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.
Aconitate decarboxylase 1, an enzyme member of the MmgE-PrpD family of proteins, has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases. Its product, itaconate, is a multifunctional metabolite shown to drive several disease states. Though extensively studied in cellulo and in vivo, this protein is biochemically and mechanistically under characterized and although a family of inhibitors has been described, no ligand-bound structures have yet been determined. In this work we present a thorough structural investigation that yielded the first ligand-bound structure of this protein family, which required the generation of artifact-free apo crystals. We also developed a novel, low-consumption, robust kinetic assay and investigated active site and allosteric mutants to further elucidate structural and dynamic activity relationships of this protein.
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/Mg2+ 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 (epiIV), thereby exposing two established WASP ubiquitylation sites. Single-residue WAS-inducing mutations with mild phenotypes all influence the same WASP-epiIV 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.
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.
Allosteric regulation in biosynthetic L-threonine deaminase (BTD) has long been attributed to effector-induced conformational changes. Here, we identify a trans-interface coupling mechanism that regulates the active site architecture of EcIlvA, the BTD homolog of Escherichia coli. Starting from a low-activity, regulatory-domain-truncated variant (EcIlvA1-335, termed CDL) that retains only the catalytic domain and the native linker region, we combined computational screening with experimental selection and directed evolution to identify a variant (designated HSG) bearing three mutations in the linker region (H322L, S328L, G334L). These mutations restore enzymatic activity to approximately two-thirds of the full-length EcIlvA level. Size-exclusion chromatography experiments suggested that the truncated CDL variant is dimeric in solution, whereas the HSG mutations restore the tetrameric form of the full length native protein. High-resolution crystal structures of CDL and HSG reveal two distinct functional states. They demonstrate that substitutions at the monomer-monomer interfaces within the dimer-of-dimers tetrameric assembly trigger inter-subunit rotation, propagating conformational changes across subunits and remodeling interactions at distal dimer-dimer interfaces. This allosteric cascade stabilizes a tetrameric assembly that hosts an open, catalytically competent active site. Molecular dynamics simulations further reveal that tetramerization in this form enhances global structural rigidity while retaining essential flexibility in the substrate-binding loops. Collectively, our findings establish that trans-interface-coupling within the dimer-of-dimers architecture of the tetrameric enzyme acts as a key allosteric mechanism in EcIlvA, providing direct structural and dynamic evidence for quaternary structure-driven allostery in a classical metabolic enzyme.
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.
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.
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 31P 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.
Accurate determination of ligand structures in protein-ligand complexes is essential for elucidating molecular recognition mechanisms and advancing structure-based drug discovery. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful technique for determining macromolecular structures; however, reliable identification of small-molecule ligands from cryo-EM maps remains challenging, particularly in the absence of accurate initial ligand models. Here, we present MLAC (MicroED-assisted Ligand structure Analysis in Complexes), an integrative framework that combines microcrystal electron diffraction (MicroED) with cryo-EM single-particle analysis (SPA). In MLAC, high-resolution ligand structures determined by MicroED from submicrometer-sized crystals are used as initial models for fitting into cryo-EM maps of protein-ligand complexes. As a proof of concept, previously reported hERG-ligand complexes were reanalyzed. MicroED structures of representative hERG ligands-astemizole, pimozide, and E-4031-were determined at resolutions of 0.66-0.92 Å and then used for model fitting and refinement. Several quantitative metrics, including Q-score, atom inclusion, model-to-map correlation coefficients, clash analysis, and Mogul analysis, together with visual inspection, indicated that MicroED-derived ligand structures can facilitate ligand modeling for astemizole and, to a lesser extent, pimozide, whereas no clear advantage was observed for E-4031. Notably, MicroED frequently revealed structural polymorphs that provided alternative ligand conformations and helped resolve modeling ambiguities, including the chair-boat conformational variability of the piperidine ring and alternative ligand placements in the hERG-astemizole complex. Collectively, these findings support MLAC as a proof-of-concept framework that provides experimentally determined starting models to complement computational ligand-generation approaches.
Bacteroides thetaiotaomicron ( B. theta ) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other Bacteroides , B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated “lid” lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named “utilisomes”), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCD dex core and an associated glycoside hydrolase (GH dex ) and surface glycan binding protein (SBGP dex ). Via X-ray crystallography we solved high-resolution structures of SBGP dex in isolation and SusD dex and GH dex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusC dex , SusD dex and GH dex , with substrate observed in both open and closed states of SusD dex . 3D variability analysis showed a minority of particles in the process of SusD dex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.
Omega-3 polyunsaturated fatty acids (PUFAs) are essential nutrients for humans and are synthesized de novo by specialized enzymes known as PUFA synthases (Pfas). The domains of these enzymes are structurally related to those of mammalian or bacterial fatty acid synthases (FAS), as well as microbial polyketide synthases (PKS). Pfas are typically composed of three polypeptides in thraustochytrids and myxobacteria, or four in marine gammaproteobacteria. The enoyl-ACP reductase (ER) domain plays a key role in PUFA synthesis by catalyzing the reduction of carbon‑carbon double bonds during modification reactions. In gammaproteobacteria, a single ER domain is present as a standalone protein (PfaD) within the megasynthase. However, in thraustochytrids ER domains are found in both PfaB (ERb) and PfaC (ERc), although their specific functional roles remain unclear. Previous studies have shown that ER domains act as homodimers in Pfas, FAS, and PKS systems. Here, we investigate the PUFA synthase from the thraustochytrid Schizochytrium sp. and demonstrate that ERb and ERc interact to form a heterodimer, as confirmed by size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and by a crystal structure solved at 2.2 Å resolution with bound flavin mononucleotide (FMN). These findings indicate that ER domains may facilitate dimerization between PfaB and PfaC. Furthermore, molecular docking and structural alignments support a ping-pong mechanism involving FMN and NADH for ERb and ERc activity. To our knowledge, this is the first reported crystal structure of a PUFA synthase ER-domain complex from thraustochytrids, providing new insights into the mechanism of action of these enzymes.
