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Infection by enterotoxigenic Escherichia coli (ETEC) causes severe watery diarrhea and dehydration in humans. Heat-labile enterotoxin (LT) is a major virulence factor produced by ETEC. LT is one of AB 5 -type toxins, such as Shiga toxin (Stx) and cholera toxin (Ctx), and the B-subunit pentamer is responsible for high affinity binding to the LT-receptor, ganglioside GM1, through multivalent interaction. In this report, we found that Glu51 of the B-subunit plays an essential role in receptor binding compared with other amino acids, such as Glu11, Arg13, and Lys91, all of which were previously shown to be involved in the binding. By targeting Glu51, we identified four tetravalent peptides that specifically bind to the B-subunit pentamer with high affinity by screening tetravalent random-peptide libraries, which were tailored to bind to the B-subunit through multivalent interaction. One of these peptides, GGR-tet, efficiently inhibited the cell-elongation phenotype and the elevation of cellular cAMP levels, both induced by LT. Furthermore, GGR-tet markedly inhibited LT-induced fluid accumulation in the mouse ileum. Thus, GGR-tet represents a novel therapeutic agent against ETEC infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and β-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and β-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and β-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the β-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and β-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-Å resolution structure of complex I from the α-proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies., (© 2024. The Author(s).)

During DNA replication, core histones that form nucleosomes on template strands are evicted and associate with newly synthesized strands to reform nucleosomes. Mcm2, a subunit of the Mcm2-7 complex, which is a core component of the replicative helicase, interacts with histones in the amino-terminal region (Mcm2N) and is involved in the parental histone recycling to lagging strands. Herein, the interaction of Mcm2N with histones was biochemically analyzed to reveal the molecular mechanisms underlying histone recycling by Mcm2N. With the addition of Mcm2N, a histone hexamer, comprising an H3-H4 tetramer and an H2A-H2B dimer, was excised from the histone octamer to form a complex with Mcm2N. The histone hexamer, but not H3-H4 tetramer was released from Mcm2N in the presence of Nap1, a histone chaperone. FACT, another histone chaperone, stabilized Mcm2N-histone hexamer complex to protect from Nap1-dependent dissociation. This study indicates cooperative histone transfer via Mcm2N and histone chaperones., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

We developed a method for ex vivo receptor encapsulation and single-molecule imaging techniques from neuronal and cardiac tissues, illustrating the method's broad applicability for measuring membrane receptor assembly. Ryanodine receptor 2 (RyR2) is a tetrameric Ca 2+ channel governing intracellular Ca 2+ dynamics, which is critical for muscle contraction. Employing GFP-RyR2 knock-in mice, we isolated individual receptor proteins in tissue-specific nanovesicles and performed subunit counting analyses to yield quantitative assessment of stoichiometric distributions across different organs. With this method, we explored the potential heterogeneity of brain-derived RyR2, which has been reported to form heteromeric assemblies with other ryanodine receptor isoforms.

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Förster resonance energy transfer (FRET) microscopy for decades. In this work, we show that MINFLUX fluorescence nanoscopy measures intramolecular distances down to 1 nanometer-and in planar projections down to 1 angstrom-directly, linearly, and with angstrom precision. Our method was validated by quantifying well-characterized 1- to 10-nanometer distances in polypeptides and proteins. Moreover, we visualized the orientations of immunoglobulin subunits, applied the method in human cells, and revealed specific configurations of a histidine kinase PAS domain dimer. Our results open the door for examining proximities and interactions by direct position measurements at the intramacromolecular scale.

Ferritin is a 24-mer protein nanocage that stores iron and regulates intracellular iron homeostasis. The nuclear receptor coactivator-4 (NCOA4) binds specifically to ferritin H subunits and facilitates the autophagic trafficking of ferritin to the lysosome for degradation and iron release. Using isothermal titration calorimetry (ITC), we studied the thermodynamics of the interactions between ferritin and the soluble fragment of NCOA4 (residues 383-522), focusing on the effects of the recently identified FeS cluster bound to NCOA4, ferritin subunit composition, and ferritin-iron loading. Our findings show that in the presence of the FeS cluster, the binding is driven by a more favorable enthalpy change and a decrease in entropy change, indicating a key role for the FeS cluster in the structural organization and stability of the complex. The ferritin iron core further enhances this association, increasing binding enthalpy and stabilizing the NCOA4-ferritin complex. The ferritin subunit composition primarily affects binding stoichiometry of the reaction based on the number of H subunits in the ferritin H/L oligomer. Our results demonstrate that both the FeS cluster and the ferritin iron core significantly affect the binding thermodynamics of the NCOA4-ferritin interactions, suggesting regulatory roles for the FeS cluster and ferritin iron content in ferritinophagy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

