| 1. |
- Burger, Pieter B., et al.
(author)
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A novel inhibitor of Plasmodium falciparum spermidine synthase: a twist in the tail
- 2015
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In: Malaria Journal. - : Springer Science and Business Media LLC. - 1475-2875. ; 14
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Journal article (peer-reviewed)abstract
- Background: Plasmodium falciparum is the most pathogenic of the human malaria parasite species and a major cause of death in Africa. It's resistance to most of the current drugs accentuates the pressing need for new chemotherapies. Polyamine metabolism of the parasite is distinct from the human pathway making it an attractive target for chemotherapeutic development. Plasmodium falciparum spermidine synthase (PfSpdS) catalyzes the synthesis of spermidine and spermine. It is a major polyamine flux-determining enzyme and spermidine is a prerequisite for the post-translational activation of P. falciparum eukaryotic translation initiation factor 5A (elF5A). The most potent inhibitors of eukaryotic SpdS's are not specific for PfSpdS. Methods: 'Dynamic' receptor-based pharmacophore models were generated from published crystal structures of SpdS with different ligands. This approach takes into account the inherent flexibility of the active site, which reduces the entropic penalties associated with ligand binding. Four dynamic pharmacophore models were developed and two inhibitors, (1R, 4R)-(N1-(3-aminopropyl)-trans-cyclohexane-1,4-diamine (compound 8) and an analogue, N-(3-aminopropyl)-cyclohexylamine (compound 9), were identified. Results: A crystal structure containing compound 8 was solved and confirmed the in silico prediction that its aminopropyl chain traverses the catalytic centre in the presence of the byproduct of catalysis, 5'-methylthioadenosine. The IC50 value of compound 9 is in the same range as that of the most potent inhibitors of PfSpdS, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and 4MCHA and 100-fold lower than that of compound 8. Compound 9 was originally identified as a mammalian spermine synthase inhibitor and does not inhibit mammalian SpdS. This implied that these two compounds bind in an orientation where their aminopropyl chains face the putrescine binding site in the presence of the substrate, decarboxylated S-adenosylmethionine. The higher binding affinity and lower receptor strain energy of compound 9 compared to compound 8 in the reversed orientation explained their different IC50 values. Conclusion: The specific inhibition of PfSpdS by compound 9 is enabled by its binding in the additional cavity normally occupied by spermidine when spermine is synthesized. This is the first time that a spermine synthase inhibitor is shown to inhibit PfSpdS, which provides new avenues to explore for the development of novel inhibitors of PfSpdS.
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| 2. |
- Galchenkova, Marina, et al.
(author)
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Radiation damage in a hemoglobin crystal studied with an X-ray free-electron laser
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Other publication (other academic/artistic)abstract
- Radiation damage is a topic since the dawn of X-ray crystallography, and has gained new importance in the era of X-ray free-electron lasers (XFELs), due to their unprecedented brilliance and pulse duration. One of the driving questions has been how short the XFEL pulse has to be for the structural information to be ”damage free”. Here we compare data from Serial Femtosecond Crystallography (SFX) experiments conducted with a 3 fs and a 10 fs X-ray pulse. We conclude that even if the estimated displacement of atoms in the sample is an order of magnitude larger in the case of the 10 fs experiment, the displacement is still too small to affect the experimental data at a resolution relevant for structural determination.
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| 3. |
- Johansson, Renzo, et al.
(author)
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High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin
- 2010
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:20, s. 4265-4277
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Journal article (peer-reviewed)abstract
- The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.
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| 4. |
- Manzoni, Francesco, et al.
(author)
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Perdeuteration, crystallization, data collection and comparison of five neutron diffraction data sets of complexes of human galectin-3C
- 2016
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In: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 72:11, s. 1194-1202
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Journal article (peer-reviewed)abstract
- Galectin-3 is an important protein in molecular signalling events involving carbohydrate recognition, and an understanding of the hydrogen-bonding patterns in the carbohydrate-binding site of its C-terminal domain (galectin-3C) is important for the development of new potent inhibitors. The authors are studying these patterns using neutron crystallography. Here, the production of perdeuterated human galectin-3C and successive improvement in crystal size by the development of a crystal-growth protocol involving feeding of the crystallization drops are described. The larger crystals resulted in improved data quality and reduced data-collection times. Furthermore, protocols for complete removal of the lactose that is necessary for the production of large crystals of apo galectin-3C suitable for neutron diffraction are described. Five data sets have been collected at three different neutron sources from galectin-3C crystals of various volumes. It was possible to merge two of these to generate an almost complete neutron data set for the galectin-3C-lactose complex. These data sets provide insights into the crystal volumes and data-collection times necessary for the same system at sources with different technologies and data-collection strategies, and these insights are applicable to other systems.Perdeuteration, purification and the growth of large crystals of the carbohydrate-recognition domain of galectin-3C are described. Five neutron diffraction data sets have been collected at four neutron sources; these are compared and two are merged.
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| 5. |
- Sprenger, Janina, et al.
(author)
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Binding and inhibition of spermidine synthase from Plasmodium falciparum and implications for in vitro inhibitor testing
- 2016
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:9
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Journal article (peer-reviewed)abstract
- The aminopropyltransferase spermidine synthase (SpdS) is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS) transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet) site must be occupied before the aminopropyl acceptor (putrescine) site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD-IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism.
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| 6. |
- Sprenger, Janina, et al.
