Peptidyl-tRNA hydrolase (Pth2) hydrolyzes peptidyl-tRNA, an immature product of aborted translation, into peptide and tRNA, thereby maintaining cellular protein synthesis through peptide release and tRNA recycling. Here, we present the crystal structure of Pth2 from Candidatus Lokiarchaeum sp. GC14_75 (LokiPth2) at 2.12 Å resolution. This is the first structure of Pth2 from a lineage within Promethearchaeati, a kingdom of archaea closely related to eukaryotes. The structure reveals that LokiPth2 forms a homodimer and closely resembles Pth2 structures from other species. However, LokiPth2 exhibits two prominent structural differences: a short helix around the catalytic center, which is absent in other Pth2s, and a distinct orientation of the C-terminal helix. Detailed comparative structural analysis suggests that these regions may regulate enzymatic activity and substrate binding, respectively. Furthermore, the corresponding regions in other Pth2s also exhibit high flexibility, suggesting that similar mechanisms may be conserved among Pth2s. To gain insights into the growth environment of Candidatus Lokiarchaeum sp. GC14_75, we assess the optimal temperature for the catalytic reaction of LokiPth2, which suggests that Candidatus Lokiarchaeum sp. GC14_75 inhabits moderately thermophilic environments.
The Anticalin CL31d, an engineered lipocalin protein previously designed to specifically bind rare-earth and related metal ions as chelate complexes with p-NH2-Bn-CHX-A″-DTPA (DTPA-NH2), was subjected to structural and binding studies with a series of 11 different MIII·DTPA-NH2 complexes. These complexes include various lanthanide and main-group metal(III) ions whose radioisotopes are useful in nuclear medicine, with ionic radii ranging from 0.62 to 1.03 Å. Binding activities of the Anticalin for the MIII·DTPA-NH2 complexes were quantified by fluorescence titration (probing the intrinsic Tyr/Trp emission), revealing Kd values of 0.8-2.4 nM for most of the lanthanide ions investigated (rion = 0.75-0.96 Å), but showing markedly reduced affinities towards the small and large main-group metal ions Ga3+ (Kd = 15.3 nM) and Bi3+ (Kd = 47.8 nM), respectively. The crystal structures of six representative MIII·DTPA-NH2 complexes bound to the Anticalin were solved at high resolution (1.5-1.8 Å) using synchrotron X-ray diffraction. Superposition onto the previously described Anticalin CL31 with bound Y3+·DTPA-SCN indicated an essentially invariant conformation both for the binding protein and its metal-chelate ligands, including conserved hydrogen bonds, and a surprisingly uniform ninefold metal coordination via five carboxylate groups, three N atoms and one water molecule. However, there were two exceptions: the small Sc3+ ion appeared to be coordinated only eightfold with the DTPA-NH2 chelator but lacking the water ligand, whereas no metal electron density was observed for the Ga3+ ion, in line with its known noncanonical DTPA complex geometry. Interestingly, in this case a water molecule was detected at the expected position of the central metal ion within the protein-bound DTPA-NH2 chelator. Our investigation of the influence of chelate geometry on complex stability establishes the Anticalin CL31d as a small and robust universal binding protein for medically relevant MIII·DTPA complexes with surprisingly broad tolerance towards varying ionic radii, thus enabling flexible radionuclide-targeting strategies in nuclear medicine.
The Gly/N-degron pathway is a branch of the proteasomal degradation pathway that specifically targets proteins initiated with an N-terminal glycine. The E3 ligase substrate adaptors ZYG11B and ZER1 have been identified as being responsible for recognizing the target proteins of the Gly/N-degron pathway. Previously, it has been shown that the Gly/N-degron pathway activates the human NLRP1 inflammasome by degrading the autoinhibitory N-terminal fragment of NLRP1 after cleavage by the enteroviral 3C protease. However, the recognition of the NLRP1 Gly/N-degron is not yet fully understood. Here, we determined the X-ray crystal structure of ZER1 bound to the NLRP1 Gly/N-degron at a resolution of 2.2 Å. The structural information revealed that ZER1 uses its ARM repeats to form a conserved cavity that engages the N-terminal glycine (G1) through hydrogen bonds to Asp556, Asn597 and Glu600. Structural comparisons show a shared recognition mode for Gly/N-degrons despite subtle differences in side-chain interactions. However, ZER1 exhibits weaker affinity for the NLRP1 Gly/N-degron than ZYG11B, likely due to distinct local environments surrounding position 3. This study elucidates the molecular basis of NLRP1 recognition by ZER1 and provides insights into targeting this pathway in inflammatory diseases.