l-threonine aldolase (LTA) catalyzes the synthesis of β-hydroxy-α-amino acids, which are important chiral intermediates widely used in the fields of pharmaceuticals and pesticides. However, the limited thermostability of LTA hinders its industrial application. Furthermore, the trade-off between thermostability and activity presents a challenge in the thermostability engineering of this enzyme. This study proposes a strategy to regulate the rigidity of LTA's V-shaped subunit by modifying its opening and hinge regions, distant from the active center, aiming to mitigate the trade-off. With LTA from Bacillus nealsonii as targeted enzyme, a total of 25 residues in these two regions were investigated by directed evolution. Finally, mutant G85A/M207L/A12C was obtained, showing significantly enhanced thermostability with a 20 °C increase in T 50 60 to 66 °C, and specific activity elevated by 34 % at the optimum temperature. Molecular dynamics simulations showed that the newly formed hydrophobicity and hydrogen bonds improved the thermostability and boosted proton transfer efficiency. This work enhances the thermostability of LTA while preventing the loss of activity. It opens new avenues for the thermostability engineering of other industrially relevant enzymes with active center located at the interface of subunits or domains., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

T cells in jawed vertebrates comprise two lineages, αβ T cells and γδ T cells, defined by the antigen receptors they express-that is, αβ and γδ T cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring that γδ TCRs recognize more structurally diverse ligands 1 . Nevertheless, the receptors use shared CD3 subunits to initiate signalling. Whereas the structural organization of αβ TCRs is understood 2,3 , the architecture of γδ TCRs is unknown. Here, we used cryogenic electron microscopy to determine the structure of a fully assembled, MR1-reactive, human Vγ8Vδ3 TCR-CD3δγε 2 ζ 2 complex bound by anti-CD3ε antibody Fab fragments 4,5 . The arrangement of CD3 subunits in γδ and αβ TCRs is conserved and, although the transmembrane α-helices of the TCR-γδ and -αβ subunits differ markedly in sequence, packing of the eight transmembrane-helix bundles is similar. However, in contrast to the apparently rigid αβ TCR 2,3,6 , the γδ TCR exhibits considerable conformational heterogeneity owing to the ligand-binding TCR-γδ subunits being tethered to the CD3 subunits by their transmembrane regions only. Reducing this conformational heterogeneity by transfer of the Vγ8Vδ3 TCR variable domains to an αβ TCR enhanced receptor signalling, suggesting that γδ TCR organization reflects a compromise between efficient signalling and the ability to engage structurally diverse ligands. Our findings reveal the marked structural plasticity of the TCR on evolutionary timescales, and recast it as a highly versatile receptor capable of initiating signalling as either a rigid or flexible structure., (© 2024. The Author(s).)

The 2-oxoglutarate dehydrogenase complex (OGDHc) orchestrates a critical reaction regulating the TCA cycle. Although the structure of each OGDHc subunit has been solved, the architecture of the intact complex and inter-subunit interactions still remain unknown. Here we report the assembly of native, intact OGDHc from Sus scrofa heart tissue using cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and subtomogram averaging (STA) to discern native structures of the whole complex and each subunit. Our cryo-EM analyses revealed the E2o cubic core structure comprising eight homotrimers at 3.3-Å resolution. More importantly, the numbers, positions and orientations of each OGDHc subunit were determined by cryo-ET and the STA structures of the core were resolved at 7.9-Å with the peripheral subunits reaching nanometer resolution. Although the distribution of the peripheral subunits E1o and E3 vary among complexes, they demonstrate a certain regularity within the position and orientation. Moreover, we analyzed and validated the interactions between each subunit, and determined the flexible binding mode for E1o, E2o and E3, resulting in a proposed model of Sus scrofa OGDHc. Together, our results reveal distinctive factors driving the architecture of the intact, native OGDHc., (© 2024. The Author(s).)

