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

The α/β-hydrolase fold superfamily includes esterases and hydroxynitrile lyases which, despite catalyzing different reactions, share a Ser-His-Asp catalytic triad. We report a 1.99 Å resolution crystal structure of HNL6V, an engineered variant of hydroxynitrile lyase from Hevea brasiliensis (HbHNL) containing seven amino-acid substitutions (T11G, E79H, C81L, H103V, N104A, G176S and K236M). The structure reveals that HNL6V maintains the characteristic α/β-hydrolase fold while exhibiting systematic shifts in backbone and catalytic atom positions. Compared with wild-type HbHNL, the C^α positions in HNL6V differ by a mean of 0.2 ± 0.1 Å, representing a statistically significant displacement. Importantly, the catalytic triad and oxyanion-hole atoms have moved 0.2-0.8 Å closer to their corresponding positions in SABP2, although they remain 0.3-1.1 Å from fully achieving the configuration of SABP2. The substitutions also increase local flexibility, particularly in the lid domain covering the active site. This structural characterization demonstrates that targeted amino-acid substitutions can systematically shift catalytic geometries towards those of evolutionarily related enzymes.

The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.

Histone deacetylase inhibitors (HDACi) are widely used in cancer therapy but often suffer from off-target effects due to their pan-inhibitory activity towards zinc-dependent enzymes. Vorinostat (SAHA), a hydroxamate-based HDACi, has been shown to lack isoform selectivity, potentially leading to unintended interactions with other metalloenzymes. Here, we report high-resolution crystal structures of SAHA bound to human carbonic anhydrase II (CA II) and a carbonic anhydrase IX (CA IX) active-site mimic. Structures determined at room temperature and 100 K revealed two distinct SAHA conformers in both CA II and the CA IX mimic, with the hydroxamate moiety displacing the zinc-bound water and adopting either a tetrahedral or pentahedral coordination to Zn²⁺. Differences in hydrophobic interactions were observed between CA II and the CA IX mimic due to the F131V amino-acid difference between the two enzymes. SwissDock modeling accurately predicted the SAHA binding orientations observed in crystallography. Thermal shift assays using nanoDSF showed minimal stabilization of either CA by SAHA, in contrast to the potent CA inhibitor acetazolamide. Binding-energy calculations suggest that SAHA may bind carbonic anhydrases with affinities comparable to its HDAC targets. These findings highlight potential off-target binding of SAHA to carbonic anhydrases, which may contribute to its clinical side effects. The results also suggest that hydroxamates may serve as a nonsulfonamide scaffold for novel CA inhibitors, although isoform selectivity remains a challenge.

Virulence protein J (VirJ) is a periplasmic protein encoded by the bacterial pathogen Brucella abortus and is important for its virulence. The VirJ homologue AcvB from Agrobacterium tumefaciens was found to be a lysyl-phosphatidylglycerol hydrolase that contains two domains, D1 and D2. Interestingly, both VirJ and AcvB are associated with the type IV secretion system (T4SS) activity in the respective bacteria. To date, no structural information is available for these proteins, limiting our understanding of their function. Here, we have purified, crystallized and determined the crystal structure of the N-terminal domain 1 of VirJ (VirJ^D1) at a resolution of 1.7 Å. Our structural analysis shows that VirJ^D1 adopts an α/β-hydrolase fold but lacks the characteristic catalytic triad. The structure presented here may help to decipher the function of VirJ in Brucella spp. and other bacterial pathogens, as well as its contribution to the T4SS function.

Here, we report the crystal structure of Escherichia coli glucokinase (GLK), which has phosphate bound in the cleft between the α and β domains adjacent to the active site. A ternary complex consisting of GLK, glucose and phosphate is also reported in this work. Diffraction data were collected at 2.63 Å resolution for the phospate-bound form (R_work/R_free = 0.191/0.230) and at 2.54 Å resolution for the ternary complex (R_work/R_free = 0.202/0.258), both at 297 K. A B-factor analysis of the phosphate-bound GLK structure revealed consistently lower values for phosphate-interacting basic residues in the α4, α5 and α9 helices, while significant root-mean-square deviation (r.m.s.d.) spikes indicated flexibility in regions preceding β1 and within the loop between the β5 and β6 sheets of the α domain. In the ternary complex, phosphate is bound adjacent to glucose, and the B factors for the α4, α5 and α9 helices were further reduced, while r.m.s.d. spikes were observed at the end of the β10 sheet and within the α6 helix of the β-domain. This structural characterization suggests that phosphate could influence the activity of GLK by altering glucose binding and modulating interactions with a loop-interacting regulatory protein.

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.