Current Research in Structural Biology

Directional anisotropy in electron density provides key information about chemical bonding that is not readily accessible from conventional electron-density maps. Here, a model-independent framework is presented for decomposing experimental structure factors into angular components using spherical harmonics. Reciprocal-space projection onto spherical harmonics followed by standard Fourier synthesis yields angularly filtered density maps. The{ell} = 0 component captures the isotropic part of the density, while the{ell} = 1 components resemble px, py and pz-like dipolar functions that highlight directional electronic structure. Applications to high-resolution datasets, including urea, the Gly-Ala dipeptide and a 0.97 [A]{beta}-lactamase structure, reveal chemically interpretable dipolar features associated with carbonyl and amide bonds, N-H interactions and aromatic{pi} systems. Quantitative analysis using bond-centred sampling demonstrates stable dipolar signatures that remain detectable under moderate resolution truncation. These results establish spherical-harmonic angular decomposition as a practical framework for extracting directional electronic information from crystallographic electron-density maps. SynopsisAngular decomposition of experimental structure factors reveals dipolar anisotropy and directional electron-density features that are directly meaningful for chemical interpretation.

Protein phosphatase 2A containing the B56{delta} regulatory subunit (PP2A-B56{delta}) is a critical signaling enzyme whose dysregulation is associated with cancer, neurodegenerative disorders, and Jordan's syndrome, a severe intellectual disability disorder caused by mutations in B56{delta}. Unlike other PP2A holoenzymes, PP2A-B56{delta} is regulated through a unique dual autoinhibition mechanism in which the N- and C-arms occlude the catalytic site while a substrate-mimicking short linear motif (SLiM) blocks the substrate-binding pocket. Although disease-associated mutations have been shown to alter enzyme activity, the molecular mechanism underlying activation of PP2A-B56{delta} and the effects of pathogenic mutations remain poorly understood. Here, we combined cryo-electron microscopy (cryo-EM), enhanced-sampling molecular dynamics (MD) simulations, Markov state model (MSM) construction, and transition-state analysis using Transition State identification via Dispersion and vAriational principle Regularized neural networks (TS-DAR) to characterize the conformational landscape of the disease variant E198K. Our cryo-EM analysis identified two distinct structures of E198K: an inactive closed-form with the N/C-arms resolved and an active loose-form in which the N/C-arms become highly flexible and could not be fully resolved. These structures therefore established that activation is governed by conformational changes of the N/C-arms but did not reveal the underlying mechanism. Starting from the inactive closed-form, we generated over 1,600 trajectories with an average length of 1,260 ns combined for E198K and wild-type (WT) PP2A-B56{delta}. TS-DAR identified four metastable states and two major activation pathways connecting inactive and active conformations. We found that activation occurs through progressive loosening of the N/C-arm interface while maintaining the overall holoenzyme architecture, rather than a complete opening of the interface. This mechanism exposes both the catalytic site and substrate-binding pocket. Comparison of E198K and WT revealed that the disease-associated mutation shifts the conformational equilibrium toward active states while leaving the transition-state ensemble largely unchanged. Mechanistically, E198K disrupts a salt-bridge network and weakens interactions between the internal loop and the C-arm that normally stabilize active-site occlusion. The resulting increase in C-arm mobility promotes active-site exposure and explains the elevated catalytic activity of the mutant. Together, these findings establish a previously uncharacterized activation mechanism for PP2A-B56{delta} and provide an atomic-level explanation for how the pathogenic E198K mutation allosterically promotes holoenzyme activation.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson's disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90/cell division cycle 37/FK506-binding protein 51 (HSP90/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine-proline-phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.

