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At least 19 recordsLinked to original sources

PubMed2026

Crystal structures of Parechovirus A1 3Dpol reveal a mechanism of conformational stabilization in +ssRNA virus RNA-dependent RNA polymerase.

Parechovirus A1 (PeV A1) 3Dpol is an RNA-dependent RNA polymerase responsible for replication of the virus genome. We solved crystal structures of PeV A1 3Dpol structure in complex with GTP and in apo-state at 1.8-2.0 Å resolutions. In the 3Dpol-GTP complex, the conformation of the conserved motif B loop was stabilized by zinc ion coordination by cysteine residues. Apo-state structures of PeV A1 3Dpol showed significant conformational flexibility in the motif B loop, in the absence of zinc. While one of the conformational states of apo-3Dpol was similar to the 3Dpol-GTP complex structure, the alternative apo-3Dpol conformation showed a 4.3 Å movement of the motif B loop out of the active site cavity relative to the complex of 3Dpol with GTP. We propose that PeV A1 3Dpol activity is regulated by conformational stabilization of the motif B loop by zinc coordination.

Crystal structure↗

PubMed2001

Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2.

Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.

Amino Acid Sequence↗

PubMed2024

Assessing nanobody interaction with SARS-CoV-2 Nsp9.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

Viral Nonstructural Proteins↗

PubMed2013

Structural insights into adeno-associated virus serotype 5.

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.

Capsid Proteins↗

PubMed2025

Pharmacologic interrogation of USP28 cellular function in p53 signaling.

Deubiquitinating enzymes (DUBs) are crucial regulators of ubiquitin signaling and protein degradation that remain incompletely understood in part due to the lack of high-quality chemical probes. To address this challenge, we developed CAS-010, a low nanomolar, ubiquitin-competitive inhibitor of USP28 that demonstrates preferential activity against USP28 over other DUBs, while also exhibiting some activity against the closely related USP25. We rationalized our SAR trends and observed selectivity using a crystal structure of USP28 in complex with an inhibitor. We validated on-target effects of CAS-010 on the negative regulation of p53 transactivation in the wild-type setting. We demonstrated that CAS-010 disrupts the 53BP1-USP28 interaction, and more broadly showed that USP28 catalytic activity contributes to this key interaction. Taken together, CAS-010 and the accompanying negative control compound WPT-086 and inhibitor-resistant mutant provide well-validated tools for further characterizing the role of USP28 in p53-mediated effect on cell cycle control and cell fate.

Tumor Suppressor Protein p53↗

PubMed2026

Rapidly evolving aphid gall effector proteins exhibit saposin-like folds.

Many insects manipulate plants by injecting effector proteins. In one extreme example of this molecular "hijacking", Hormaphis cornu aphids inject bicycle proteins into Hamamelis virginiana (Witch Hazel), contributing to the development of novel organs called galls. Bicycle proteins share no amino acid sequence similarity with proteins of known function. Here, we report the crystal structures of two divergent bicycle proteins. Both proteins contain saposin-like folds: one with multiple disulfide bonds exhibits a helix swap; the other has no disulfide bonds and possesses two tandem domains. To explore the structural evolution of bicycle proteins, we predicted bicycle protein structures with Alphafold2 (AF2). While AF2 did not recover the two experimental structures using existing databases, it succeeded after we provided multiple sequence alignments (MSAs) containing protein sequences encoded in new genome sequences from closely related aphid species. Using this customized approach at scale, we generated 2400 high-confidence predictions for bicycle proteins from seven aphid species. This dataset revealed that bicycle proteins without cysteines are outliers in fold space and appear to have evolved from ancestral proteins with disulfide-bonded saposin-like folds. While all bicycle proteins contain predicted saposin-like folds, they display a vast diversity of structural and physicochemical properties. While this diversity thwarts prediction of conserved functions encoded in structure, it suggests that bicycle proteins have evolved to target diverse plant processes and/or to evade plant immune surveillance.

AlphaFold predictions↗

PubMed2026

Genetic and biochemical screens identify MGAT1 as a druggable glycosyltransferase target in STK11-mutant lung cancer.

