategy to further investigate the T. brucei RRP44 nuclease in pre-rRNA maturation. TbRRP44 contains both a PIN and a RNB domain whose homologues are found in association with the exosome complex. We found that the exonucleolytic activity of the RNB domain as well as the physical presence of the PIN domain are essential for TbRRP44 function, while a catalytic site mutation in the PIN domain has no detectable effect on cell growth. A new endonucleolytic cleavage site in ITS1 was identified. In addition to the 5.8S rRNA 3'-end maturation, TbRRP44 is required for degradation of the excised 5'-ETS and for removal of part of ITS1 during maturation of the 18S rRNA 3'-end. TbRRP44 deficiency leads to accumulation of many LSU intermediate precursors, most of them not detected in control cells. TbRRP44 is also required for U3 snoRNA and spliced leader processing, indicating that TbRRP44 may have a wide role in RNA processing in T. brucei.

Trypanosoma brucei RRP44: a versatile enzyme for processing structured and non-structured RNA substrates.

Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We have determined the crystal structure of the exoribonucleolytic module of TbRRP44 in an active conformation, revealing novel details of the catalytic mechanism of the RNB domain. For the first time, the position of the second magnesium involved in the two-metal-ion mechanism was determined for a member of the RNase II family. In vitro, TbRRP44 acts preferentially on non-structured uridine-rich RNA substrates. However, we demonstrated for the first time that both TbRRP44 and its homologue from Saccharomyces cerevisiae can also degrade structured substrates without 3'-end overhang, suggesting that Rrp44/Dis3 ribonucleases may be involved in degradation of a wider panel of RNA than has been assumed. Interestingly, deletion of TbRRP44 PIN domain impairs RNA binding to different extents, depending on the type of substrate.

Functional EGF domain of the human neuregulin 1α produced in Escherichia coli with accurate disulfide bonds.

BACKGROUND: Neuregulins comprise a large family of growth factors containing an epidermal growth factor (EGF) domain. NRG1 acts in signaling pathways involved in proliferation, apoptosis, migration, differentiation, and adhesion of many normal cell types and in human diseases. The EGF domain of NRG1 mediates signaling by interaction with members of the ErbB family of receptors. Easy access to correctly folded hNRG1α EGF domain can be a valuable tool to investigate its function in different cell types. MATERIALS AND METHODS: The EGF domain of hNRG1α was produced in Escherichia coli in fusion with TrxA and purified after cleavage of TrxA. Conformation and stability analyses were performed by using biophysical methods and the disulfide bonds were mapped by mass spectrometry. The activity of the hNRG1α EGF domain was demonstrated in cell proliferation and migration assays. RESULTS: Approximately 3.3 mg of hNRG1α EGF domain were obtained starting from a 0.5 L of E. coli culture. Correct formation of the three disulfide bonds was demonstrated by mass spectrometry with high accuracy. Heat denaturation assays monitored by circular dichroism and dynamic light scattering revealed that it is a highly stable protein. The recombinant EGF domain of hNRG1α purified in this work is highly active, inducing cell proliferation at concentration as low as 0.05 ng/mL. It induces also cell migration as demonstrated by a gap closure assay. CONCLUSION: The EGF domain of hNRG1α was produced in E. coli with the correct disulfide bonds and presented high stimulation of HeLa cell proliferation and NDFH cell migration.

Engineering defensin α-helix to produce high-affinity SARS-CoV-2 spike protein binding ligands.

The binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 mimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2-5 kDa) and highly stable proteins containing solvent-exposed alpha-helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha-helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS-CoV-2 spike protein. The engineered proteins (h-deface2, p-deface2, and p-deface2-MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high-affinity binding to the spike protein with apparent Kd values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing of high-affinity ligands.

A toolkit for recombinant production of seven human EGF family growth factors in active conformation.

Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.

De novo crystal structure determination of double stranded RNA binding domain using only the sulfur anomalous diffraction in SAD phasing.

Single-wavelength anomalous dispersion (SAD)-phasing using sulfur as the unique anomalous scatterer is a powerful method to solve the phase problem in protein crystallography. However, it is not yet widely used by non-expert crystallographers. We report here the structure determination of the double stranded RNA binding domain of human dihydrouridine synthase using the sulfur-SAD method and highly redundant data collected at 1.8 â€‹Å ("off-edge"), at which the estimated overall anomalous signal was 1.08%. High multiplicity data were collected on a single crystal rotated along the ϕ or ω axis at different κ angles, with the primary beam intensity being attenuated from 50% to 95%, compared to data collection at 0.98 â€‹Å, to reduce radiation damage. SHELXD succeeded to locate 14 out 15 sulfur sites only using the data sets recorded with highest beam attenuation, which provided phases sufficient for structure solving. In an attempt to stimulate the use of sulfur-SAD phasing by a broader community of crystallographers, we describe our experimental strategy together with a compilation of previous successful cases, suggesting that sulfur-SAD phasing should be attempted for determining the de novo structure of any protein with average sulfur content diffracting better than 3 â€‹Å resolution.

