Acta crystallographica. Section F, Structural biology communications最新文献

Nucleotide-bound crystal structures of SARS-CoV-2 NSP13 in ADP- and ATP-bound states were resolved to 1.8 and 1.9 Å, respectively. The ADP-bound model captures a state immediately following ATP hydrolysis, with both ADP and orthophosphate still present in the active site. Further comparative analysis revealed that crystal packing influences NSP13 by stabilizing the nucleotide-binding site, underscoring the importance of accounting for these effects in structure-based drug design targeting NSP13.

Menaquinones (vitamin K₂) are a family of redox-active small lipophilic molecules that serve as vital electron carriers in many bacterial electron-transport pathways. The ThDP-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase (MenD) catalyses the first irreversible step in bacterial classical menaquinone biosynthesis via a series of reactions involving covalent ThDP-bound intermediates. We report structures of MenD from the pathogen Listeria monocytogenes (LmoMenD) in its ThDP cofactor-bound and in-crystallo captured intermediate I-bound forms. Analysis of the structures revealed that LmoMenD adopts the typical three-domain ThDP-dependent fold observed for MenD orthologs, while a combination of structure, size-exclusion chromatography, mass photometry and small-angle X-ray scattering analysis showed that the enzyme has a homotetrameric quaternary structure. While both of the ligand-bound structures reported here were very similar, comparison with an apo form from the PDB revealed a closing down of the active site in the ligand-bound forms, with more complete models suggesting lower levels of disorder around key regions of the active site that interface with ThDP or the captured intermediate. Enzyme kinetics characterization showed the enzyme was active and enabled allosteric inhibition to be measured. There was weak inhibition of enzyme activity in the presence of 1,4-dihydroxy-2-naphthoic acid, an allosteric regulator of Mycobacterium tuberculosis MenD and downstream metabolite in the menaquinone-biosynthesis pathway.

Hepatoma-derived growth factor-related protein 2 (HRP-2) is a member of the HDGF-related protein family, which has been linked to multiple malignancies. A defining feature of this protein family is the presence of an N-terminal PWWP domain, which enables binding to nucleosomes carrying a dimethylation or trimethylation marker on residue Lys36 of histone H3. To support the rational design of small-molecule drugs that bind to the PWWP domain, crystallographic fragment screening was chosen. A critical requirement for such screening is the ability to reliably produce large batches of high-quality crystals, ideally grown under low-salt conditions to prevent the precipitation of drug-like fragments during crystal soaking. Initial crystallization of the wild-type (WT) HRP-2 PWWP domain only produced crystals under high-salt conditions and these significantly lost diffraction quality over two weeks. It was hypothesized that these complications were caused by oxidation of the solvent-exposed Cys64 residue. To overcome these difficulties, a Cys64Ser mutant was produced. This mutation revealed a substantially improved crystallization propensity, as eight crystal forms could be obtained and resolved versus two forms for the WT. Moreover, the mutant crystals could be grown in PEG-based low ionic strength conditions which are optimal for fragment soaking. Finally, the crystals did not lose their diffraction quality for up to six months. Importantly, systematic analysis of all obtained X-ray structures revealed that the Cys64/Ser64 residue lies at a key lattice interface which is conserved across all crystal forms. This suggests that even minor chemical changes at this position could disrupt important intermolecular contacts, explaining the demonstrated major benefit of the introduced mutation. The presented data underpin the substitution of surface-exposed cysteines as a general strategy to enhance protein crystallization and diffraction quality. Ultimately, the results presented here were pivotal to the successful execution of the crystallographic fragment-screening campaign with the HRP-2 PWWP domain.

Fascin1 proteins are a family of globular proteins with actin-bundling activity that cross-link actin filaments together, allowing the formation of actin-rich structures involved in cell migration and adhesion, such as filopodia, invadopodia, stress fibers, micro-spikes and podocytes. The overexpression of human fascin1 has been linked to tumor progression in most human cancers, particularly during the epithelial–mesenchymal transition, making it a promising biomarker for cancer metastasis and a major target for the development of novel cancer therapies. X-ray crystallography has been instrumental in human fascin1-inhibition research since it provides detailed insights into the structure of the protein and its interactions with small-molecule inhibitors. This technique has allowed the characterization of a range of molecular conformations in which the protein naturally exists. However, human fascin1 has never been fully modeled until now. To the best of our knowledge, this study presents the first full-length structure of human fascin1 in which both copies are fully resolved. Comparison of this structure with the available wild-type and complexed structures provides new insights into the conformational plasticity of fascin1 that will facilitate subsequent studies on human fascin1 in the context of drug design for cancer-related therapies.

Sucrose phosphorylases are essential enzymes regulating sucrose metabolism, and it has been shown that a loop rearrangement is essential to their catalytic cycle. Crystal structures of only six sucrose phosphorylase enzymes are available. Here, we present the crystal structure of a sucrose phosphorylase from a proteobacterium, Alteromonas mediterranea, at 2.15 Å resolution. The available sucrose phosphorylase structures have shown that an important conformational change occurs during the catalytic cycle or upon mutagenesis. Interestingly, our data present clear indications of the two major conformations in the same crystal.

rsCherry was one of the first reversibly photoswitchable variants to be developed from mCherry. However, its practical applications have been limited due to several inherent drawbacks. We have recently shown that the purified protein undergoes oxygen-induced chromophore degradation in solution, resulting in the progressive loss of its fluorescence and color. In this work, we present four crystal structures of rsCherry that exhibit varying degrees of degradation. Our structural analysis indicates that oxygen-induced degradation of rsCherry predominantly affects the chromophore without altering the protein backbone. Changes were only observed in the conformation of Lys70, confirming the crucial role of this residue in chromophore damage in rsCherry. Overall, this study provides valuable insights into the structural changes triggered by oxygen exposure in rsCherry, offering suggestions for the development of stable red fluorescent proteins with improved resistance to oxidative damage.

Unlike humans, Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, has a de novo histidine-biosynthesis pathway. The enzyme imidazole glycerol phosphate dehydratase (IGPD), which catalyses the conversion of imidazole glycerol phosphate to imidazole acetol phosphate, has been studied extensively from various organisms and has become a major target for the development of antibacterial, antiweed and antifungal small molecules. In our previous studies, we have shown that in crystals IGPD forms a 24-mer oligomeric state in which the monomers are arranged in 432 symmetry. In order to gain insights into the oligomeric state of Mtb IGPD in solution, we determined cryogenic sample electron microscopy (cryo-EM) structures of apo IGPD at 2.2 and 3.1 Å resolution. In addition, we also determined the cryo-EM structure of IGPD in the presence of 3-amino-1,2,4-triazole (ATZ) to 2.8 Å resolution. The results of this work, which corroborate those from the crystallographic studies, indicate that IGPD forms a homo-oligomeric structure in solution comprising of 24 subunits. ATZ binds in the active-site pocket of the enzyme, which is located at the interface of three monomers and tethers 24 ATZ molecules. The results of this study suggest that cryo-EM, in addition to being a rapidly evolving and complementary imaging technology for elucidating 3D structures of biological macromolecules, can be useful in pinpointing the mode of binding small molecules of low mass (here ∼85 Da) and mapping protein-ligand interactions, which could assist in the design of accurate (high-potency) inhibitors.