Checkpoint inhibitors are standard-of-care therapies for non-small cell lung cancer (NSCLC), but their efficacy is limited in tumors with STK11 mutations, highlighting the need for new therapeutic strategies. Here, we performed complementary in vivo and in vitro CRISPR-Cas9 functional genomic screens to identify genes whose loss restores sensitivity to anti-PD-1 therapy. We found that loss of MGAT1, a Golgi glycosyltransferase critical for the maturation of high-mannose N-glycans into hybrid and complex glycan structures, reversed resistance to anti-PD-1 treatment in syngeneic mouse tumor models harboring STK11 mutations. Parallel co-culture screens with antigen-matched CD8+ T cells further showed that disruption of N-glycosylation strongly sensitized tumor cells to T cell-mediated killing. Genetic rescue studies demonstrated that this immune-evasion phenotype depends on MGAT1 catalytic activity, supporting direct biochemical interrogation of the enzyme. Using purified human MGAT1 and a UDP-Glo™ glycosyltransferase assay, we established a tractable screening platform and performed a 500,000-compound biochemical high-throughput screen, identifying an initial hit (compound 1; IC50 = 197 μM). Subsequent medicinal chemistry optimization delivered progressively more potent analogs, including TNG-9333 (0.814 μM) and TNG-2673 (0.043 μM) and represented a >1000-fold improvement in biochemical potency from the starting hit. Crystal structures of human MGAT1 in apo, UDP-bound, UDP-GlcNAc-bound, and inhibitor-bound states, together with SPR and DSF analyses, revealed that this chemical series engages a previously unrecognized allosteric pocket and inhibits MGAT1 through a UDP-noncompetitive mechanism. Collectively, our work implicates N-glycosylation as a key mediator of immune evasion and establishes MGAT1 as a ligandable, structurally tractable target for small-molecule drug discovery.

CRISPR/Cas9 target discovery↗

PubMed2022

Atomic model of vesicular stomatitis virus and mechanism of assembly.

Like other negative-strand RNA viruses (NSVs) such as influenza and rabies, vesicular stomatitis virus (VSV) has a three-layered organization: a layer of matrix protein (M) resides between the glycoprotein (G)-studded membrane envelope and the nucleocapsid, which is composed of the nucleocapsid protein (N) and the encapsidated genomic RNA. Lack of in situ atomic structures of these viral components has limited mechanistic understanding of assembling the bullet-shaped virion. Here, by cryoEM and sub-particle reconstruction, we have determined the in situ structures of M and N inside VSV at 3.47 Å resolution. In the virion, N and M sites have a stoichiometry of 1:2. The in situ structures of both N and M differ from their crystal structures in their N-terminal segments and oligomerization loops. N-RNA, N-N, and N-M-M interactions govern the formation of the capsid. A double layer of M contributes to packaging of the helical nucleocapsid: the inner M (IM) joins neighboring turns of the N helix, while the outer M (OM) contacts G and the membrane envelope. The pseudo-crystalline organization of G is further mapped by cryoET. The mechanism of VSV assembly is delineated by the network interactions of these viral components.

Animals↗

PubMed2004

Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery.

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90s conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.

Binding Sites↗

PubMed2025

Insights into the Catalytic Activity of a Metagenome-Derived Urethanase.

The discovery of urethanases shows an opportunity to access the biotechnological recycling of polyurethane-based plastics (PURs), widely used in the manufacture of everyday materials. However, the mechanistic understanding of these enzymes remains under debate. In this work, we report a QM/MM-based mechanistic study of the metagenome-derived urethanase UMG-SP2 catalyzing the degradation of a urethane-like model compound, 4-nitrophenyl benzylcarbamate (pNC). A high-quality structural model generated with AlphaFold2, prior to the availability of the crystal structure, accurately captured the Ser-Ser-Lys catalytic triad characteristic of amidase signature enzymes. Highly accurate constant-pH nonequilibrium molecular dynamics and Monte Carlo (neMD/MC) simulations provided the full titration curve of active site Lys, explaining the need for alkaline media for the enzyme to be active. The generation of the free energy landscape, obtained by means of free energy perturbation methods with the M06-2X DFT functional describing the QM region of the full system, reveals an esterase-like three-step mechanism of UMG-SP2, i.e., acylation, hydrolysis, and decarboxylation, with all steps being kinetically feasible. Our computational results show very good agreement with experimental kinetic data, with a calculated free energy barrier of 21.2 kcal·mol-1 for the rate-determining step compared to 22.9 kcal·mol-1 derived from the experimentally measured turnover frequency (TOF). The present results also open the door for the final decarboxylation occurring in the solution after the release of the product of the hydrolysis step or within the active site. These findings provide an atomistic insight into the urethanase function and establish a robust framework for the future design of biocatalysts targeting polyurethane degradation.

Metagenome↗

PubMed2026

Assessing Metal Ion Assignment Accuracy in Protein Data Bank Models via Elemental Spectroscopy.