The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners.

The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5' end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in Leishmania species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in L. infantum. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in Trypanosoma brucei, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the Leishmania-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in Leishmania (W45) did not substantially impair the identified interactions. Finally, the crystal structure of Leishmania EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its Trypanosoma orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.

Insights into Glucose-6-phosphate Allosteric Activation of ß-Glucosidase A.

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. ß-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa ß-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of G6P-ß-glucosidase A interactions have not yet been described so far. We investigated the molecular details of G6P binding into B. polymyxa ß-glucosidase A through in silico docking using the site identification by ligand competitive saturation technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift toward the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of ß-glucosidase A.

Crystal structure of the MIF4G domain of the Trypanosoma cruzi translation initiation factor EIF4G5.

Kinetoplastida, a class of early-diverging eukaryotes that includes pathogenic Trypanosoma and Leishmania species, display key differences in their translation machinery compared with multicellular eukaryotes. One of these differences involves a larger number of genes encoding eIF4E and eIF4G homologs and the interaction pattern between the translation initiation factors. eIF4G is a scaffold protein which interacts with the mRNA cap-binding factor eIF4E, the poly(A)-binding protein, the RNA helicase eIF4A and the eIF3 complex. It contains the so-called middle domain of eIF4G (MIF4G), a multipurpose adaptor involved in different protein-protein and protein-RNA complexes. Here, the crystal structure of the MIF4G domain of T. cruzi EIF4G5 is described at 2.4 Šresolution, which is the first three-dimensional structure of a trypanosomatid MIF4G domain to be reported. Structural comparison with IF4G homologs from other eukaryotes and other MIF4G-containing proteins reveals differences that may account for the specific interaction mechanisms of MIF4G despite its highly conserved overall fold.

Crystal structure of the Trypanosoma cruzi EIF4E5 translation factor homologue in complex with mRNA cap-4.

Association of the initiation factor eIF4E with the mRNA cap structure is a key step for translation. Trypanosomatids present six eIF4E homologues, showing a low conservation and also differing significantly from the IF4Es of multicellular eukaryotes. On the mRNA side, while in most eukaryotes the mRNA contains cap-0 (7-methyl-GTP), the trypanosomatid mRNA features a cap-4, which is formed by a cap-0, followed by the AACU sequence containing 2'-O-ribose methylations and base methylations on nucleotides 1 and 4. The studies on eIF4E-cap-4 interaction have been hindered by the difficulty to synthesize this rather elaborated cap-4 sequence. To overcome this problem, we applied a liquid-phase oligonucleotide synthesis strategy and describe for the first time the crystal structure of a trypanosomatid eIF4E (T. cruzi EIF4E5) in complex with cap-4. The TcEIF4E5-cap-4 structure allowed a detailed description of the binding mechanism, revealing the interaction mode for the AACU sequence, with the bases packed in a parallel stacking conformation and involved, together with the methyl groups, in hydrophobic contacts with the protein. This binding mechanism evidences a distinct cap interaction mode in comparison with previously described eIF4E structures and may account for the difference of TcEIF4E5-cap-4 dissociation constant in comparison with other eIF4E homologues.

Trypanosoma brucei RRP44 is involved in an early stage of large ribosomal subunit RNA maturation.

Ribosomal RNA precursors undergo a series of structural and chemical modifications to generate matured RNA molecules that will comprise ribosomes. This maturation process involves a large set of accessory proteins as well as ribonucleases, responsible for removal of the external and internal transcribed spacers from the pre-rRNA. Early-diverging eukaryotes belonging to the Kinetoplastida class display several unique characteristics, in particular in terms of RNA synthesis and maturation. These peculiarities include the rRNA biogenesis and the extensive fragmentation of the large ribosomal subunit (LSU) rRNA. The role of specific endo- and exonucleases in the maturation of the unusual rRNA precursor of trypanosomatids remains largely unknown. One of the nucleases involved in rRNA processing is Rrp44, an exosome associated ribonuclease in yeast, which is involved in several metabolic RNA pathways. Here, we investigated the function of Trypanosoma brucei RRP44 orthologue (TbRRP44) in rRNA processing. Our results revealed that TbRRP44 depletion causes unusual polysome profile and accumulation of the complete LSU rRNA precursor, in addition to 5.8S maturation impairment. We also determined the crystal structure of TbRRP44 endonucleolytic domain. Structural comparison with Saccharomyces cerevisiae Rrp44 revealed differences in the catalytic site and substitutions of surface residues, which could provide molecular bases for the lack of interaction of RRP44 with the exosome complex in T. brucei.