Accurate assessment of ligand coordinate-density consistency across different resolutions remains challenging in macromolecular crystallography. We introduce the atomic Box Correlation Coefficient (aBCC), an atom-level metric for evaluating the consistency between ligand atomic coordinates and electron density in a resolution-standardized framework. To predict aBCC values from electron-density maps, we developed QAEmap, a machine-learning model based on three-dimensional convolutional neural networks (3D-CNNs). The model was trained using Fourier-truncated electron-density maps and corresponding ligand coordinates generated from high-resolution structures in the Protein Data Bank. It was evaluated using both Fourier-truncated electron-density maps and experimentally determined PDB structures. was evaluated using both Fourier-truncated electron-density maps and experimentally determined PDB structures.The prediction accuracy gradually decreased with decreasing resolution, but remained reliable up to [~]3.5 [A]. These results demonstrate that aBCC enables resolution-standardized atom-wise evaluation of coordinate-density consistency across different resolutions and provide a foundation for further development and refinement of machine learning-based coordinate validation. SynopsisWe introduce the atomic box correlation coefficient (aBCC), a machine learning-based metric for the resolution-standardized atom-level evaluation of ligand coordinate-density consistency in crystallographic structures. aBCC provides a common framework for assessing and communicating the local coordinate reliability between structural biologists and researchers in structure-based drug discovery.

Ferritins are ubiquitous iron homeostasis proteins found across the tree of life that form conserved 24-subunit cages with octahedral (4-3-2) symmetry. New types of ferritins and ferritin-like proteins are being continuously discovered, such as mini-bacterioferritins, which form smaller shells of 12 subunits, and double-ferritin-fold proteins, which act as ferroxidases but do not form shells. Here, we describe double-ferritin-fold proteins from Asgard archaea, dubbed dFTNs, and determine Cryo-EM structure of a representative from Candidatus Heimdallarchaeum endolithica. The protein forms a dodecameric shell with tetrahedral (2-3) symmetry. N-terminal (NTD) and C-terminal (CTD) domains are bridged by an ordered linker and are related by two-fold rotational pseudosymmetry. C-terminal -helix (helix E) that forms the four-fold channel in classic ferritins is repositioned to be the helix 2 out of 5 ferritin domain -helices in dFTN, with two such helices from NTD and two helices from CTD forming a pseudo-four-fold symmetry structural element. Four three-fold channels are formed by NTDs, and four other such channels are formed by CTDs. The overall arrangement of dFTN ferritin domains is similar to that of protomers in classic ferritin shells. Altogether, our findings expand the range of known ferritin family proteins and provide insight into Asgard archaea iron metabolism.

Eukaryotic translation initiation is tightly regulated by interactions among translation initiation factors (eIFs) that ensure accurate start codon selection. The translation regulator, eIF5 mimic protein 1 (5MP1) contributes to this process by competing with eIF5 for binding to eIF2, thereby increasing the stringency of translation initiation. Despite its important regulatory role and emerging involvement in tumorigenesis, structural information on human 5MP1 remains limited. Here, we report the near-complete backbone and partial side-chain NMR resonance assignments of the C-terminal domain of human 5MP1 (residues 250-419), carrying a W404E substitution that disrupts dimerization. The WT protein forms a dimer at NMR concentrations, which increases the effective size of the protein and also causes disappearance of peaks corresponding to aminoacids at the dimer interface due to conformational exchange. Backbone resonance assignments were completed for 96.4% of the non-proline residues. Secondary structure was analyzed using Chemical Shift Index (CSI) and compared with the AlphaFold structural model. Regions of disagreement between the experimental and computational secondary structure assignments were further examined using 15N-NOESY-HSQC spectra, allowing experimental validation of local structural features. While the AlphaFold model accurately reproduces the overall fold of the 5MP1 C-terminal domain, several localized discrepancies were identified, particularly near the N- and C-terminal regions of the domain, where experimental NMR data support alternative secondary structure assignments. These resonance assignments and experimentally validated structural features provide a foundation for future investigations of the molecular interactions, dynamics, and functions of 5MP1 in translation initiation.