Accurate representation of metal ions in macromolecular structures is critical for chemical interpretation, computational modeling, and machine-learning methods that rely on Protein Data Bank (PDB) entries. However, the elemental identity of metals modeled in crystallographic structures is often inferred indirectly and rarely validated experimentally. Here, we combine Particle Induced X-ray Emission (PIXE) and X-ray Fluorescence Spectroscopy (XRFS) to determine the elemental composition of protein samples used to generate 70 deposited metalloprotein crystal structures. By analyzing the original protein material employed for crystallization, but before the addition of crystallization buffer solutions, we assess whether the modeled metal ions in deposited structures are consistent with experimentally detectable elemental content. We find that in a majority of cases, the metals modeled in the corresponding PDB entries are inconsistent with the metals present in the protein samples before crystallization, or that additional metals are present but not represented in the structural models. Spectroscopic results were integrated with automated crystallographic validation metrics, including real-space Z-difference (RSZD) analysis and systematic rerefinement, to evaluate atomic-number mismatch at metal sites. PIXE and XRFS show strong agreement for dominant elemental signals and provide complementary, scalable approaches for identifying suspect metal assignments. This work does not address physiological or functional metalation but instead highlights a widespread data integrity issue in deposited macromolecular structures, PDB-wide. These results establish an experimentally corroborated link between elemental identity and crystallographic validation metrics, enabling the large-scale detection of chemically inconsistent annotations in structural databases used for computational modeling and machine learning.

Databases, Protein↗

PubMed2026

Structure-based drug design of small-molecule c-Myc G-quadruplex binders.

The c-Myc oncogene is crucial in tumorigenesis. Although it is a promising therapeutic target, its protein lacks a conventional drug-binding pocket, making it traditionally "undruggable". Recent studies show that the c-Myc promoter can form a G-quadruplex (G4) structure, which suppresses transcription and offers a new strategy for indirect inhibition. In this study, structure-based virtual screening was performed using the c-Myc G4 crystal structure to screen the ChemDiv compound library, aiming to identify small molecules that bind to the G4 structure. Candidate compounds were evaluated in preliminary in vitro assays for biological activity. The results showed that Y502-3888 binds to the c-Myc G4 and downregulates c-Myc expression at both mRNA and protein levels. Collectively, these findings support the potential of Y502-3888 as a c-Myc G4 binder for the treatment of multiple myeloma (MM), providing a foundation for future development of anticancer agents targeting the c-Myc G4.

G-Quadruplexes↗

PubMed2024

Structure and Methyl-lysine Binding Selectivity of the HUSH Complex Subunit MPP8.

The Human Silencing Hub (HUSH) guards the genome from the pathogenic effects of retroelement expression. Composed of MPP8, TASOR, and Periphilin-1, HUSH recognizes actively transcribed retrotransposed sequences by the presence of long (>1.5-kb) nascent transcripts without introns. HUSH recruits effectors that alter chromatin structure, degrade transcripts, and deposit transcriptionally repressive epigenetic marks. Here, we report the crystal structure of the C-terminal domain (CTD) of MPP8 necessary for HUSH activity. The MPP8 CTD consists of five ankyrin repeats followed by a domain with structural homology to the PINIT domains of Siz/PIAS-family SUMO E3 ligases. AlphaFold3 modeling of the MPP8-TASOR complex predicts that a SPOC domain and a domain with a novel fold in TASOR form extended interaction interfaces with the MPP8 CTD. Point mutations at these interfaces resulted in loss of HUSH-dependent transcriptional repression in a cell-based reporter assay, validating the AlphaFold3 model. The MPP8 chromodomain, known to bind the repressive mark H3K9me3, bound with similar or higher affinity to sequences in the H3K9 methyltransferase subunits SETDB1, ATF7IP, G9a, and GLP. Hence, MPP8 promotes heterochromatinization by recruiting H3K9 methyltransferases. Our work identifies novel structural elements in MPP8 required for HUSH complex assembly and silencing, thereby fulfilling vital functions in controlling retrotransposons.

Humans↗

PubMed2026

Structural Characterization and Engineering of a GH134 β-Mannanase from Aspergillus nidulans for Enhancement of Activity and Stability.

Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.