Complete amino-acid sequence, crystallization and preliminary X-ray diffraction studies of leucurolysin-a, a nonhaemorrhagic metalloproteinase from Bothrops leucurus snake venom.

Leucurolysin-a (leuc-a) is a class P-I snake-venom metalloproteinase isolated from the venom of the South American snake Bothrops leucurus (white-tailed jararaca). The mature protein is composed of 202 amino-acid residues in a single polypeptide chain. It contains a blocked N-terminus and is not glycosylated. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood. Unlike some other venom fibrinolytic metalloproteinases, leuc-a has no haemorrhagic activity. Leuc-a was sequenced and was crystallized using the hanging-drop vapour-diffusion technique. Crystals were obtained using PEG 6000 or PEG 1500. Diffraction data to 1.80 and 1.60 A resolution were collected from two crystals (free enzyme and the endogenous ligand-protein complex, respectively). They both belonged to space group P2(1)2(1)2(1), with very similar unit-cell parameters (a = 44.0, b = 56.2, c = 76.3 A for the free-enzyme crystal).

Crystallization and preliminary X-ray diffraction analysis of Q4DV70 from Trypanosoma cruzi, a hypothetical protein with a putative thioredoxin domain.

Q4DV70 is annotated in the Trypanosoma cruzi CL Brener genome as a hypothetical protein with a predicted thioredoxin-like fold, although the catalytic cysteine residues that are conserved in typical oxidoreductases are replaced by serine residues. Gene-expression analysis indicates that this protein is differentially expressed during the T. cruzi life cycle, suggesting that it plays an important role during T. cruzi development. The gene coding for Q4DV70 was cloned and the protein was overexpressed in Escherichia coli with an N-terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method. A diffraction data set was collected to 1.50 A resolution from a single crystal grown in 25% PEG 1500, 200 mM sodium thiocyanate pH 6.9, 10 mM phenol and 10% ethylene glycol. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.04, b = 50.32, c = 61.18 A. The Q4DV70 structure was solved by molecular replacement using protein disulfide isomerase from yeast (PDB code 2b5e) as a search model. Initial refinement of the model indicated that the solution was correct. These data are being used for refinement of the model of Q4DV70.

Structural aspects of the distinct biochemical properties of glutaredoxin 1 and glutaredoxin 2 from Saccharomyces cerevisiae.

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower K(M) for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGrx2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 A, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function.

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 A resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

Crystallization and preliminary X-ray analysis of a decameric form of cytosolic thioredoxin peroxidase 1 (Tsa1), C47S mutant, from Saccharomyces cerevisiae.

Saccharomyces cerevisiae cytosolic thioredoxin peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His(6)-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 A resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress.

Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation.

BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix-turn-helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 A, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit.

Crystal structure of chagasin, the endogenous cysteine-protease inhibitor from Trypanosoma cruzi.

Trypanosoma cruzi chagasin belongs to a recently discovered family of cysteine protease inhibitors found in lower eukaryotes and prokaryotes but not in mammals. Chagasin binds tightly to cruzain, the major lysosomal T. cruzi cysteine protease, involved with infectivity and survival of the parasite in mammalian host cells. In the scope of a project to characterize proteins diferentially expressed during T. cruzi metacyclogenesis, we have determined the crystal structure of chagasin, which is now the first X-ray structure of a chagasin-like cysteine protease inhibitor to be reported. The structure was solved by the SIRAS method and refined at 1.7A resolution and a comparison with the two NMR structures available revealed some differences in the loops involved in binding to cysteine proteases. The highly flexible loop 4 could be entirely modeled and residues 29-33 from loop 2 form a 3(10)-helix structure that may be important to stabilize the loop conformation. Chagasin crystal structure was docked to the highest resolution structure available of cruzain and a model of chagasin-cruzain interaction was analyzed. The knowledge of the chagasin crystal structure may contribute to the elucidation of the molecular mechanism involved in the inhibition of cruzain and other T. cruzi cysteine proteases.