Structural Basis Research Articles

Structural basis of antimicrobial membrane coat assembly by human GBP1.

Guanylate-binding proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) mediating host defense against intracellular pathogens. Their antimicrobial activity hinges on their ability to self-associate and coat pathogen-associated compartments or cytosolic bacteria. Coat formation depends on GTPase activity but how nucleotide binding and hydrolysis prime coat formation remains unclear. Here, we report the cryo-electron microscopy structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and describe the molecular ultrastructure of the GBP1 coat on liposomes and bacterial lipopolysaccharide membranes. Conformational changes of the middle and GTPase effector domains expose the isoprenylated C terminus for membrane association. The α-helical middle domains form a parallel, crossover arrangement essential for coat formation and position the extended effector domain for intercalation into the lipopolysaccharide layer of gram-negative membranes. Nucleotide binding and hydrolysis create oligomeric scaffolds with contractile abilities that promote membrane extrusion and fragmentation. Our data offer a structural and mechanistic framework for understanding GBP1 effector functions in intracellular immunity.

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Design and implementation of corrosion-resistant multitasking cell stainers.

Compared with machine staining, traditional manual staining faces various problems, such as a low preparation success rate, low efficiency, and harm to the human body due to corrosive gases. Therefore, a stainer that is of low cost, has strong corrosion resistance, and is suitable for small-batch preparation should be developed. In this study, by choosing a rotary scheme as the structural basis, a reusable container cover and a master-slave manipulator cooperation scheme are developed, which greatly improve the space utilization rate. Through material selection and structural design, in the designed stainer, effective protection against strong acids with high volatility and permeability is realized, thereby eliminating the corrosion issue. Using the designed splitting-running algorithm for the dyeing procedure, simultaneous multistaining is realized, which significantly improves the staining efficiency. Compared with large-sized stainers, the cost of the proposed stainer is very low, which will help popularize early screening for cervical cancer in low- and middle-income countries.

SEAs for seas: Strategic environmental assessment for more strategic and environmentally-oriented marine spatial planning processes

The foreseen, and already ongoing, substantial increase in offshore renewable energy and corresponding infrastructures urges policymakers and scholars to carefully consider corresponding long-term environmental effects on the marine environment. Strategic Environmental Assessment (SEA) is becoming a standard tool for Marine Spatial Planning (MSP) processes for enabling planners to develop more sustainable marine spatial plans. To explore how SEA can contribute to a more strategic and environmentally-oriented MSP practice, four SEA processes from four North Sea countries are examined, building on available SEA documents and semi-structured interviews. The results show that SEAs for MSP can take an exploratory or appraisal-oriented approach. Adopting an exploratory approach can open up comparatively more opportunities for SEA to contribute to MSP than an adopting an appraisal-oriented approach, but it requires an early initiation of the SEA within the larger planning process. Still, it does not imply that there is one single best method. Rather, the institutional context establishes the conditions for SEA processes to contribute to MSP practice. Particularly, the political pressures and prior sectoral policymaking for offshore renewable energy restrict what can be included in the scope of an SEA. Also, the limited knowledge on the larger dynamics of the marine environment complicate the assessment of transboundary and cumulative effects. Therefore, as future research continues working towards a robust scientific knowledge base on the marine environment, it is crucial that scholars and policymakers collaborate on a structural basis to bridge the gap between science and policy implementation.

Molecular identification and studies on genetic diversity and structure-related GC heterogeneity of Spatholobus Suberectus based on ITS2

To determine the role of internal transcribed spacer 2 (ITS2) in the identification of Spatholobus suberectus and explore the genetic diversity of S. suberectus. A total of 292 ITS2s from S. suberectus and 17 other plant species were analysed. S. suberectus was clustered separately in the phylogenetic tree. The genetic distance between species was greater than that within S. suberectus. Synonymous substitution rate (Ks) analysis revealed that ITS2 diverged the most recently within S. suberectus (Ks = 0.0022). These findings suggested that ITS2 is suitable for the identification of S. suberectus. The ITS2s were divided into 8 haplotypes and 4 evolutionary branches on the basis of secondary structure, indicating that there was variation within S. suberectus. Evolutionary analysis revealed that the GC content of paired regions (pGC) was greater than that of unpaired regions (upGC), and the pGC showed a decreasing trend, whereas the upGC remained unchanged. Single-base mutation was the main cause of base pair substitution. In both the initial state and the equilibrium state, the substitution rate of GC was higher than that of AU. The increase in the GC content was partly attributed to GC-biased gene conversion (gBGC). High GC content reflected the high recombination and mutation rates of ITS2, which is the basis for species identification and genetic diversity. We characterized the sequence and structural characteristics of S. suberectus ITS2 in detail, providing a reference and basis for the identification of S. suberectus and its products, as well as the protection and utilization of wild resources.