(author)
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Calmodulin complexes with brain and muscle creatine kinase peptides
- 2021
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In: Current Research in Structural Biology. - : Elsevier BV. - 2665-928X. ; 3, s. 121-132
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Journal article (peer-reviewed)abstract
- Calmodulin (CaM) is a ubiquitous Ca2+ sensing protein that binds to and modulates numerous target proteins and enzymes during cellular signaling processes. A large number of CaM-target complexes have been identified and structurally characterized, revealing a wide diversity of CaM-binding modes. A newly identified target is creatine kinase (CK), a central enzyme in cellular energy homeostasis. This study reports two high-resolution X-ray structures, determined to 1.24 Å and 1.43 Å resolution, of calmodulin in complex with peptides from human brain and muscle CK, respectively. Both complexes adopt a rare extended binding mode with an observed stoichiometry of 1:2 CaM:peptide, confirmed by isothermal titration calorimetry, suggesting that each CaM domain independently binds one CK peptide in a Ca2+-depended manner. While the overall binding mode is similar between the structures with muscle or brain-type CK peptides, the most significant difference is the opposite binding orientation of the peptides in the N-terminal domain. This may extrapolate into distinct binding modes and regulation of the full-length CK isoforms. The structural insights gained in this study strengthen the link between cellular energy homeostasis and Ca2+-mediated cell signaling and may shed light on ways by which cells can ‘fine tune’ their energy levels to match the spatial and temporal demands.
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| 7. |
- Sprenger, Janina, et al.
(author)
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Crystal structures of Val58Ile tryptophan repressor in a domain-swapped array in the presence and absence of l-tryptophan Sprenger Janina
- 2021
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In: Acta Crystallographica Section F: Structural Biology Communications. - 2053-230X. ; 77, s. 215-225
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Journal article (peer-reviewed)abstract
- The crystal structures of domain-swapped tryptophan repressor (TrpR) variant Val58Ile before and after soaking with the physiological ligand l-tryptophan (l-Trp) indicate that l-Trp occupies the same location in the domain-swapped form as in native dimeric TrpR and makes equivalent residue contacts. This result is unexpected because the ligand binding-site residues arise from three separate polypeptide chains in the domain-swapped form. This work represents the first published structure of a domain-swapped form of TrpR with l-Trp bound. The presented structures also show that the protein amino-terminus, whether or not it bears a disordered extension of about 20 residues, is accessible in the large solvent channels of the domain-swapped crystal form, as in the structures reported previously in this form for TrpR without N-terminal extensions. These findings inspire the exploration of l-Trp analogs and N-terminal modifications as labels to orient guest proteins that cannot otherwise be crystallized in the solvent channels of crystalline domain-swapped TrpR hosts for potential diffraction analysis.
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| 8. |
- Sprenger, Janina, et al.
(author)
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Guest-protein incorporation into solvent channels of a protein host crystal (hostal)
- 2021
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In: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 471-485
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Journal article (peer-reviewed)abstract
- Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.
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| 9. |
- Sprenger, Janina
(author)
-
Polyamine Pathway as Drug Target against Malaria
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Malaria, caused by the protozoan parasite Plasmodium falciparum is responsible for about 600.000 death cases every year. Mainly affected are populations of subtropical countries in Africa and the largest groups of victims are children below the age of 5 years. The fast evolving drug resistances of Plasmodium against the the most pow- erful antimalarials threatens the successful containment of this disease in the future. Therefore new, cheap and powerful antimalarial are urgently needed. A better under- standing of the parasite’s unique molecular biology would help to identify new drug targets and could predict resistances. This thesis describes aspects of drug design and the parasite’s unique feature of sequence insertions within conserved proteins by studies on two enzymes of the polyamine pathway that are suggested drug targets. These enzymes are S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdS) from Plasmodium falciparum. The first part of this work describes the heterologous expression and biochemi- cal characterization of Pf AdoMetDC. The enzyme contains a 150 amino acid long Plasmodium specific insert domain, compared to its homologs. This domain is known to interact with an ornithine decarboxylase domain (ODC) in the native Pf AdoMetDC/ODC bifunctional enzyme. Using several biochemical and biophysical techniques including limited proteolysis, CPMG-NMR, UV-CD and ab-initio SAXS modeling it is shown that the quaternary structure, like that of the mammalian ho- mologs, is a dimer. Furthermore comparison of SAXS models from Pf AdoMetDC with and without the insert shows the positions of the insert domain. All together the results give new insights into the structural biology Pf AdoMetDC/ODC complex and demonstrate that the 150 amino acid insert domain mainly adopts a three-dimensional structure. The second part includes studies on Pf SpdS with the focus on inhibitor design. Several structures of the enzyme with various potential inhibitors (described earlier for homologous SpdS or newly discovered by virtual screening and rational design ap- proach) bound are presented. Using enzyme activity assays and isothermal titration calorimetry (ITC) the binding and inhibition of Pf SpdS by potential inhibitors is in- vestigated. It is demonstrated that there is discrepancy between binding and inhibition potency. Predicted inhibitors can bind to the enzyme in vitro without inhibiting the enzyme activity. A sequential binding process, suggested earlier by crystallographic data, is supported by the binding data, and is proposed to explain the discrepancies between ligand-binding affinity and inhibition. The present findings may explain the limited success of previous efforts at structure-based inhibitor design for Pf SpdS, and they may be relevant for other drug targets that follow a sequential binding process.
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| 10. |
- Sprenger, Janina, et al.
(author)
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Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design.
- 2015
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In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 71:Pt 3, s. 484-493
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Journal article (peer-reviewed)abstract
- The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.
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