BackgroundA hallmark of the eukaryotic cell is the regulated transport between the nucleus and cytoplasm, which is mediated by a multi-subunit protein assembly called the nuclear pore complex (NPC). While its overall architecture has been preserved across eukaryotes, the NPC structure varies in different organisms, which appears to have tuned its function. Outside of a handful of model systems, the NPC has not been comprehensively studied. This is particularly true of species that are not closely related to well-studied models, such as apicomplexan parasites. Indeed, the evolutionary divergence of Apicomplexa has complicated facile prediction of NPC proteins in these organisms. Because of this, the NPC components remain largely unidentified, and therefore NPC cellular function in Apicomplexa is poorly understood. Principal FindingsHere we identified, experimentally validated, and functionally characterized protein components of the NPC in the apicomplexan parasite Toxoplasma gondii. By combining proximity biotinylation with careful bioinformatic analysis, we identified 16 previously uncharacterized proteins that localize to the Toxoplasma NPC. We demonstrated 8 of these proteins are essential to parasite replication. Importantly, we defined components of the mRNA export machinery, as well as Nups required for the stability and/or assembly of specific NPC subcomplexes. Consistent with the evolutionary distance between Toxoplasma and well-studied models, the majority of our newly validated NPC components show no clear homology to NPC proteins in yeast, animals, or plants. Moreover, we demonstrated that the Toxoplasma mRNA export machinery has a distinct composition from other well-established systems. Intriguingly, Sus1, a well-defined protein of the TREX-2 and SAGA complexes, is missing from the Toxoplasma genome. In contrast, others, such as Centrin-3, have been conserved in Toxoplasma, but are not required for mRNA export in the parasite. ConclusionOur work highlights the distinct composition of multiple subcomplexes of the Toxoplasma NPC and paves the way for future studies to provide high-resolution structural information on the parasites unusual NPC architecture.

Cation-proton antiporters (CPAs) are vital for the maintenance of ionic homeostasis and normal physiology among diverse cell types. Despite recent insights into K+/H+ exchange transporters, the diversity in their structural organization and regulatory mechanisms of K+-specific CPAs are minimally understood. Here, we explore the architecture of an E. coli CPA1 K+/H+ antiporter, YcgO and its inhibition by the unphosphorylated form of PtsN, the terminal protein of a regulatory phosphorelay, using cryoEM structures at 3.4 [A] and 3.2 [A] resolution, respectively. Homodimeric YcgO bound to K+ ions in the occluded conformation, harbors additional linked cytosolic domains, RCK and CorC, to regulate the movement of the transport helices within the YcgO dimer. These domains are the sites of interaction and efflux inhibition by unphosphorylated PtsN, which interacts with the CorC domains with high affinity and allosterically augments inhibitory interactions of CorC with transport helices of YcgO. Inhibition is relieved leading to constitutive activation, upon disrupting the CorC-transport conduit interface. This study illuminates the structural basis of K+ efflux mediated through regulation of a K+/H+ antiporter in E. coli and related prokaryotes via a metabolic network involving a regulatory phosphorelay.

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron-sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD- and NAD(P)+-interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984_05830, which is annotated as a sporulation protein but possesses all six predicted cofactor-interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin-dependent reductase activity, with a wild-type kcat of 0.007 min-1--a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome-wide screen further revealed a repertoire of ferredoxin-like carriers, suggesting that C. pasteurianum distributes hydrogen-derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor-interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186-199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.

