AcrB is a member of the resistance-nodulation-cell division (RND) family of efflux transporters expressed in Escherichia coli, and it plays a crucial role in antimicrobial resistance. AcrB has been structurally characterized in a variety of states through observations using diverse methods, and by applying conditions that have not yet been explored, there is potential to reveal new structural features. In this study, we performed cryo-electron microscopy (cryo-EM) structural analysis of AcrB reconstituted into liposomes in the presence of the substrate doxorubicin (DOX). As a result, both the functional LTO state and the resting LLL state were observed on the same EM grid. In the LTO state, substrate binding was observed in the deep binding pocket of the T protomer, whereas the LLL state could be further classified into multiple subclasses. This study not only provides additional structural insight into AcrB but also highlights the importance of observing membrane proteins under a variety of conditions.

l-lysine 6-dehydrogenase (LysDH; EC 1.4.1.18) oxidatively deaminates the ε-amino group of l-lysine. Due to its high substrate specificity, LysDH serves as a valuable tool for l-lysine quantification. However, the molecular basis of this specificity has remained unclear because of the lack of substrate-bound structures. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of LysDH from the thermophilic bacterium Geobacillus stearothermophilus (GstLysDH) in the apo form at 2.9 Å resolution and in complex with NAD[+] and l-lysine at 2.5 Å resolution. GstLysDH assembles as a tetramer, which undergoes a global conformational transition upon NAD[+] binding. Structural analysis revealed that the α-carboxyl and α-amino groups of l-lysine were coordinated by oppositely charged residues, thereby orienting the ε-amino group toward the nicotinamide ring of NAD[+] and anchoring the substrate in the optimal binding mode. This precise recognition mechanism accounts for the enzyme's strict specificity for the ε-amino group of l-lysine. Furthermore, comparative structural analysis with L-phenylalanine dehydrogenase suggests that the oxidative deamination in GstLysDH proceeds through a conserved hydride transfer mechanism. Together, these insights establish a structural framework for the rational design and industrial application of LysDH and related amino acid dehydrogenases.

Co-translational membrane insertion is essential for the efficient integration of mitochondrially encoded proteins into the inner mitochondrial membrane (IMM) and is critical for respiratory chain biogenesis. Mba1 is a mitochondrial ribosome-associated protein implicated in coupling mitochondrial translation with inner-membrane protein biogenesis, but its structural basis of function remains poorly understood. Here, we determined the solution structure of mature Saccharomyces cerevisiae Mba1 (mMba1) using multidimensional nuclear magnetic resonance (NMR) spectroscopy. The structure reveals a compact α + β fold with a central hydrophobic cavity and distinct charged surface regions. Ribosome titration, paramagnetic relaxation enhancement, and Cox2-derived peptide titration identified several regions of mMba1 that are affected by these different interaction conditions. Mapping these regions onto the structure reveals spatially distinct surfaces that may contribute to ribosome association, membrane proximity, and interactions with hydrophobic peptide segments. These findings provide a structural framework for interpreting previous functional studies of Mba1 and support a working model in which Mba1 may function as a peripheral adaptor at the mitoribosome-inner membrane interface. Further structural and biochemical studies will be required to establish the molecular mechanisms underlying these interactions.

Catalases are key antioxidant enzymes that protect aerobic organisms from reactive oxygen species generated through metabolic activities and host immune responses. In Escherichia coli, the monofunctional HPII catalase (encoded by katE) forms a stable tetrameric enzyme that contributes to oxidative stress resistance. During the purification of a recombinantly produced construct of the Vibrio cholerae FrhA adhesin, we unexpectedly observed a co-purifying protein species which peptide mass spectrometry identified as E. coli HPII catalase. Initial reconstruction was limited by severe preferred orientation, with ∼98% particles adopting a dominant view that produced anisotropic reconstructions. We evaluated multiple processing strategies to mitigate this bias and found that the recently reported high-resolution ab initio reconstruction workflow provided the most effective improvement in angular sampling, yielding a 3.2 Å reconstruction with D2 symmetry. The resulting map enabled atomic model building, and comparison with existing crystallographic models showed strong overall agreement while revealing modest differences in tetrameric dimensions and surface-exposed regions that may reflect reduced lattice constraints in the cryo-EM structure. Together, this work reports the first cryo-EM structure of the E. coli HPII catalase serendipitously co-purified with an adhesin and provides a practical workflow for overcoming severe preferred orientation in single-particle cryo-EM analysis.

Encapsulins are self-assembling protein nanocompartments found in bacteria and archaea that encapsulate cargo enzymes to protect the cell from their toxic reaction products or intermediates. Developments in cryo-electron microscopy (cryo-EM) data processing strategies have enabled encapsulins and their cargo proteins to be investigated together in greater detail. In this study, we present the single particle cryo-EM structure of the Rhodospirillum rubrum encapsulin in both the presence and absence of its partner encapsulated ferritin (EncFtn). Single particle icosahedral reconstructions of empty and loaded encapsulins revealed a higher degree of conformational flexibility at the five-fold pore in the cargo loaded encapsulin. We applied a new non-point group averaging workflow to analyze the encapsulated ferritins within the encapsulin nanocompartment, to produce the first fully refined in situ atomic model of the EncFtn at 2.8 Å resolution. Masked 2D classification and particle subtraction demonstrate that cargo loading is heterogeneous in this recombinant complex, with the encapsulin able to house up to five of the decameric EncFtn complexes. Our data provides new insights into the dynamics and cargo arrangement in encapsulins and demonstrates an adaptable workflow for high resolution reconstruction of encapsulin cargoes.