NKX2-1 (thyroid transcription factor 1, TTF-1) is a homeodomain transcription factor that plays critical roles in the development and function of the thyroid, lung and forebrain. Here, we report the crystal structure of the NKX2-1 homeodomain bound to a 19 bp DNA duplex containing two palindromically arranged NK2-recognition motifs, refined to 3.26 Å resolution. The structure reveals two homeodomains bound to a single DNA duplex and demonstrates that the overall fold and DNA-binding interactions are highly conserved relative to those of NKX2-5. Comparison with NKX2-5 further shows that the amino-acid residues that differ between the two homeodomains are located away from the protein-DNA interface, suggesting that functional differences between these transcription factors are unlikely to arise from distinct DNA-recognition mechanisms. These findings provide a structural framework for understanding DNA recognition by NKX2-1 and for interpreting the effects of pathogenic variants within its homeodomain.
L-Galactose dehydrogenase plays a key role in the biosynthesis of L-ascorbic acid (vitamin C) in plants. Here, we report crystal structures of rice (Oryza sativa) L-galactose dehydrogenase in both apo and NAD+-bound forms at 1.2 and 1.8 Å resolution, respectively. Rice L-galactose dehydrogenase adopts a conserved (β/α)8-barrel fold with a well preserved NAD+-binding cleft, as in other plant L-galactose dehydrogenases. Structural comparisons revealed conformational flexibility in the first and second loop regions, which form the lid and side wall of the NAD+-binding site. These loops undergo significant rearrangement upon NAD+ binding, likely facilitating cofactor and substrate uptake. These findings suggest a flexible loop-mediated regulation of cofactor and substrate access.
Dickkopf (DKK) family proteins (DKK1-DKK4), which function as extracellular modulators of Wnt signaling, contain two cysteine-rich domains: CRD1 and CRD2. In DKK1, CRD1 modulates interaction with its receptor low-density lipoprotein receptor-related protein (LRP) 5/6, whereas CRD2 directly binds to LRP5/6. The crystal structure of human DKK4-CRD1 was determined at 1.83 Å resolution. Crystals were obtained from refolded protein expressed as inclusion bodies and belonged to space group P21, with two molecules in the asymmetric unit. Initial molecular-replacement attempts using the solution NMR structure were unsuccessful, whereas an AlphaFold2-predicted model provided a clear solution. The refined structure reveals a compact fold comprising N- and C-subdomains connected by a linker region and stabilized by five conserved disulfide bonds. The crystal structure closely resembles the AlphaFold2 model, but shows larger deviations from the NMR ensemble. ANSURR analysis and hydrogen-bond comparisons indicate that the NMR models underestimate structural rigidity, particularly in β-sheet regions, owing to fewer stabilizing hydrogen bonds. Notably, enhanced conformational variability is observed in the N-subdomain, suggesting a potential role for structural plasticity in ligand recognition.
The type VI secretion system (T6SS) is a dynamic protein nanomachine employed by Gram-negative bacteria to secrete toxic effector proteins directly into microbial competitors, eukaryotic host cells, or the environment. The T6SS is primarily involved in interbacterial antagonism, along with host-cell manipulation and resource scavenging, underscoring its central role in microbial community structuring and establishment of infection. Pseudomonas aeruginosa employs the 52 kDa adaptor protein Tla3 for the successful loading of the antibacterial phospholipase effector Tle3 onto the H2-T6SS machinery. Tla3 is reported to contain two DUF2875 domains. To date, no structural information is present for DUF2875 domain-containing adaptor proteins of the T6SS. Here, we report the 1.8 Å resolution crystal structure of the T6SS adaptor protein Tla3 from P. aeruginosa strain UCBPP-PA14. The structure shows a thiolase-like fold, with a conserved five-layered α-β-α-β-α core topology. Detailed atomic insight into the structure is significant, as it adds to the hitherto limited structural knowledge of adaptor-mediated effector-loading mechanisms in the T6SS, thereby contributing to a broader understanding of T6SS functionality.
Accurate determination of the image pixel size is critical for quantitative cryo-electron microscopy analyses, yet existing calibration methods remain under-utilized because installation barriers and workflow complexity discourage routine adoption. To fill in this gap, a web-based application, WebCalEM, was developed to transform specialized calibration procedures into an accessible routine practice. Micrographs of any specimen with a known crystalline lattice, such as gold or graphene oxide, are uploaded through a standard browser, processed entirely client-side and analyzed with real-time visualization and downloadable statistical outputs. The application is delivered as a single self-contained HTML file that runs in any modern web browser without server-side computation, a configuration that is well suited to isolated core-facility microscope workstations. Cross-standard consistency between gold and graphene oxide measurements across two microscopes and ten magnification settings yields a Bland-Altman bias of -0.005% of nominal with 95% limits of agreement of [-0.30%, +0.29%]. By delivering this workflow with no local installation, WebCalEM lowers the practical barrier to documented per-dataset magnification calibration in routine cryo-EM operation.