Structural basis of mRNA decay by the human exosome-ribosome supercomplex.

The interplay between translation and mRNA decay is widespread in human cells1-3. In quality-control pathways, exonucleolytic degradation of mRNA associated with translating ribosomes is mediated largelyby the cytoplasmic exosome4-9, which includes the exoribonuclease complex EXO10 and the helicase complex SKI238 (refs. 10-16). The helicase can extract mRNA from the ribosome and is expected to transfer it to the exoribonuclease core through a bridging factor, HBS1L3 (also known as SKI7), but the mechanisms of this molecular handover remain unclear7,17,18. Here we reveal how human EXO10 is recruited by HBS1L3 (SKI7) to an active ribosome-bound SKI238 complex. We show that rather than a sequential handover, a direct physical coupling mechanism takes place, which culminates in the formation of a cytoplasmic exosome-ribosome supercomplex. Capturing the structure during active decay reveals a continuous path in which an RNA substrate threads from the 80S ribosome through the SKI2 helicase into the exoribonuclease active site of the cytoplasmic exosome complex. The SKI3 subunit of the complex directly binds to HBS1L3 (SKI7) and also engages a surface of the 40S subunit, establishing a recognition platform in collided disomes. Exosome and ribosome thus work together as a single structural and functional unit in co-translational mRNA decay, coordinating their activities in a transient supercomplex.

A Weibo local network growth model constructed from the perspective of following-followed

Abstract In order to explore the evolution process of the Weibo local network, this study first defines four factors influencing the evolution of the Weibo network. On this basis, the BA scale-free network model was enhanced by incorporating these four factors and accounting for directionality, resulting in a Weibo local network evolution model based on user attributes and behavioral similarity. The model's validity was validated by comparing simulation results with real data. The findings indicate that the Weibo local network exhibits both small-world characteristics and distinctive features. The results show that the Weibo local network exhibits both small-world characteristics and distinctive properties. The in-degree distribution follows a mixed pattern of exponential and power-law distributions, the degree-degree shows isomatching, and both the in-degree centrality and eigenvector centrality values are relatively low. This research contributes to our understanding of user behaviour in the Weibo network, and provides a structural basis for exploring the impact of Weibo network structure on information dissemination.

Structural basis for antibiotic transport and inhibition in PepT2

The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 family transporters recognize and discriminate between different drug classes and dietary peptides remains unclear, hampering efforts to improve antibiotic pharmacokinetics through targeted drug design and delivery. Here, we present cryo-EM structures of the proton-coupled peptide transporter, PepT2 from Rattus norvegicus, in complex with the widely used beta-lactam antibiotics cefadroxil, amoxicillin and cloxacillin. Our structures, combined with pharmacophore mapping, molecular dynamics simulations and biochemical assays, establish the mechanism of beta-lactam antibiotic recognition and the important role of protonation in drug binding and transport.

Structural basis of μ-opioid receptor targeting by a nanobody antagonist

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbE’s antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

Structural basis and molecular mechanisms of Cl- transmembrane transport in cardiomyocytes

The intracellular concentration of chlorine anions ([Cl-]i), the equilibrium potential for chlorine anions (ECl) and transmembrane chloride currents (ICl) are the factors that significantly influence the electrophysiological properties of excitable tissue, including the myocardium. Several types of chloride (anion) conductance have been identified in the heart. In recent years, a number of transmembrane proteins demonstrating chloride conductance have been identified (CFTR, ClC, TMEM16, LRRC8), and the expression of these macromolecules in cardiac tissue has been confirmed. Accumulated data allow for establishing a molecular substrate for some chloride anion currents (ICl,PKA, ICl,ir, ICl,vol, ICl,swell, ICl,Ca, Ito2) detected in the heart. Furthermore, the molecular mechanisms regulating [Cl-]i and ECl through chloride cotransporters (KCC, NKCC1) and chloride-bicarbonate exchangers have been established. The variety of structures determining chloride transmembrane conductivity and the complexity of molecular mechanisms regulating chloride homeostasis underlie the complex effects of activation of chloride transporters in the pacemaker, conduction system and working myocardium of the heart. This review discusses the structural, biophysical properties and molecular regulation of chloride transporter protein complexes identified in the myocardium. The review also covers the mechanisms by which chloride transmembrane transport influences the bioelectrical activity of cardiomyocytes.

