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

Collagen lysyl hydroxylases catalyze the hydroxylation of collagen lysine residues during collagen synthesis in animals and mimiviruses. Lysyl hydroxylation is crucial for collagen fibrogenesis and function. We previously demonstrated that recombinant mimiviral and human collagen lysyl hydroxylases, isolated from bacterial and mammalian cells, have Fe²⁺ in their active sites, suggesting that lysyl hydroxylases have a high affinity for Fe²⁺. We found that Fe²⁺ binding stabilizes lysyl hydroxylase dimers, although the underlying mechanism remains unclear. Crystal structure analysis of mimiviral lysyl hydroxylase revealed that Fe²⁺ is coordinated by a 2His–1Asp (His825/His877/Asp827) triad, with a nearby highly conserved histidine residue (His869) involved in an alternative 2His–1Asp triad (His869/His877/Asp827). This unique structural architecture suggests that the alternative 2His–1Asp triad may also bind Fe²⁺. To investigate whether the alternative 2His–1Asp triad binds Fe²⁺ and how Fe²⁺ binding regulates lysyl hydroxylase dimerization, we crystallized the mimiviral lysyl hydroxylase mutant His825Ala, which lacks one 2His–1Asp (His825/His877/Asp827) triad but retains the alternative triad (His869/His877/Asp827). Despite providing Fe²⁺ during crystallization, we found no electron density near the alternative 2His–1Asp triad in the His825Ala mutant, indicating that the alternative 2His–1Asp triad does not bind Fe²⁺ with high affinity. Although the His825Ala mutant forms a dimer similar to the wild-type enzyme, conformational changes occur in residues near Ala825, including Leu873, which is critical for dimerization. These structural findings provide new insights into the function and regulation of collagen lysyl hydroxylases.

A lectin-like protein was discovered in Agaricus bisporus as part of the mushroom tyrosinase complex. The protein has a β-trefoil fold, which is typical of the ricin B-like-type lectin family. The structure of the recombinant protein has been elucidated, and its specific sugar-binding affinity towards mannose and mannitol has also been reported; therefore, the protein was named A. bisporus mannose-binding protein (Abmb). Although the sugar-binding site of Abmb is predicted to be close to the C-terminus, the sugar-binding site has not yet been determined. In this study, a variant of recombinant Abmb with a longer C-terminal region including a 6×His-tag was constructed and its structure was solved at 1.51 and 2.34 Å resolution in an orthorhombic and a monoclinic space group, respectively. The overall structure showed a β-trefoil fold as previously reported; however, several surface loop regions including the C-terminal region showed high flexibility. In addition, a glycan-search assay of this variant showed weak binding affinity towards β-d-galactose but no affinity towards α-d-mannose. The plasticity of the C-terminal tail could be related to the differences in the carbohydrate-binding affinity of Abmb.

Entamoeba histolytica causes amebiasis, a neglected disease that kills ∼100 000 people globally each year. Due to emerging drug resistance, E. histolytica is one of the target organisms for structure-based drug discovery by the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Purification, crystallization and three structures of the putative drug target endoribonuclease L-PSP from E. histolytica (EhL-PSP) are presented. EhL-PSP has a two-layer α/β-sandwich with structural homology to endoribonuclease L-PSP. All three structures reveal the prototypical YjgF/YER057c/UK114 family trimer topology with accessible allosteric active sites. Citrate molecules from the crystallization solution are bound to the allosteric site in two of the three reported structures. The large allosteric site of EhL-PSP is well conserved with bacterial YjgF/YER057c/UK114 family members and could be targeted for inhibition, drug discovery or repurposing.

DNA ligases are foundational molecular-biological tools used for cloning and sequencing workflows, and are essential replicative enzymes for all cellular life forms as well as many viruses and bacteriophage. There is considerable interest in structurally and functionally characterizing novel DNA ligases and profiling their suitability for molecular-biological applications. Here, we report the crystal structure of the ATP-dependent DNA ligase from the Rhizobium phage vB_RleM_P10VF bound to a nicked DNA duplex determined to 2.2 Å resolution. The enzyme crystallized in the DNA-encircling conformation, arrested as a step 2 intermediate in the catalytic cycle with the adenylating cofactor transferred to the 5′-phosphate of the DNA nick. The overall structure of the DNA ligase closely resembles that of the T4 DNA ligase, including an α-helical globular DNA-binding domain. Several secondary-structural elements are abbreviated in the P10VF DNA ligase relative to the T4 DNA ligase enzyme, which may account for its lower specific activity, especially on DNA substrates containing double-stranded breaks.