Bacterial cellulose (BC) is the glucose polymer produced by bacterial metabolism. The bacterial cellulose synthase (BCS) is the key enzyme for catalyzing the formation of BC. The cooperation between different submits of BCS is necessary for the intracellular formation and extracellular secretion of BC. This review summarized the BC-producing strains and the differences of BCS among different strains. Furthermore, we detailed the BC synthesis mechanism, the interactions between BCS subunits, and the relationship between the structural characteristics of strains and the formation of highly ordered fiber structures. A comprehensive insight into the mechanism of BC synthesis and secretion will supply more strategies for optimizing the BC synthesis via methods of synthetic biology.

Type-A γ-aminobutyric acid (GABA A ) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABA A receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor's distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABA A receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABA A receptor α1, β2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and β2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABA A receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits' modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABA A receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases.

The yeast pre1-1 (β4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with β2- and β5-propeptides, which together fill most of the antechambers between the α- and β-rings. The β5-propeptide is unprocessed and separates Ump1 from β6 and β7. The β2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and β3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, β2 is activated first enabling processing of β1-, β6-, and β7-propeptides. Subsequent maturation of β5 and β1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2., (© 2024 Mark et al.)

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein-protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO 2 F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which are crucial for AMPAR clustering at synaptic sites. However, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit complexed with TARP γ2 in lipid environments. HS-AFM videos of GluA2-γ2 in the resting and activated/open states revealed fluctuations in NTD dimers. Conversely, in the desensitized/closed state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we observed individual NTD dimers transitioning into monomers, with extended monomeric states in the activated/open state. Molecular dynamics simulations provided further support, confirming the energetic stability of the monomeric NTD states within lipids. This NTD-dimer splitting resulted in subunit exchange between the receptors and increased the number of interaction sites with synaptic protein neuronal pentraxin 1 (NP1). Moreover, our HS-AFM studies revealed that NP1 forms a ring-shaped octamer through N-terminal disulfide bonds and binds to the tip of the NTD. These findings suggest a molecular mechanism in which NP1, upon forming an octamer, is secreted into the synaptic region and binds to the tip of the GluA2 NTD, thereby bridging and clustering multiple AMPARs. Thus, our findings illuminate the critical role of NTD dynamics in the synaptic clustering of AMPARs and contribute valuable insights into the fundamental processes of synaptic transmission.

Immunoglobulin E (IgE) plays a pivotal role in allergic responses 1,2 . The high-affinity IgE receptor, FcεRI, found on mast cells and basophils, is central to the effector functions of IgE. FcεRI is a tetrameric complex, comprising FcεRIα, FcεRIβ and a homodimer of FcRγ (originally known as FcεRIγ), with FcεRIα recognizing the Fc region of IgE (Fcε) and FcεRIβ-FcRγ facilitating signal transduction 3 . Additionally, FcRγ is a crucial component of other immunoglobulin receptors, including those for IgG (FcγRI and FcγRIIIA) and IgA (FcαRI) 4-8 . However, the molecular basis of FcεRI assembly and the structure of FcRγ have remained elusive. Here we elucidate the cryogenic electron microscopy structure of the Fcε-FcεRI complex. FcεRIα has an essential role in the receptor's assembly, interacting with FcεRIβ and both FcRγ subunits. FcεRIβ is structured as a compact four-helix bundle, similar to the B cell antigen CD20. The FcRγ dimer exhibits an asymmetric architecture, and coils with the transmembrane region of FcεRIα to form a three-helix bundle. A cholesterol-like molecule enhances the interaction between FcεRIβ and the FcεRIα-FcRγ complex. Our mutagenesis analyses further indicate similarities between the interaction of FcRγ with FcεRIα and FcγRIIIA, but differences in that with FcαRI. These findings deepen our understanding of the signalling mechanisms of FcεRI and offer insights into the functionality of other immune receptors dependent on FcRγ., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES 7 ) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES 7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES 7 ) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES 7 and CMC-GroES 7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein complexes are fundamental to all cellular processes, so understanding their evolutionary history and assembly processes is important. Gene duplication followed by divergence is considered a primary mechanism for diversifying protein complexes. Nonetheless, to what extent assembly of present-day paralogous complexes has been constrained by their long evolutionary pathways and how cross-complex interference is avoided remain unanswered questions. Subunits of protein complexes are often stabilized upon complex formation, whereas unincorporated subunits are degraded. How such cooperative stability influences protein complex assembly also remains unclear. Here, we demonstrate that subcomplexes determined by cooperative stabilization interactions serve as building blocks for protein complex assembly. We further develop a protein stability-guided method to compare the assembly processes of paralogous complexes in cellulo. Our findings support that oligomeric state and the structural organization of paralogous complexes can be maintained even if their assembly processes are rearranged. Our results indicate that divergent assembly processes by paralogous complexes not only enable the complexes to evolve new functions, but also reinforce their segregation by establishing incompatibility against deleterious hybrid assemblies., (© 2024. The Author(s).)