Structural insights into KSHV-GPCR constitutive activation and CXCL1 chemokine recognition

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a viral G protein-coupled receptor, KSHV-GPCR, that contributes to KSHV immune evasion and pathogenesis of Kaposi's sarcoma. KSHV-GPCR shares a high similarity with CXC chemokine receptors CXCR2 and can be activated by selected chemokine ligands. Like other herpesvirus-encoded GPCRs, KSHV-GPCR is characterized by its constitutive activity by coupling to various G proteins. We investigated the structural basis of ligand-dependent and constitutive activation of KSHV-GPCR, obtaining high-resolution cryo-EM structures of KSHV-GPCR-Gi complexes with and without the bound CXCL1 chemokine. Analysis of the apo-KSHV-GPCR-Gi structure (2.81 Å) unraveled the involvement of extracellular loop 2 in constitutive activation of the receptor. In comparison, the CXCL1-bound KSHV-GPCR-Gi structure (3.01 Å) showed a two-site binding mode and provided detailed information of CXCL1 binding to a chemokine receptor. The dual activation mechanism employed by KSHV-GPCR represents an evolutionary adaptation for immune evasion and contributes to the pathogenesis of Kaposi's sarcoma. Together with results from functional assays that confirmed the structural models, these findings may help to develop therapeutic strategies for KSHV infection.

Structural basis for Vipp1 membrane binding: from loose coats and carpets to ring and rod assemblies.

Vesicle-inducing protein in plastids 1 (Vipp1) is critical for thylakoid membrane biogenesis and maintenance. Although Vipp1 has recently been identified as a member of the endosomal sorting complexes required for transport III superfamily, it is still unknown how Vipp1 remodels membranes. Here, we present cryo-electron microscopy structures of Synechocystis Vipp1 interacting with membranes: seven structures of helical and stacked-ring assemblies at 5-7-Å resolution engulfing membranes and three carpet structures covering lipid vesicles at ~20-Å resolution using subtomogram averaging. By analyzing ten structures of N-terminally truncated Vipp1, we show that helix α0 is essential for membrane tubulation and forms the membrane-anchoring domain of Vipp1. Lastly, using a conformation-restrained Vipp1 mutant, we reduced the structural plasticity of Vipp1 and determined two structures of Vipp1 at 3.0-Å resolution, resolving the molecular details of membrane-anchoring and intersubunit contacts of helix α0. Our data reveal membrane curvature-dependent structural transitions from carpets to rings and rods, some of which are capable of inducing and/or stabilizing high local membrane curvature triggering membrane fusion.

The discovery and structural basis of two distinct state-dependent inhibitors of BamA

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins.

Structural basis for receptor-binding domain mobility of the spike in SARS-CoV-2 BA.2.86 and JN.1

Since 2019, SARS-CoV-2 has undergone mutations, resulting in pandemic and epidemic waves. The SARS-CoV-2 spike protein, crucial for cellular entry, binds to the ACE2 receptor exclusively when its receptor-binding domain (RBD) adopts the up-conformation. However, whether ACE2 also interacts with the RBD in the down-conformation to facilitate the conformational shift to RBD-up remains unclear. Herein, we present the structures of the BA.2.86 and the JN.1 spike proteins bound to ACE2. Notably, we successfully observed the ACE2-bound down-RBD, indicating an intermediate structure before the RBD-up conformation. The wider and mobile angle of RBDs in the up-state provides space for ACE2 to interact with the down-RBD, facilitating the transition to the RBD-up state. The K356T, but not N354-linked glycan, contributes to both of infectivity and neutralizing-antibody evasion in BA.2.86. These structural insights the spike-protein dynamics would help understand the mechanisms underlying SARS-CoV-2 infection and its neutralization.

8523 An Unconventional Estrogen Receptor Conformation Induces SUMO Expression In Breast Cancer Cells

Abstract Disclosure: G.R. Hancock: None. K. Young: None. G.L. Greene: None. S.W. Fanning: None. An Unconventional Estrogen Receptor Conformation Induces SUMO Expression in Breast Cancer Cells Govinda Hancock, Kristen Young, Geoffrey L. Greene, Sean W. Fanning Competitive allosteric regulators, selective estrogen receptor modulators (SERMs)/degraders (SERDs) differentially manipulate receptor structure to treat and prevent metastatic breast cancers. However, their utility is limited by the ∼40% rate of disease progression due to acquired therapy resistance. In this study, a new SERM based on a tetrahydro-6-isoquinoline (T6I) scaffold reveals the relationship between ERα conformation and therapeutic transcriptional pathways in drug-resistant breast cancers. Optimization of structure-activity relationships produced unique lead compound that engages transcriptional programs similar to the clinically approved SERD fulvestrant. Notably, both drugs induce the expression of small ubiquitin-like modifier-1 (SUMO1), in opposition to the pathogenic hormone 17β-estradiol. SUMOylation is a post-translational modification affecting protein-protein interactions, localization, stability and functional properties. High expression of SUMO1 is associated with better patient prognosis in ERα+ disease. SUMOylation in breast cancer will be elucidated through understanding how it’s induced, its structural basis, and its role in therapeutic response of antiestrogen treatments. Presentation: 6/3/2024