The cyclic oligonucleotide-based antiphage signaling system (CBASS) employs diverse effector proteins to trigger abortive infection upon phage invasion, yet structural information on membrane-associated CBASS effectors remains limited. CD-NTase-associated protein 16 from Enterococcus faecalis (EfCap16) is a predicted CBASS effector comprising an N-terminal transmembrane region and a C-terminal Nudix-like domain. Here, we report the recombinant expression, purification, crystallization and X-ray crystal structure of the cytosolic Nudix domain of EfCap16 (PDB entry 22lg) in space group P1211. The protein was purified to homogeneity and crystallized, and its structure was determined at 1.6 Å resolution. The EfCap16 Nudix domain shares structural similarity with the Nudix superfamily and contains a conserved Nudix motif. This structure provides a reference framework for future biochemical and functional studies of transmembrane CBASS effectors.
Aeropyrum pernix is a hyperthermophilic archaeon that possesses three proliferating cell nuclear antigen (PCNA) isoforms (ApePCNA1, ApePCNA2 and ApePCNA3) that form a heterotrimeric sliding clamp. To gain more detailed structural insights into this heterotrimeric assembly, we determined the crystal structures of ApePCNA1 and ApePCNA2. ApePCNA1 was crystallized under a new condition, and the 1.60 Å resolution structure revealed a unique nonproline cis-peptide bond between Arg187 and Arg188, which was not deeply discussed in a previous report. The structure of ApePCNA2 was determined at 2.17 Å resolution, and it forms a typical homotrimeric ring. In the cubic crystal form, its crystal packing shows an intriguing tetrahedral assembly of four trimers. Modeling the ApePCNA1-ApePCNA2-ApePCNA3 heterotrimer based on these structures suggests that the cis-peptide in ApePCNA1 induces significant steric hindrance at the subunit interface, leading to a symmetry-broken or distorted ring conformation rather than the canonical pseudo-threefold-symmetric assembly.
The papain-like protease (PLpro) of SARS-CoV-2 is part of the multi-domain nonstructural protein 3 (NSP3) and consists of two domains: a ubiquitin-like domain 2 (Ubl2) and a protease domain. PLpro plays a crucial role in the replication cycle of SARS-CoV-2, facilitating host immune-system evasion and the formation of double-membrane vesicles where replication occurs. While the function of the Ubl2 domain is still not clear, it is critical for the stability and the functional efficiency of PLpro. Despite its predicted inherent flexibility, nearly all SARS-CoV-1 and SARS-CoV-2 PLpro crystal structures deposited in the Protein Data Bank show the Ubl2 domain in a highly similar, closed conformation against the catalytic domain. Here, we present a crystal structure of PLpro exhibiting Ubl2 in two distinct conformations: the well characterized closed state, where Ubl2 is positioned near the PLpro domain, and an as yet uncharacterized open state, where Ubl2 is displaced by 4 Å from the PLpro core. This conformational variability in our structure appears to be related to the occupancy of a zinc ion within the zinc-finger domain. These results provide new insights into the flexibility of Ubl2, suggesting potential avenues for targeting and harnessing this dynamic behaviour for drug discovery.
Plasmodium vivax is a major cause of malaria globally and has recently been transmitted locally in the USA. P. vivax produces homologs of host proteins, including cytokines such as macrophage migration inhibitory factor (MIF). MIF regulates both adaptive and innate immune responses and contributes to the pathogenesis of parasitic infections, including malaria. Plasma concentrations of P. vivax MIF (PvMIF) correlate with the severity of P. vivax malaria. Plasmodium spp. MIFs have been recognized as candidate malaria vaccines. PvMIF, like other protozoan MIFs, binds to host CD74 and can suppress host MIF-CD74 signaling. The production, crystallization and 1.8 Å resolution structure of PvMIF (PDB entry 9b0m, pdb_00009b0m) are reported. PvMIF crystallized in space group P63 with a single molecule in the asymmetric unit. The biological unit of PvMIF is the prototypical MIF trimer.