Paenibacillus sp. str. FPU-7 (P. FPU-7), a chitinolytic bacterium, efficiently degrades chitin and uses the solute-binding proteins (SBPs) NagB1 and NagB2 on the cell surface to facilitate the uptake and intracellular transport of chitooligosaccharides. SBPs are essential components of the bacterial carbohydrate transport system and play key roles in carbon source acquisition. However, they exhibit diverse substrate specificities with ligands yet to be identified. Predicting SBP ligands solely from amino acid sequences remains a significant challenge. In addition to encoding NagB1 and NagB2, the genome of P. FPU-7 encodes several SBPs that are potentially involved in carbohydrate import. In the present study, we identified a novel SBP, designated PsMBP, in the P. FPU-7 genome and confirmed its mRNA expression. Genes adjacent to psmbp encode transmembrane domains, suggesting that PsMBP functions as part of an ABC transporter complex. We also characterized the sugar-binding specificity of PsMBP using biochemical analysis. PsMBP exhibited binding affinities for various α-glucosaccharides, including maltose, trehalose, isomaltose, sucrose, maltotriose, and maltotetraose. In particular, it showed high binding affinity for both maltose and trehalose. Furthermore, we determined the crystal structure of PsMBP in its ligand-free form and in complexes with different saccharides at resolutions of 1.4-2.1 Å. The structures revealed the molecular basis for α-glucosaccharide recognition by PsMBP. Overall, our findings advance the understanding of bacterial carbohydrate transport mechanisms and provide a foundation for developing efficient transport systems and new microbial biotechnological applications.
OprM is the outer membrane channel component of resistance-nodulation-division (RND) efflux pumps in Pseudomonas aeruginosa, a high-priority human pathogen. OprM-dependent pumps play diverse roles in bacterial physiology and mediate resistance to critical antibiotic classes, including fluoroquinolones, aminoglycosides, and broad-spectrum β-lactam/β-lactamase inhibitor combinations such as ceftazidime/avibactam. As the outer membrane component of the tripartite efflux system, OprM gates substrate export through an iris-like periplasmic pore that opens upon association with its inner membrane partners. Here we present a single-particle cryo-EM structure of OprM at 2.08 Å, the highest resolution OprM structure to date. The structure reveals a closed leucine-lined periplasmic pore, consistent with previous X-ray structures. A detailed structure-based comparison of the closed and open states reveals the conformational changes accompanying periplasmic pore opening, including iris-like twisting of the coiled-coil helices, displacement of the gating Leu429 residues, and a broader reorganization of salt bridges than previously described. Conservation of the MexA-binding interface across multiple Mex proteins suggests a shared mechanism of OprM engagement. Three lipopolysaccharide (LPS) molecules are resolved at the β-barrel interface, bridging adjacent OprM monomers through a combination of hydrophobic interactions with the fatty acyl chains and specific polar contacts with the KDO, heptose, and phosphate groups. This binding mode resembles that of other generic β-barrels and is expected to be preserved in vivo. Together, these findings provide the most detailed structural characterization of OprM to date, offering new insights into its gating mechanism and membrane interactions with implications for the development of efflux pump inhibitors.
Henneguya piaractus is a myxozoan parasite infecting the gills of Piaractus mesopotamicus, yet its cellular biology remains poorly understood. Here, we investigated mitochondrial organization and the occurrence of autophagy-related processes using an integrated approach combining confocal laser microscopy and transmission electron microscopy. SSU rDNA sequencing (1546 bp) confirmed species identity, showing 99.7% similarity to available H. piaractus sequences. Confocal microscopy revealed clear labeling of nuclei, polar capsules, and valves, whereas no signal indicative of mitochondrial activity was detected in mature myxospores. Ultrastructural analysis showed a plasmodium surrounded by a single membrane with numerous pinocytotic channels and mitochondria with well-developed cristae in the ectoplasmic region. Sporogenesis occurred asynchronously at the periphery, where sporoblasts and immature myxospores were observed. This region also exhibited double-membrane vesicles consistent with autophagosome-like structures, as well as phagophore-like membranes associated with damaged mitochondria. In addition, mitochondria-endoplasmic reticulum contact sites (MERCs) were identified. In contrast, centrally located mature myxospores contained mitochondria lacking cristae. Together, these findings indicate stage-dependent mitochondrial remodeling and suggest an autophagy-related process, possibly involving mitochondrial degradation. However, as these observations are based primarily on morphological evidence, the involvement of canonical autophagy pathways requires further molecular confirmation. This study provides novel insights into organelle dynamics in H. piaractus and contributes to a better understanding of cellular adaptations in myxozoan parasites.