NVA1309 is a non-brain penetrant next-generation gabapentinoid shown to bind Cavα2δ at R243 within a triple Arginine motif forming the binding site for gabapentin and pregabalin. In this study we have compared the effects of NVA1309 with Mirogabalin, a gabapentinoid drug with higher affinity for the voltage-gated calcium channel subunit Cavα2δ-1 than pregabalin which is approved for post-herpetic neuralgia in Japan, Korea and Taiwan. Both NVA1309 and mirogabalin inhibit Cav2.2 currents in vitro and decrease Cav2.2 plasma membrane expression with higher efficacy than pregabalin. Mutagenesis of the classical binding residue arginine R243 and the newly identified binding residue lysine K615 reverse the effect of mirogabalin on Cav2.2 current, but not that of NVA1309., (© 2024. The Author(s).)

Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe 2+ at binuclear iron ferroxidase centers (FOC) and store the Fe 3+ in their interior, thus protecting cells from unwanted Fe 3+ /Fe 2+ redox cycling and storing iron at concentrations far above the solubility of Fe 3+ . Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe 2+ to Fe 3+ , while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe 3+ by transferring electrons via the heme, enabling Fe 2+ mobilization to the cytosol for incorporation in metabolism., (© 2024. The Author(s).)

Glutamate transmission and activation of ionotropic glutamate receptors are the fundamental means by which neurons control their excitability and neuroplasticity 1 . The N-methyl-D-aspartate receptor (NMDAR) is unique among all ligand-gated channels, requiring two ligands-glutamate and glycine-for activation. These receptors function as heterotetrameric ion channels, with the channel opening dependent on the simultaneous binding of glycine and glutamate to the extracellular ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, respectively 2,3 . The exact molecular mechanism for channel gating by the two ligands has been unclear, particularly without structures representing the open channel and apo states. Here we show that the channel gate opening requires tension in the linker connecting the LBD and transmembrane domain (TMD) and rotation of the extracellular domain relative to the TMD. Using electron cryomicroscopy, we captured the structure of the GluN1-GluN2B (GluN1-2B) NMDAR in its open state bound to a positive allosteric modulator. This process rotates and bends the pore-forming helices in GluN1 and GluN2B, altering the symmetry of the TMD channel from pseudofourfold to twofold. Structures of GluN1-2B NMDAR in apo and single-liganded states showed that binding of either glycine or glutamate alone leads to distinct GluN1-2B dimer arrangements but insufficient tension in the LBD-TMD linker for channel opening. This mechanistic framework identifies a key determinant for channel gating and a potential pharmacological strategy for modulating NMDAR activity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The ionotropic glutamate receptors (iGluRs) are comprised of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-d-aspartate receptor, kainate, and delta subtypes and are pivotal in neuronal plasticity. Recent structural studies on AMPA receptors reveal intricate conformational changes during activation and desensitization elucidating the steps from agonist binding to channel opening and desensitization. Additionally, interactions with auxiliary subunits, including transmembrane AMPA-receptor regulatory proteins, germ-cell-specific gene 1-like protein, and cornichon homologs, intricately modulate AMPA receptors. We discuss the recent high-resolution structures of these complexes that unveil stoichiometry, subunit positioning, and differences in specific side-chain interactions that influence these functional modulations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Vasanthi Jayaraman reports financial support was provided by National Institute of General Medical Sciences. Cuauhtemoc U. Gonzalez reports was provided by National Institute of Neurological Disorders and Stroke. Vasanthi Jayaraman reports a relationship with Biophysical Journal that includes: board membership. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications 1 . Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms 2-4 . Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the β-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Polyploid wheats include a group of tetraploids known as Timopheevii (A u A u GG), which are represented by two subspecies: Triticum timopheevii ssp. timopheevii (cultivated) and Triticum timopheevii ssp. araraticum (wild). The combined use of electrophoretic (SDS-PAGE) and chromatographic (RP-HPLC) techniques carried out on high-molecular-weight glutenin subunits (HMW-GSs) permitted the association of different x- and y-type subunits to the A and G genomes and the assessment of allelic variation present at corresponding loci. The results also revealed that in both subspecies, accessions are present that possess expressed y-type subunits at the Glu-A1 locus. Genes corresponding to these subunits were amplified and amplicons corresponding to x- and y-type genes associated with the A genome were detected in all accessions, including those without expressed x- and y-type subunits. The comparison with genes of polyploid wheats confirmed the structural characteristics of typical y-type genes, with the presence of seven cysteine residues and with hexapeptide and nonapeptide repeat motifs. The identification of wild and cultivated T. timopheevii with both x- and y-type glutenin subunits at the Glu-A1 and Glu-G1 loci represents a useful source for the modification of the allelic composition of HMW-GSs in cultivated wheats with the ultimate objective of improving technological properties.