Rio Kinase 1 (RioK1) is an anti-cancer target for colorectal cancer. In pursuit of selective inhibitors of RioK1, small drug-like molecules were identified using computer-aided drug design (CADD). CADD made use of a 3D crystal structure of human RioK1 bound to ADP/Mg[2+] and a fragment-based computational method termed Site-Identification by Ligand Competitive Saturation (SILCS). Compounds identified via SILCS were selected based on predicted binding affinities and were experimentally confirmed to bind a RioK1 homolog from Archaeoglobus fulgidus via biophysical methods. One newly designed scaffold molecule, KPSH02, had its X-ray crystal structure determined in complex with afRioK1. The structure confirmed that KPSH02 occupies the adenine binding region seen in the Toyocamycin-afRioK1 structure, while also occupying the divalent metal-ion site observed in the hsRioK1-ADP structure. This structure thereby provides novel insights that may be exploited for the design of selective RioK1 inhibitors that may be useful in the future for targeting RioK1 in cancer.

The complex formed between Wiskott-Aldrich syndrome protein (WASP) and WASP-Interacting Protein (WIP) is a potent regulator of cytoskeletal changes in hematopoietic cells. Mutations in the WASP N-terminal domain cause the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Using NMR we determine the structure of the WASP/WIP complex and provide a first molecular view of this key biochemical junction. The central feature of this complex is the extensive binding interface formed by four WIP epitopes that wrap around the canonical EVH1 domain. Phosphoregulation of the WIP chaperone function occurs on two tyrosine residues, and not a distal serine residue as suggested earlier, and involves selective dissociation of the fourth epitope (epi[IV]), thereby exposing two established WASP ubiquitylation sites. Single-residue WAS-inducing mutations with mild phenotypes all influence the same WASP-epi[IV] interface, suggesting this is the molecular mechanism behind WAS. This structural viewpoint of WASP/WIP biology creates a much-needed molecular context for understanding hematopoietic cytoskeletal regulation in homeostasis and in WAS/XLT and is expected to be invaluable in the search for new therapeutic approaches to these rare diseases.

Solute carrier proteins (SLCs) are essential membrane protein transporters of small solutes. Among them, the SLC12 family is known to facilitate transport of ions. Members of the SLC12 family ensure cell homeostasis by co-transporting chloride alongside sodium and/or potassium across the plasma membrane. The majority of SLC12 proteins are well described, and a recent surge in structural studies facilitated by cryo-electron microscopy revealed molecular details of their function. These include multiple conformations of the transporters, covering a range of functional states and providing a window into the ion transport mechanism. Yet, only limited knowledge exists regarding their dimerization or higher order oligomerization and its role in regulation, despite SLC12 proteins consistently operating as dimers. In this review, we highlight the structural knowledgebase established in recent years and summarize the varying dimerization mechanisms. Altogether, it is becoming increasingly clear that large conformational changes in dimeric arrangements deserve attention, alongside other understudied areas like lipid interactions, nucleotide or N-terminal binding to the dimerization domains, and potential roles of the less described member SLC12A9.

AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH predominantly forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AFG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function.

DNA methyltransferase DNMT3A is a key enzyme responsible for establishing DNA methylation patterns during mammalian development. T-cell leukemia/lymphoma 1 A (TCL1A) is a proto-oncogene expressed mainly in embryonic and fetal tissues, as well as in specific lymphocyte populations. In this study, we determined the structure of the murine DNMT3A-TCL1A complex using single-particle cryo-electron microscopy. The complex adopts a linear conformation, with two TCL1A dimers bound to the catalytic domain of DNMT3A to form a heterohexamer. TCL1A competitively binds to the same structural interface on DNMT3A as DNMT3L, but produces an inhibitory-rather than an activating-effect on the catalytic activity of DNMT3A. Furthermore, comparative analysis with previously reported assembly modes of murine TCL1A revealed that the TCL1A dimer complex we resolved adopts distinct molecular conformations and interaction mechanisms. Our findings elucidate the allosteric mechanism by which murine TCL1A inhibits DNMT3A activity, providing a structural basis for understanding mammalian epigenetic reprogramming.