Background: Comparisons between independently predicted wild-type and missense-variant protein structures can generate mechanistic hypotheses, but small apparent differences may reflect model-selection variability rather than mutation-specific effects. Methods: Human mitochondrial DNA polymerase gamma (POLG; UniProt P54098) variants p.Arg627Gln (R627Q) and p.Trp748Ser (W748S) were evaluated using five AlphaFold2-PTM network-model outputs per condition generated with one random seed under matched ColabFold settings. Ten pairwise wild type comparisons at each site described between-network model-selection variability. Variant effects were summarized across five within-network wild-type-versus-variant comparisons using rotation-invariant local C-alpha pair distances and local displacement after global and local alignment. Because these comparison designs differ, the wild-type distribution was used as context rather than a mutation-effect null. Wild-type cryo-EM structure 9GGF was used for contact and interface mapping. Experimental A467T and G848S structures 9GGE and 9GGC provided contextual benchmarks. Results: R627Q measurements fell within the range of between-network wild-type differences: its median mean local pair-distance change was 0.170 angstrom, compared with a wild-type median of 0.170 angstrom, and its locally aligned displacement was 0.265 versus 0.248 angstrom. W748S showed higher median values (0.168 versus 0.132 angstrom for pair-distance change; 0.236 versus 0.182 angstrom for locally aligned displacement), but the ranges overlapped and the comparison-design asymmetry precluded a calibrated mutation-effect percentile. Experimental A467T and G848S comparisons produced local changes of similar magnitude. In 9GGF, R627 and W748 directly shared a local microenvironment, with a minimum heavy-atom distance of 3.53 angstrom. R627 also formed short polar-contact candidates with D629 and D743, whereas W748 occupied a hydrophobic packing environment containing Y622 and F750. Both sites were more than 18 angstrom from nucleic acid, more than 30 angstrom from POLG2, and more than 33 angstrom from PZL-A in a ligand-bound structure. Conclusions: Available AlphaFold2 comparisons do not establish a mutation-specific structural deformation for either variant. Experimental-structure mapping supports testable physicochemical hypotheses involving a shared R627-W748 microenvironment - loss of an arginine-centered polar network for R627Q and disruption of a buried aromatic environment for W748S - but not direct DNA, POLG2, or PZL-A contact mechanisms. Matched control substitutions and independent seeds are required to calibrate small mutation-associated structural deltas.

Motivation: Continuing advances in genome and metagenome sequencing expand the number of identified conserved protein families that remain functionally uncharacterized and contain domains of unknown function (DUFs). Functional-association resources such as STRING provide biological context, but mostly do not distinguish indirect association from physical interaction. We assessed whether AlphaFold 3 complex prediction, combined with STRING evidence and domain-level analysis of interfaces and interaction partners, can help identify and characterize DUF-containing proteins. Results: We generated four structural-prediction cohorts from STRING associations involving DUF-containing proteins and evaluated the predicted complexes using interface ipSAE, average pLDDT and buried surface area. An L2-regularized logistic regression model was trained on an initial cohort of predictions from high-confidence STRING associations to prioritize DUF-containing candidates likely to produce structurally confident AlphaFold 3 complexes. The model was then applied across all 12,535 organisms represented in STRING v12.0, followed by grouping into DUF-family and partner-architecture modules, covering 2,076 unique DUF families. The final L2-model screen contained 12,298 successfully modelled protein pairs, including 1,208 (9.82%) complexes meeting a strict-confidence criterion and 2,433 (19.78%) meeting a more liberal confidence criterion. Two examples suggest roles for DUF4130 in nucleic-acid-associated radical-SAM biology and DUF5819 in a bacterial system related to vitamin-K-dependent carboxylation. Availability and implementation: Predicted structures and associated metadata are available through Zenodo at https://doi.org/10.5281/zenodo.21875362. The model implementation and code used to generate the analyses and figures are available at https://github.com/linoriep/Proteome-scale-structure-prediction-of-DUF-containing-protein-protein-interactions.

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, reside within a specialized compartment known as the parasitophorous vacuole (PV) during their intracellular life cycle. The PV membrane (PVM), which derives from the host plasma membrane upon invasion, serves as a selective barrier that permits nutrient acquisition while shielding the parasite from host defense mechanisms. Although the protein composition of the PVM has been studied extensively, its lipid organization remains poorly understood. Using the quick-freeze, freeze-fracture replica labeling (QF-FRL) method, we quantitatively analyzed the transbilayer distribution of phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and GM3 ganglioside in the PVM of T. gondii and P. falciparum. Unlike host cell plasma membranes, where these lipids exhibit strict asymmetry--PtdSer and PtdEtn confined to the cytoplasmic leaflet and GM3 to the exoplasmic leaflet--we found that all three lipids were symmetrically distributed across both leaflets of the PVM. This striking loss of lipid asymmetry suggests that the PVM undergoes profound remodeling during infection. The presence of PtdSer and PtdEtn in the luminal leaflet may facilitate the binding of perforin-like proteins (PLP1s) during egress. These findings reveal a unique feature of the PVM that redefines our understanding of host-parasite membrane biology.

Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all--helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

Human cytochrome P450 2C9 (CYP2C9) is a hepatic microsomal enzyme involved in the oxidative metabolism of clinically important drugs, but the structural organization of its oligomeric assemblies outside crystallographic packing environments remains poorly understood. Here, we report the cryo-EM structure of human CYP2C9 determined under aqueous, membrane-free conditions at 3.31 Angstrom resolution. The structure reveals a C2-symmetric hexameric assembly organized as a dimer of trimers. Individual protomers retain the conserved P450 fold and heme-binding architecture observed in previously reported crystal structures, indicating that assembly formation does not substantially perturb the catalytic core. The hexamer is stabilized by defined intra-trimer interfaces involving the N-terminal region and residues around Trp212 and Phe482, together with inter-trimer interfaces involving Leu71 and the 220-227 loop. These interfaces are distinct from the crystal packing contacts observed in CYP2C9 crystal structures, demonstrating that the assembly is not a simple recapitulation of crystallographic packing. Notably, the inter-trimer interface is located near the FG-loop-containing surface previously implicated in membrane association. This suggests that the observed hexamer may represent a membrane-free association of two trimers through membrane-related surfaces, whereas the trimeric arrangement itself may be compatible with membrane-associated organization. The structure therefore provides a framework for investigating how trimer formation, membrane interaction and local conformational changes in the FG-loop region may influence CYP2C9 function.

Structural biology has advanced antimicrobial discovery by enabling drug-target identification and validation and supporting structure-guided inhibitor design. However, Mycobacterium leprae (M. leprae), the obligate intracellular bacillus that causes leprosy (Hansens disease), remains structurally under-characterised. Only 10 Protein Data Bank (PDB) entries represent seven unique proteins within a proteome encoded by 1,603 protein-coding genes. To address this gap, we present HANSEN (https://hansen-leprosy.medschl.cam.ac.uk/home), an integrated structural and functional resource containing computationally predicted three-dimensional models across the M. leprae proteome. Monomeric and oligomeric models were generated using complementary structure-prediction methods, including AlphaFold 3, Boltz-1, Chai-1, and Boltz-2. Models were annotated with predicted Local Distance Difference Test (pLDDT) scores and predicted aligned error (PAE) values. Ligand-binding pockets were predicted using AF2BIND, P2Rank, and fpocket, and ligands from the best-matching PDB templates were modelled within oligomeric complexes. Residue-level B-cell epitope propensity was estimated using DiscoTope-3.0, and ProteomeLM-derived essentiality scores were calculated for each protein. These features were integrated into a relational web database with interactive visualisation through Mol*. We also ranked all 1,603 proteins using a Target Priority Score ranging from 0 to 100. The score combines ProteomeLM-derived essentiality with pocket and AF2BIND predictions, functional annotations, and Boltz-2-associated measures of model quality and tractability. The essentiality model used a logistic-regression head trained on Mycobacterium tuberculosis (M. tuberculosis) Tn-seq labels. It achieved an AUROC of 0.84 in homology-grouped M. tuberculosis cross-validation and a transfer AUROC of 0.78 against the orthologue-aligned M. leprae reference set. Proteins were assigned to four tiers, ranging from high priority to exploratory candidates. Together, HANSEN provides a practical resource for generating and prioritising experimentally testable hypotheses for M. leprae target discovery and structure-guided drug development. TeaserPredicted structures and druggability annotations for the whole M. leprae proteome in an open resource. Key pointsO_LIHANSEN provides a proteome-wide structural resource for M. leprae, integrating predicted monomeric and oligomeric models with confidence metrics and functional annotations. C_LIO_LIHANSEN integrates UniProt annotations, ligand and cofactor associations, structure-based predictions of small-molecule binding pockets, B-cell epitope propensity, gene-essentiality estimates and multi-parameter target prioritisation within a single, protein-centric interface for the proteome of M. leprae. C_LIO_LIThe resource further incorporates a dedicated analytical module for Oxford Nanopore MinION amplicon-sequencing data, enabling the identification of mutations within drug-resistance-determining regions that confer antimicrobial resistance (AMR) in M. leprae. C_LIO_LIBenchmarking against available experimental structures, together with cross-method concordance analyses, supports the use of pLDDT, PAE and agreement between prediction methods as complementary indicators of model reliability. C_LIO_LIIntegrated target prioritisation produced a ranked set of candidate proteins, including established mycobacterial drug targets, to support experimental hypothesis generation for leprosy drug discovery. C_LI