The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.

GABA A receptors (GABA A Rs) are neurotransmitter-gated ion channels critical for inhibitory synaptic transmission as well as the molecular target for benzodiazepines (BZDs), one of the most widely prescribed class of psychotropic drugs today. Despite structural insight into the conformations underlying functional channel states, the detailed molecular interactions involved in conformational transitions and the physical basis for their modulation by BZDs are not fully understood. We previously identified that alanine substitution at the central residue in the α1 subunit M2-M3 linker (V279A) enhances the efficiency of linkage between the BZD site and the pore gate. Here, we expand on this work by investigating the effect of alanine substitutions at the analogous positions in the M2-M3 linkers of β2 (I275A) and γ2 (V290A) subunits, which together with α1 comprise typical heteromeric α1β2γ2 synaptic GABA A Rs. We find that these mutations confer subunit-specific effects on the intrinsic pore closed-open equilibrium and its modulation by the BZD diazepam (DZ). The mutations α1(V279A) or γ2(V290A) bias the channel toward a closed conformation, whereas β2(I275A) biases the channel toward an open conformation to the extent that the channel becomes leaky and opens spontaneously in the absence of agonist. In contrast, only α1(V279A) enhances the efficiency of DZ-to-pore linkage, whereas mutations in the other two subunits have no effect. These observations show that the central residue in the M2-M3 linkers of distinct subunits in synaptic α1β2γ2 GABA A Rs contribute asymmetrically to the intrinsic closed-open equilibrium and its modulation by DZ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many ion channels are known to behave as an allosteric protein, coupling environmental stimuli captured by specialized sensing domains to the opening of a central pore. The classic Monod-Wyman-Changeux (MWC) model, originally proposed to describe binding of gas molecules to hemoglobin, has been widely used as a framework for analyzing ion channel gating. Here, we address the issue of how accurately the MWC model predicts activation of the capsaicin receptor TRPV1 by vanilloids. Taking advantage of a concatemeric design that makes it possible to lock TRPV1 in states with zero to four bound vanilloid molecules, we showed quantitatively that the overall gating behavior is satisfactorily predicted by the MWC model. There is, however, a small yet detectable subunit position effect: ligand binding to two kitty-corner subunits is 0.3-0.4 kcal/mol more effective in inducing opening than binding to two neighbor subunits. This difference-less than 10% of the overall energetic contribution from ligand binding-might be due to the restriction on subunit arrangement imposed by the planar membrane; if this is the case, then the position effect is not expected in hemoglobin, in which each subunit is related equivalently to all the other subunits., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase 1,2 . However, BDQ also inhibits human ATP synthase 3 . At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Most insects, including human-targeting mosquitoes, detect odors through odorant-activated ion channel complexes consisting of a divergent odorant-binding subunit (OR) and a conserved co-receptor subunit (Orco). As a basis for understanding how odorants activate these heteromeric receptors, we report here cryo-electron microscopy structures of two different heteromeric odorant receptor complexes containing ORs from disease-vector mosquitos Aedes aegypti or Anopheles gambiae . These structures reveal an unexpected stoichiometry of one OR to three Orco subunits. Comparison of structures in odorant-bound and unbound states indicates that odorant binding to the sole OR subunit is sufficient to open the channel pore, suggesting a mechanism of OR activation and a conceptual framework for understanding evolution of insect odorant receptor sensitivity.