γ-Aminobutyric acid aminotransferase (GABA-T) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes γ-aminobutyric acid (GABA) degradation in the mitochondrial GABA shunt. Plant GABA-Ts correspond to EC 2.6.1.96 and differ from mammalian and bacterial GABA-Ts (EC 2.6.1.19) in substrate specificity; however, their structural basis has remained unclear. Here, we report the crystal structure of GABA aminotransferase from Arabidopsis thaliana (AtGABA-T) at 2.0 Å resolution. Structural comparison using Foldseek indicates that AtGABA-T belongs to the class III aminotransferase family. Comparison with a class III ω-aminotransferase suggests that Arg423 located in the C-terminal region of AtGABA-T functions as the primary anchor for the carboxyl group of GABA, together with residues forming a tunnel-shaped substrate-access pathway. In contrast to nonplant GABA aminotransferases, which utilize a conserved N-terminal arginine for substrate recognition, AtGABA-T employs a distinct C-terminal arginine. These findings provide direct structural evidence for the classification of AtGABA-T as a class III aminotransferase and reveal a distinct mode of substrate recognition in AtGABA-T.
Insomnia is a widespread sleep disorder that significantly impairs quality of life and imposes a societal burden. Although benzodiazepines and Z-drugs are commonly used for treating insomnia, these drugs often have side effects, such as excessive muscle relaxation and dependency. The orexin signaling pathway has emerged as a promising therapeutic target for insomnia, with dual orexin receptor antagonists (DORAs) offering an alternative approach to treatment. To reduce the potential of these drugs for next-day residual effects, we developed vornorexant, a novel DORA with a high receptor affinity and a short elimination half-life. In this study, we investigated the molecular interactions of this drug with human orexin receptors through crystal structure analysis of its binding to orexin type 2 receptor (OX2R) and a docking simulation of the drug with orexin type 1 receptor (OX1R). The crystal structure of the OX2R-vornorexant complex revealed a conserved U-shaped conformation stabilized by hydrophobic and hydrogen-bonding interactions, including key contacts with Asn324, His350 and Pro131. OX1R-vornorexant docking simulations indicated a similar binding mode to OX1R, with no steric hindrance observed, supporting a balanced dual antagonism. These results provide a structural basis for the high-affinity dual antagonism of vornorexant and offer insights for the design of orexin receptor antagonists.
The structure of spectrin-like repeat 24 of human dystrophin was determined at 2.5 Å effective resolution. The structure exhibits a three-helix bundle fold, common to all spectrin-repeat family members, and shares a high degree of homology with existing structures of spectrin-like repeat 1 from dystrophin and utrophin. The structure provides molecular details of the atomic interactions that stabilize the repeat, including hydrophobic interactions and inter-helix and intra-helix salt bridges. AlphaFold models of the repeat are in excellent agreement with the structure, showing an all-atom r.m.s.d. of 1.13 Å. Accurate modeling of SR24 supports AlphaFold modeling of all 24 of the dystrophin spectrin-like repeats and the use of these models in predicting the molecular determinants of dystrophin stability, a key aspect of its biological function as a structural protein that cross-links actin filaments to the dystrophin-glycoprotein complex to mediate a mechanical connection between the cytoskeleton and the extracellular matrix.
Extracellular S100 proteins act as alarmins and trigger pro-inflammatory signaling cascades by activating cognate cell-surface receptors such as the receptor for advanced glycation end-products (RAGE), thereby contributing to both normal and pathological inflammation depending on the physiological context. These ligand-receptor interactions occur in an oxidative environment that is known to induce post-translational modifications, notably on the cysteine residues present in S100 proteins, giving rise to disulfide-crosslinked S100 species. The fine molecular architecture of these S100 covalent assemblies and their impact on the interaction of S100 with RAGE remains poorly characterized, as most in vitro studies employ cysteine variants or reducing conditions. In this study, a thorough analysis of cysteine conservation within the whole S100 family shows an enriched presence of cysteines in the second half of helix H4, with a hotspot for cysteine occupancy at position 84. Following the introduction of a cysteine at this conserved position in S100A6, SDS-PAGE analysis under nonreducing conditions shows a noteworthy amount of covalent S100A6 Y84C dimer in solution, and the structural analysis of the resulting complex with the RAGE ectodomain reveals the formation of a covalent Cys84-Cys84 linkage between the two S100A6 protomers, thus stabilizing the dimeric conformation of RAGE-bound S100A6. Modeling of other S100 proteins that naturally bear a Cys84 in the RAGE-bound conformation suggests that this covalent S100 dimer architecture may be adopted by other members of the family previously reported to form disulfide-crosslinked species. Altogether, these findings provide a first possible model for S100 covalent homodimerization that is fully compatible with RAGE binding.