DNA replication is tightly regulated to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea partly achieve this regulation by strictly controlling the activation of hexameric minichromosome maintenance (MCM) helicase rings that unwind DNA during its replication. In eukaryotes, MCM activation critically relies on the sequential recruitment of the essential factors Cdc45 and a tetrameric GINS complex at the onset of the S-phase to generate a larger CMG complex. We present the crystal structure of the tetrameric GINS complex from the archaeal organism Saccharolobus solfataricus (Sso) to reveal a core structure that is highly similar to the previously determined GINS core structures of other eukaryotes and archaea. Using molecular modeling, we illustrate that a subdomain of SsoGINS would need to move to accommodate known interactions of the archaeal GINS complex and to generate a SsoCMG complex analogous to that of eukaryotes.

We report structures of the Mycobacterium tuberculosis isoprenyl diphosphate synthase Rv2173 in three forms: apo and two substrate-bound forms [isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)]. The protein possesses a canonical all-α-helical trans-isoprenyl diphosphate synthase fold that is dimeric in each form. There are some differences between the structures: the IPP-bound form shows IPP bound in the DMAPP/allylic substrate-binding site with three divalent metal ions bound around the IPP and the complete C-terminus closing around the active site, while the apo and DMAPP-bound forms are more open, with some of the C-terminal region disordered, supporting suggestions that the C-terminus is important in substrate entry/product exit. In the DMAPP form DMAPP occupies the expected allylic substrate site, but only two metal ions are associated with the binding, with the DMAPP diphosphates adopting a slightly different binding pose compared with IPP in the same site, and the third metal-binding site is unoccupied. In no case is the IPP binding site occupied by IPP. There has been some uncertainty regarding product length for Rv2173, with variable lengths being reported. In the structures reported here, the `capping' residue at the bottom of the binding cavity is tryptophan and comparison with other IPP synthases suggests that the structure of Rv2173 is most consistent with a C₁₀-C₁₅ final product size.

Nature is a rich and largely untapped reservoir of small molecules, the latter historically being the main source of new drugs. Three-dimensional structures of proteins in complex with small-molecule ligands represent key information to progress drug-discovery projects, in particular in the hit-to-lead phase. High-throughput crystallography has been of extensive use in recent years, especially to obtain crystallographic complexes of synthetic ligands and fragments. However, the process of discovering novel bioactive natural products has experienced limitations that have long prevented large drug-discovery programs using this outstanding source of molecules. Recent technologies have contributed to the re-emergence of natural products in modern drug discovery. We present the use of high-throughput protein crystallography to directly capture bioactive natural products from unpurified biota chemical samples using protein crystals. These routines, which are currently in use at the Brazilian Centre for Research in Energy and Materials (CNPEM), are introduced with a description of crystal preparation, automated data collection and processing at the MANACÁ beamline (Sirius, LNLS, CNPEM), along with case examples of bioactive natural product capture using protein crystals. The usefulness of this pipeline, which accelerates the discovery and structural elucidation of both known and previously unknown bioactive natural products, paves the way for the development of innovative therapeutic agents, thus contributing to the new era of natural product-based drug discovery.

A newly designed protein featuring a rare left-handed βαβ motif has successfully been crystallized and characterized by preliminary X-ray diffraction. The computational design was conducted using a combination of Rosetta BluePrintBDR, ProteinMPNN and AlphaFold2, generating eight candidates based on predicted stability and folding accuracy. The final construct was expressed, purified and crystallized in space group P2₁. Complete X-ray diffraction data were collected on the BL2S1 beamline at the Aichi Synchrotron and processed to 1.95 Å resolution. Despite multiple attempts, molecular replacement using the AlphaFold2 model did not yield a conclusive solution, suggesting that alternative phasing methods or refined modeling approaches will be needed. This work highlights both the promise and the challenges of pushing protein biodesign into underexplored structural motifs and provides a foundation for future structural and functional investigations.

Piperine has been investigated for a diverse array of biological effects, including a potential role in modulating peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor that plays a pivotal role in regulating lipid and glucose metabolism. This study conducted a comprehensive co-crystallographic analysis of the complex of piperine with the PPARγ ligand-binding domain (PPARγ-LBD), with the objective of elucidating the precise binding interactions of piperine. The co-crystal structure revealed that piperine binds within the ligand-binding pocket of PPARγ-LBD via hydrogen-bonding and hydrophobic interactions with residues of the ligand-binding site. Notably, in contrast to conventional full agonists, piperine does not directly stabilize helix H12. This could contribute to the comparatively weaker agonistic activity of piperine. The results of this study also suggest that piperine binding facilitates a role as a partial agonist or even an antagonist under certain physiological conditions. Collectively, these findings contribute to a greater understanding of the manner in which piperine modulates PPARγ function and its potential as a therapeutic candidate for the treatment of metabolic disorders. Given its natural origin and relatively minimal side-effect profile, piperine and its derivatives could be promising alternatives to synthetic PPARγ modulators such as thiazolidinediones, which have significant side effects.