Insects detect and discriminate a diverse array of chemicals using odorant receptors (ORs), which are ligand-gated ion channels comprising a divergent odorant-sensing OR and a conserved odorant receptor co-receptor (Orco). In this work, we report structures of the Ap OR5-Orco heterocomplex from the pea aphid Acyrthosiphon pisum alone and bound to its known activating ligand, geranyl acetate. In these structures, three Ap Orco subunits serve as scaffold components that cannot bind the ligand and remain relatively unchanged. Upon ligand binding, the pore-forming helix S7b of Ap OR5 shifts outward from the central pore axis, causing an asymmetrical pore opening for ion influx. Our study provides insights into odorant recognition and channel gating of the OR-Orco heterocomplex and offers structural resources to support development of innovative insecticides and repellents for pest control.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are opened in an allosteric manner by membrane hyperpolarization and cyclic nucleotides such as cAMP. Because of conflicting reports from experimental studies on whether cAMP binding to the four available binding sites in the channel tetramer operates cooperatively in gating, we employ here a computational approach as a promising route to examine ligand-induced conformational changes after binding to individual sites. By combining an elastic network model (ENM) with linear response theory (LRT) for modeling the apo-holo transition of the cyclic nucleotide-binding domain (CNBD) in HCN channels, we observe a distinct pattern of cooperativity matching the "positive-negative-positive" cooperativity reported from functional studies. This cooperativity pattern is highly conserved among HCN subtypes (HCN4, HCN1), but only to a lesser extent visible in structurally related channels, which are only gated by voltage (KAT1) or cyclic nucleotides (TAX4). This suggests an inherent cooperativity between subunits in HCN channels as part of a ligand-triggered gating mechanism in these channels.

Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex., Competing Interests: Declaration of interests All authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets., (© 2024. The Author(s).)