Mechanical Characteristics of Deep Excavation Support Structure with Asymmetric Load on Ground Surface

The purpose of this paper is to capture the mechanical response of the support structure of deep excavation subject asymmetric load. A two-dimensional (2D) numerical analysis model was established by taking a pipe gallery deep excavation subject to asymmetric load as an example. The numerical analysis results were in good agreement with the measured data, thus verified the validity of the numerical model. On this basis, the stress and displacement of support structure caused by the change in foundation asymmetric load were studied. According to the numerical results, horizontal displacement of the diaphragm wall (DW) was dominant, and the maximum horizontal displacement of the DW was 7.54 mm when the deep excavation was completed. With the increase in asymmetric load, the left wall displacement continued to increase, while the displacement of the right DW continued to decrease, and the maximum horizontal wall displacement occurred near the excavation face. The DW was the main bending component, and the maximum wall bending moment when the deep excavation was completed was 173.5 kN·m. The maximum wall bending moment increased with the increase in asymmetric load, and the maximum wall bending moment on the left of the deep excavation was greater than that on the right. The inner support sustained the main component of axial force, with the axial force peaking at 1051.8 kN when the deep excavation was completed. The axial force of the inner support increased with increasing the asymmetric load, and the axial force of the second inner support was obviously greater than that of the first inner support. This research has a positive effect on the design and optimization of deep excavation support structure subject to asymmetric load on ground surface.

Structural basis of α-latrotoxin transition to a cation-selective pore

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca2+-conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report cryoEM structures of the vertebrate-specific α-LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles rearrange and together form a highly stable, 15 nm long, cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data give insight into a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications.

Syntactic Parallelism in Muglan

This article explores the syntactic parallelism in Mulan. The data are carried out from the secondary sources. The qualitative and descriptive method is used for analysis. The sixteen sentences are taken from the source and their targets were noted along with the transliteration. The selected sentences were analyzed on the basis of structure, functions and voice. On the basis of syntactic parallelism, the findings have been drawn on the basis of voice, sentence types and sentence function. Most of the Nepali active voices in the source text (ST) have been translated into English active voices in the target text (TT). Therefore, the tendency of active (ST) into active (TT) was very high. Throughout the sampled data, Nepali passive voice (ST) into English passive voice (TT) has very low. Similarly on the basis of sentence types, simple sentences in the ST translated into simple sentences in the TT has higher tendency as compared to compound sentences in the ST translated into compound sentences in the TT. The complex sentences in the ST translated into complex sentences in the TT have highest tendency. Likewise on the basis of sentence function, the declarative sentences in the ST into declarative sentences in the TT has highest tendency. Interrogative sentences in the ST into interrogative sentences in the TT has higher tendency than exclamatory sentences in the ST into exclamatory in the TT.The imperative sentence (ST) into imperative sentence (TT) has lowest tendency.

Structural basis of thiamine transport and drug recognition by SLC19A3

Thiamine (vitamin B1) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane, which is mainly mediated by the two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters lead to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report seven cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enable us to capture different states of SLC19A3’s transport cycle, revealing the molecular details of thiamine recognition and transport. We identify seven previously unknown drug interactions of SLC19A3 and present structures of the transporter in complex with the inhibitors fedratinib, amprolium and hydroxychloroquine. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.

LYCHOS is a human hybrid of a plant-like PIN transporter and a GPCR.

Lysosomes have crucial roles in regulating eukaryotic metabolism and cell growth by acting as signalling platforms to sense and respond to changes in nutrient and energy availability1. LYCHOS (GPR155) is a lysosomal transmembrane protein that functions as a cholesterol sensor, facilitating the cholesterol-dependent activation of the master protein kinase mechanistic target of rapamycin complex 1 (mTORC1)2. However, the structural basis of LYCHOS assembly and activity remains unclear. Here we determine several high-resolution cryo-electron microscopy structures of human LYCHOS, revealing a homodimeric transmembrane assembly of a transporter-like domain fused to a G-protein-coupled receptor (GPCR) domain. The class B2-like GPCR domain is captured in the apo state and packs against the surface of the transporter-like domain, providing an unusual example of a GPCR as a domain in a larger transmembrane assembly. Cholesterol sensing is mediated by a conserved cholesterol-binding motif, positioned between the GPCR and transporter domains. We reveal that the LYCHOS transporter-like domain is an orthologue of the plant PIN-FORMED (PIN) auxin transporter family, and has greater structural similarity to plant auxin transporters than to known human transporters. Activity assays support a model in which the LYCHOS transporter and GPCR domains coordinate to sense cholesterol and regulate mTORC1 activation.