Mosaic penA alleles encoding highly mutated variants of penicillin-binding protein 2 (PBP2) are the principal determinants of ceftriaxone resistance in Neisseria gonorrhoeae. Resistance-associated mutations in PBP2 from the ceftriaxone-resistant strain H041 restrict formation of the inward conformation of the {beta}3-{beta}4 loop associated with efficient acylation, but how {beta}-lactam recognition is coupled to this conformational switch is unknown. Because the conserved active-site residue Tyr422 interacts with the R1 substituent of {beta}-lactams, we investigated its role in coupling ligand recognition and acylation activity. Mutation of Tyr422 to Ala lowered acylation rates by up to 120-fold for cefoperazone and piperacillin, whereas acylation rates of ceftriaxone increased 4-fold. Unexpectedly, the crystal structure of the Y422A mutant acylated by ceftriaxone revealed that the {beta}3-{beta}4 loop had adopted the inward, high-activity conformation, despite position 422 being spatially distant from the loop. Transformation experiments showed that cell viability requires a tyrosine at position 422, indicating the residue is essential for transpeptidase function. Together, these findings reveal an energetic coupling between an active-site residue in PBP2 and a conformational switch whose equilibrium is altered by resistance mutations. The previously observed higher activity of {beta}-lactams containing extended R1 groups is consistent with stronger interactions with Tyr422 that favor the conformational switch. Molecular modeling suggests that such groups enhance activity by mimicking the iso-Glu region of the pentapeptide substrate. Overall, we propose that access to the high-activity state of PBP2 where the {beta}3-{beta}4 loop is inward is regulated by interactions between Tyr422 and {beta}-lactam R1 groups, and that resistance mutations function by tilting the balance toward a lower activity state.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

Macropinocytosis is an evolutionarily conserved process from unicellular eukaryotes to mammals. This process relies on actin-rich protrusions beneath the plasma membrane to form a macropinocytic cup. Traditionally, V ATPase has been best studied for its role in late endosomal acidification. However, the specific roles of V-ATPase interaction with cytoskeletal machinery and its crosstalk with lipids in the formation of macropinocytic cups have not been investigated. Here, we uncover an unprecedented role of V-ATPase in shaping the macropinocytic cup in the pathogenic amoeba Entamoeba histolytica. Our results showed that the Entamoeba V-ATPase complex is associated with F-actin at macropinocytic cups. Moreover, we found that the V-ATPase complex is dynamically recruited to the macropinocytic cup and dissociates from the macropinosome. We further demonstrated that the V-ATPase V1B subunit directly binds to actin and uniquely promotes actin polymerization. The pH biosensor phosphatidic acid, revealed abundant V-ATPase complex and cytoskeleton associated proteins in its interactome. Surprisingly, the V1B subunit showed reproducible binding to phosphatidic acid and this interaction modulates actin polymerization. Collectively, our work highlights the novel role of V-ATPase in directly driving actin polymerization, in conjunction with phosphatidic acid, to shape the macropinocytic cup.