N-methyl-D-aspartate receptors (NMDARs) play essential roles in vital aspects of brain functions. NMDARs mediate clinical features of neurological diseases and thus, represent a potential therapeutic target for their treatments. Many findings implicated the GluN2B subunit of NMDARs in various neurological disorders including epilepsy, ischemic brain damage, and neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, Huntington's chorea, and amyotrophic lateral sclerosis. Although a large amount of information is growing consistently on the importance of GluN2B subunit, however, limited recent data is available on how subunit-selective ligands impact NMDAR functions, which blunts the ability to render the diagnosis or craft novel treatments tailored to patients. To bridge this gap, we have focused on and summarized recently reported GluN2B selective ligands as emerging subunit-selective antagonists and modulators of NMDAR. Herein, we have also presented an overview of the structure-function relationship for potential GluN2B/NMDAR ligands with their binding sites and connection to CNS functionalities. Understanding of design rules and roles of GluN2B selective compounds will provide the link to medicinal chemists and neuroscientists to explore novel neurotherapeutic strategies against dysfunctions of glutamatergic neurotransmission., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Mediator complex is a key component that bridges various transcription activators and RNA polymerase during eukaryotic transcription initiation. The Arabidopsis thaliana Med25 (aMed25), a subunit of the Mediator complex, plays important roles in regulating hormone signaling, biotic and abiotic stress responses and plant development by interacting with a variety of transcription factors through its activator-interacting domain (ACID). However, the recognition mechanism of aMed25-ACID for various transcription factors remains unknown. Here, we report the nearly complete 1 H, 13 C, and 15 N backbone and side chain resonance assignments of aMED25-ACID (residues 551-681). TALOS-N analysis revealed that aMED25-ACID structure is comprised of three α-helices and seven β-strands, which lacks the C-terminal α-helix existing in the human MED25-ACID. This study lays a foundation for further research on the structure-function relationship of aMED25-ACID., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The primary photochemical reaction of photosynthesis in green sulfur bacteria occurs in the homodimer PscA core proteins by a special chlorophyll pair. The light induced excited state of the special pair producing P 840 + is rapidly reduced by electron transfer from one of the two PscC subunits. Molecular dynamics (MD) simulations are combined with bioinformatic tools herein to provide structural and dynamic insight into the complex between the two PscA core proteins and the two PscC subunits. The microscopic dynamic model involves extensive sampling at atomic resolution and at a cumulative time-scale of 22µs and reveals well defined protein-protein interactions. The membrane complex is composed of the two PscA and the two PscC subunits and macroscopic connections are revealed within a putative electron transfer pathway from the PscC subunit to the special pair P 840 located within the PscA subunits. Our results provide a structural basis for understanding the electron transport to the homodimer RC of the green sulfur bacteria. The MD based approach can provide the basis to further probe the PscA-PscC complex dynamics and observe electron transfer therein at the quantum level. Furthermore, the transmembrane helices of the different PscC subunits exert distinct dynamics in the complex., (© 2024. The Author(s).)

N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The qualities of wheat dough are influenced by the high-molecular-weight glutenin subunits (HMW-GS), a critical component of wheat gluten protein. However, it is still unknown how HMW-GS silencing affects the aggregation characteristics of dough. Two groups of near-isogenic wheat were used to study the effects of HMW-GS silencing on dough aggregation characteristics, dough texture characteristics, and dough microstructure. It was observed that the content of gliadin in LH-11 strain significantly increased compared to the wild-type (WT). Additionally, the amount of glutenin macropolymer and the glutenin/gliadin both decreased. The aggregation characteristics and rheological characteristics of the dough in LH-11 strain were significantly reduced, and the content of β-sheet in the dough was significantly reduced. The HMW-GS silencing resulted in a reduction in the aggregation of the gluten network in the dough, which related to the alteration of the secondary and microstructure of the gluten., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the cytoplasm, the processing of short noncoding RNA, and the breakdown of RNA fragments. We determined two crystal structures of exosome components from Thermoplasma acidophilum ( Taci ): one with a resolution of 2.3 Å that reveals the central components ( Taci Rrp41 and Taci Rrp42), and another with a resolution of 3.5 Å that displays the whole exosome ( Taci Rrp41, Taci Rrp42, and Taci Rrp4). The fundamental exosome structure revealed the presence of a heterodimeric complex consisting of Taci Rrp41 and Taci Rrp42. The structure comprises nine subunits, with Taci Rrp41 and Taci Rrp42 arranged in a circular configuration, while Taci Rrp4 is located at the apex. The RNA degradation capabilities of the Taci Rrp4:41:42 complex were verified by RNA degradation assays, consistent with prior findings in other archaeal exosomes. The resemblance between archaeal exosomes and bacterial PNPase suggests a common mechanism for RNA degradation. Despite sharing comparable topologies, the surface charge distributions of Taci Rrp4 and other archaea structures are surprisingly distinct. Different RNA breakdown substrates may be responsible for this variation. These newfound structural findings enhance our comprehension of RNA processing and degradation in biological systems.

