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.
In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS).
Plasmodium falciparum like other organisms is dependent on polyamines for proliferation.Polyamine biosynthesis in these parasites is regulated by a unique bifunctional Sadenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC). Only limited biochemical and structural information is available on the bifunctional enzyme due to the low levels and impurity of an instable recombinantly expressed protein from the native gene. Here we describe the high level expression of stable monofunctional PfAdoMetDC from a codon-harmonised construct, which permitted its biochemical characterisation indicating similar catalytic properties to AdoMetDCs of orthologous parasites. In the absence of structural data, far-UV CD showed that at least on secondary structure level, PfAdoMetDC corresponds well to that of the human protein. The kinetic properties of the monofunctional enzyme were also found to be different from that of PfAdoMetDC/ODC as mainly evidenced by an increased K m . We deduced that complex formation of PfAdoMetDC and PfODC could enable coordinated modulation of the decarboxylase activities since there is a convergence of their k cat and lowering of their K m . Such coordination results in the aligned production of decarboxylated AdoMet and putrescine for the subsequent synthesis of spermidine. Furthermore, based on the results obtained in this study we propose a new AdoMetDC subclass for plasmodial AdoMetDCs.
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 datacollection strategies, and these insights are applicable to other systems.
SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the host’s innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 Å and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 Å, show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.
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