Aspergillus nidulans↗

PubMed2026

Dual solvent cavities and hydrogen-bond networks define the chromophore environment in a far-red/orange-sensing cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are bilin-binding photoreceptors that exhibit remarkable spectral diversity and mediate light-dependent signaling in cyanobacteria. Far-red/orange-sensing CBCRs (froCBCRs) have attracted interest because of their unusually red-shifted absorption properties, yet structural information for their illuminated states has been lacking. Here, we report the first high-resolution (1.8 Å) crystal structure of the orange-absorbing (Po) state of the froCBCR ToFrO from Tolypothrix sp. PCC 7910. The structure reveals a compact, cyclic bilin configuration and water-mediated hydrogen-bonding networks within two solvent-accessible cavities. Within the GAF domain, the D-ring remains nearly perpendicular to the planar A-to-C ring system through interactions involving a flexible loop region. Comparative analyses of cryogenic synchrotron and room-temperature X-ray free-electron laser (XFEL) structures, together with molecular dynamics (MD) simulations, revealed alternative Met636 conformations associated with dynamic water exchange through the solvent-accessible cavity. Site-directed mutagenesis of cavity-lining and water-interacting residues resulted in modest spectral shifts. By contrast, mutations of two Trp residues, participating in π-π stacking with the D-ring and likely imposing a steric constraint near the A-ring, resulted in substantial blue and red shifts in the dark and illuminated states, respectively. Together with the observed chromophore geometry, these findings indicate that the spectral properties of ToFrO are governed by chromophore conformation and its direct interaction with surrounding residues through hydrogen-bonding, electrostatic, and π-π interactions. These results further suggest that cavity-mediated solvent organization contributes to stabilizing the local structural environment surrounding the chromophore and adjacent protein backbone. Collectively, these findings elucidate the structural basis for photoconversion and spectral tuning in froCBCRs.

Cyanobacteria↗

PubMed2026

Allelic variation alters expression and antigen presentation of MR1 allomorphs.

The major histocompatibility complex (MHC) class I-related protein 1 (MR1) presents vitamin B-derived metabolites to mucosal-associated invariant T (MAIT) and other T cells. There is limited polymorphism of MR1, the functional impact of which is not understood. We examined the impact of allelic variation of MR1 on the expression, structure and function of the known MR1 allomorphs. The expression and function of MR1∗02, MR1∗03, and MR1∗06 were similar to the canonical MR1∗01. Crystal structures of four MR1 allomorphs show that their polymorphisms do not impact the three-dimensional fold of MR1. Despite the binding of 5-OP-RU to MR1∗05 and its cell surface upregulation, this allomorph was severely impaired in its ability to activate primary MAIT cells. This phenotype was controlled by two (His90Gln and Glu52Gly) of its three polymorphisms, which led to the loss of structurally stabilizing interactions. When cells expressing the MR1 allomorphs were infected with herpes simplex virus type 1 (HSV-1), the nascent expression of all allomorphs was severely impaired, but surface expression of MR1∗04:01 and MR1∗04:02 was relatively less impacted. Hence, MR1 allelic variation alters the expression and function of the MR1∗04 and MR1∗05 allomorphs, with implications for MAIT cell and diverse MR1-reactive T cell immunity.

Humans↗

PubMed2026

Structural insights into histone mimicry by the small hepatitis delta antigen.

Hepatitis delta virus (HDV) is a satellite RNA virus that requires hepatitis B virus (HBV) for propagation but replicates its genome independently in the nucleus. The small form of the hepatitis delta antigen (S-HDAg) is essential for replication and is regulated by post-translational modifications. Acetylation at lysine 72 (K72ac) enables S-HDAg to interact with the bromodomain (BRD) of the host chromatin remodeler bromodomain adjacent to zinc finger domain protein 2B (BAZ2B) to promote viral replication. However, the structural basis for this interaction has remained elusive. Here, we provide structural and biophysical insights into this interaction through quantitative binding assays and X-ray crystallography. Isothermal titration calorimetry revealed that BRDs of BAZ2B and its close homolog BAZ2A bind to the viral peptide weakly, with BAZ2A-BRD exhibiting a modestly higher affinity. The crystal structure of BAZ2A-BRD in complex with the S-HDAg-K72ac peptide demonstrates an inverted binding orientation relative to canonical histone ligands, rationalizing the weak interaction. Mutagenesis studies confirmed the critical binding interface both in vitro and in cells. These findings elucidate the molecular mechanism by which HDV co-opts host BAZ2 bromodomains via a unique, weak-affinity interaction, providing a structural framework for understanding viral replication.

Hepatitis delta Antigens↗

PubMed2026

TFAM organizes DNA into compact higher order structures.

TFAM (Transcription Factor A, Mitochondrial) is an essential human protein that plays two key roles in mitochondrial DNA (mtDNA) homeostasis. TFAM acts as a transcription factor that specifically binds to promoter regions, but it is also solely responsible for organizing mtDNA into nucleoids by nonspecifically covering the entire genome. Many studies have addressed TFAM in transcription regulation, but its role as a genome organizing entity is not well characterized. The current understanding of how TFAM compacts DNA into nucleoids is based on crystal structures of a TFAM monomer bound to short fragments of DNA (22-28 bp). However, this does not adequately reflect the biological role of TFAM in organizing the nucleoid where multiple TFAM molecules oligomerize on the 16.5 kb genome to form the nucleoid. Here, we present a biochemical and structural analysis of TFAM oligomerization on longer DNA. Our results show that TFAM compacts longer segments of DNA into higher order complexes that are homogenous yet exhibit continuous conformational dynamics.

Major classification: Biological sciences↗