Arginase is a metal-dependent metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea and is widely distributed across animals, bacteria, fungi and protozoa. Here, we report the first three-dimensional crystal structure of a cold-active arginase from the psychrophilic yeast Glaciozyma antarctica (GaArg). The apo structure was solved at 2.35 Å resolution in space group H3 by molecular replacement using an AlphaFold-generated model. GaArg adopts a conserved αβα sandwich fold similar to previously characterized arginases. The crystal structure reveals four regions lacking interpretable electron density, three of which are located near the entrance to the active site. Despite conservation of the identities and the coordination geometry of metal-binding residues, the apo GaArg structure exhibits broadly conserved ligand-binding residue orientations relative to homologous arginases, with minor conformational differences and a disordered loop corresponding to a substrate-interacting region. Analytical size-exclusion chromatography and multimer prediction support the hexameric assembly of GaArg in solution. Quantitative analysis of intramolecular interactions indicates that GaArg contains fewer hydrogen bonds than mesophilic and thermophilic homologues, while its salt-bridge content is comparable to that of the mesophilic enzyme but lower than that of the thermophilic homologue. These features are consistent with a modest reduction in structural rigidity associated with cold adaptation. Ligand- and metal-bound structures will be required to establish their contributions to cold-adaptation.
In this study, we report the crystal structures of K214Q and K216Q variants of Escherichia coli glucokinase (ecGLK), each of which is bound to phosphate in the active-site cleft. The structure of the K214Q variant was determined at 2.70 Å resolution and refined with an Rwork and Rfree of 0.140 and 0.190, respectively, while that of the K216Q variant was determined at 2.44 Å resolution with an Rwork and Rfree of 0.178 and 0.225, respectively. Both variants adopt an open conformation and maintain phosphate-binding interactions similar to the wild-type ecGLK. Structural comparison of the K214Q variant revealed large backbone deviations in the 214-224 α-helix, increased disorder in the loops surrounding the glucose-binding cleft and outward shifts of Asn99, Asp100, His160 and Glu187. Our previous study demonstrated that lysine acetylation at Lys214 and Lys216 impaired the activity of ecGLK, and here we show that acetylation mimics produced domain shifts, indicating those of lysine residues that could be essential for stabilizing the glucose-binding region of ecGLK.
Snake-venom phospholipases A2 (PLA2s) are small, structurally conserved enzymes that contribute significantly to the pathophysiology of envenomation. Here, we report the purification and crystal structure of an Asp49-PLA2 isolated from the venom of Lachesis muta, a pit viper from the Peruvian Amazon. The enzyme was purified using ion-exchange and size-exclusion chromatography and exhibited phospholipase activity in a dose- and time-dependent egg-yolk degradation assay. Pure protein crystals were obtained in space group P6222 and diffracted to 2.36 Å resolution, with two molecules in the asymmetric unit. The structure reveals the canonical fold of catalytically active group II PLA2s, with a bound Ca2+ ion and a MES molecule in the active site of one monomer. Seven disulfide bonds stabilize the structure, although one bridge typically associated with the β-hairpin is absent and is replaced by a salt bridge as in other viperid PLA2s. PISA analysis suggests a potential tetrameric assembly composed of two AB dimers generating an interface between two A subunits (A-A'). Electrostatic surface mapping reveals a notable positively charged channel at the A-A' interface, like that seen for a basic PLA2 homodimer from Crotalus durissus terrificus in which the two active sites lie accessible to the membrane. This study presents the first structural and enzymatic analysis of an Asp49-PLA2 from L. muta and provides insights into its oligomeric assembly, electrostatic landscape and potential adaptations relevant to its role in venom toxicity.
Pentatricopeptide repeat (PPR) proteins constitute the largest family of RNA-binding proteins in land plants, playing crucial roles in organellar gene expression through sequence-specific RNA recognition. Including the first PPR crystal structure in 2013, 30 PPR and 83 ribosomal PPR crystal or cryo-EM structures have been deposited in the Protein Data Bank, encompassing both native and designer proteins in apo and RNA-bound states. This comprehensive survey catalogues all PPR crystal structures, analysing their crystallographic properties, structural features and contributions to understanding PPR function. Key insights include the elucidation of the PPR recognition code, the characterization of dramatic conformational changes upon RNA binding and the structural basis for the modular architecture that enables programmable RNA targeting. Designer PPR structures have validated and extended principles derived from native proteins, demonstrating their utility as synthetic biology tools. This structural catalogue provides crystallographers and plant biologists with a comprehensive resource for understanding PPR protein architecture and function.