Kolaviron, flaunted for its anticancer potential, has driven our exploration of its mechanism of interaction with DNA, and further shedding light on its therapeutic promise. Herein, the binding efficacy and thermodynamics of kolaflavanone, a component of kolaviron, with double-stranded calf thymus DNA (ctDNA), were investigated using a range of spectroscopic techniques and molecular docking simulations. Syllogistically, spectroscopic techniques, viscometric analysis, and thermal denaturation revealed that kolaflavanone binds to the double-stranded ctDNA minor groove via electrostatic interactions, with binding constants of 10[5] L·mol[-1]. The interaction was both enthalpically and entropically driven, with a ΔH value of -18.16 kJ/mol and a ΔS value of 38.60 J/mol/K at 25 °C. This resulted in subtle but negligible structural perturbations of the ctDNA native B-conformation. ctDNA has one binding site for the flavonoid. Molecular docking and dynamics simulation analyses confirmed that kolaflavanone binds to the minor groove of DNA with a preference for the A-T region and a binding affinity of -7.4 kcal/mol. The conjugate's stability, conferred by minor groove binding and non-intercalative interactions, suggests a promising strategy for designing DNA-targeted therapeutics with enhanced stability and tumor-specific gene regulation potential.

Electrostatic interactions between arginines and phosphates are central to numerous biological processes. Here, using an integrated approach combining mutagenesis, activity measurements, molecular dynamics (MD) simulations, and NMR, we demonstrate that arginines present in the C-terminal domain of Biotin protein ligase (BPL) are critical for biotinyl-5'-AMP formation. Using NMR-based assays and a group I BPL from Leishmania major (LmBPL), we selectively monitored the first biotinylation step, i.e., formation of biotinyl-5'-AMP from biotin and ATP. The distinct chemical shifts of ATP and AMP enabled us to quantitatively measure the amount of biotinyl-5'-AMP formed by the wild-type enzyme and a C-terminal domain deletion mutant. The mutant displayed remarkably low biotinyl-5'AMP formation compared to the wild-type enzyme. MD simulations of the apo- and ATP-bound forms of LmBPL further identified key interactions between the C-terminal domain arginines (R224, R229) and the γ-phosphate of ATP. The in silico predictions were validated by biochemical studies using R224A, R229A, and R224A/R229A mutants, which displayed remarkably lower biotinyl-5'-AMP formation compared to the wild-type enzyme. Using pyrophosphate as a γ-phosphate mimic, and [31]P NMR as a probe, we demonstrate pyrophosphate binding to the wild-type LmBPL but not to the arginine mutants. Consistent with this, biotinyl-5'-AMP formation was completely inhibited by preincubation with pyrophosphate. Taken together, our findings establish a critical role for the C-terminal domain arginines in recognizing ATP phosphates during biotinylation. Extrapolating these findings to other group I and bifunctional group II BPLs, our study reveals a broadly conserved role for the C-terminal domain arginines in regulating biotinylation across the BPL family.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas-cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling and might be particularly beneficial in BSL-3 or BSL-4 laboratories where minimizing physical presence is essential for biosafety reasons.

The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We inserted the soluble protein AmpC β-lactamase into the third intracellular loop (ICL3) of ultra-thermostable β1-adrenoceptor (β1AR) via chimeric helix fusions. Biochemical and biophysical characterization showed that the resulting fusion protein after expression, solubilization and purification was monodisperse and able to bind the known β1AR weak partial agonist cyanopindolol, and the antagonist propranolol. The protein particles comprised sufficient mass and discernable structural features to elucidate its cryo-EM structure in complex with cyanopindolol without any natural (G-proteins, arrestins) or artificial (Nanobodies, DARPins) binding partners, to an overall resolution of 4.2 Å. The seven-helix architecture and helix eight, as well as both GPCR - AmpC β-lactamase connections are clearly resolved. β1AR is in an inactive-like conformation. 3D variability analysis revealed significant flexibility between the two protein domains and within the GPCR helices. The map contains clear density for the cyanopindolol. The fusion protein geometry is expected to be compatible with a subset of other class A GPCRs exhibiting suitable architecture. For receptors meeting these geometric requirements, this approach may facilitate cryo-EM structure determination of GPCR-ligand complexes in an inactive-like state. In addition, it could support structural studies of GPCRs in the absence of ligands.

Myriapora truncata and Smittina cervicornis, two common Bryozoa in the Mediterranean Sea form erect calcitic colonies with similar ultrastructures characterised by platy and granular calcite grains. At the submicron scale, the mineralized parts of the colonies show abundant organic inclusions and irregular calcite grains with undulating diffraction contrasts, indicating that crystallization progressed via particle attachment. High Resolution Electron Diffraction analyses show areas consisting of amorphous calcium carbonate (ACC) in Myriapora truncata colony walls. In Smittina cervicornis colony walls we identified calcite III in micrometre-sized areas adjacent to the organic cuticula in otherwise calcitic walls. Calcite III is a CaCO3 polymorph stable at high pressures and its occurrence at ambient conditions here suggests that it is a precursor to calcite along a stepwise crystallization pathway, stabilized by organic molecules. Our results establish that Bryozoa, like many other marine calcifiers, form their colonies via stepwise crystallization pathways rather than crystallizing calcite directly from seawater. Mg/Ca and U/Ca ratios measured in situ along the growth axis of the colony display regular peaks and troughs which could be interpreted as seasonal temperature signals and translate to growth rates of 1 to 2 mm per year for the measured branch. We suggest that these data represent a proof of concept and illustrate that Mg/Ca and U/Ca ratios could be developed into a reliable method to be added to the toolkit for measuring colony growth rates in Bryozoa.