The inositol 1,4,5-triphosphate receptor (IP 3 R) mediates Ca release in many cell types and is pivotal to a wide range of cellular processes. High-resolution cryoelectron microscopy studies have provided new structural details of IP 3 R type 1 (IP 3 R1), showing that channel function is determined by the movement of various domains within and between each of its four subunits. Channel properties are regulated by ligands, such as Ca and IP3, which bind at specific sites and control the interactions between these domains. However, it is not known how the various ligand-binding sites on IP 3 R1 interact to control the opening of the channel. In this study, we present a coarse-grained model of IP 3 R1 that accounts for the channel architecture and the location of specific Ca- and IP3-binding sites. This computational model accounts for the domain-domain interactions within and between the four subunits that form IP 3 R1, and it also describes how ligand binding regulates these interactions. Using a kinetic model, we explore how two Ca-binding sites on the cytosolic side of the channel interact with the IP3-binding site to regulate the channel open probability. Our primary finding is that the bell-shaped open probability of IP 3 R1 provides constraints on the relative strength of these regulatory binding sites. In particular, we argue that a specific Ca-binding site, whose function has not yet been established, is very likely a channel antagonist. Additionally, we apply our model to show that domain-domain interactions between neighboring subunits exert control over channel cooperativity and dictate the nonlinear response of the channel to Ca concentration. This suggests that specific domain-domain interactions play a pivotal role in maintaining the channel's stability, and a disruption of these interactions may underlie disease states associated with Ca dysregulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding β5 and β2 subunits., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Porphyromonas gingivalis, an anaerobic CFB (Cytophaga, Fusobacterium, and Bacteroides) group bacterium, is the keystone pathogen of periodontitis and has been implicated in various systemic diseases. Increased antibiotic resistance and lack of effective antibiotics necessitate a search for new intervention strategies. Here we report a 3.5 Å resolution cryo-EM structure of P. gingivalis RNA polymerase (RNAP). The structure displays new structural features in its ω subunit and multiple domains in β and β' subunits, which differ from their counterparts in other bacterial RNAPs. Superimpositions with E. coli RNAP holoenzyme and initiation complex further suggest that its ω subunit may contact the σ4 domain, thereby possibly contributing to the assembly and stabilization of initiation complexes. In addition to revealing the unique features of P. gingivalis RNAP, our work offers a framework for future studies of transcription regulation in this important pathogen, as well as for structure-based drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The membrane F o factor of ATP synthase is highly sensitive to mutations in the proton half-channel leading to the functional blocking of the entire protein. To identify functionally important amino acids for the proton transport, we performed molecular dynamic simulations on the selected mutants of the membrane part of the bacterial F o F 1 -ATP synthase embedded in a native lipid bilayer: there were nine different mutations of a -subunit residues ( a E219, a H245, a N214, a Q252) in the inlet half-channel. The structure proved to be stable to these mutations, although some of them ( a H245Y and a Q252L) resulted in minor conformational changes. a H245 and a N214 were crucial for proton transport as they directly facilitated H + transfer. The substitutions with nonpolar amino acids disrupted the transfer chain and water molecules or neighboring polar side chains could not replace them effectively. a E219 and a Q252 appeared not to be determinative for proton translocation, since an alternative pathway involving a chain of water molecules could compensate the ability of H + transmembrane movement when they were substituted. Thus, mutations of conserved polar residues significantly affected hydration levels, leading to drastic changes in the occupancy and capacity of the structural water molecule clusters (W1-W3), up to their complete disappearance and consequently to the proton transfer chain disruption.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript 1 , but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation 2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements., (© 2024. The Author(s).)

The molecular weight (MW) of an enzyme is a critical parameter in enzyme-constrained models (ecModels). It is determined by two factors: the presence of subunits and the abundance of each subunit. Although the number of subunits (NS) can potentially be obtained from UniProt, this information is not readily available for most proteins. In this study, we addressed this gap by extracting and curating subunit information from the UniProt database to establish a robust benchmark dataset. Subsequently, we propose a novel model named DeepSub, which leverages the protein language model and Bi-directional Gated Recurrent Unit (GRU), to predict NS in homo-oligomers solely based on protein sequences. DeepSub demonstrates remarkable accuracy, achieving an accuracy rate as high as 0.967, surpassing the performance of QUEEN. To validate the effectiveness of DeepSub, we performed predictions for protein homo-oligomers that have been reported in the literature but are not documented in the UniProt database. Examples include homoserine dehydrogenase from Corynebacterium glutamicum , Matrilin-4 from Mus musculus and Homo sapiens , and the Multimerins protein family from M. musculus and H. sapiens . The predicted results align closely with the reported findings in the literature, underscoring the reliability and utility of DeepSub.