Cryo-electron Microscopy Structure Research Articles

Architecture and activation of single-pass transmembrane receptor guanylyl cyclase.

The heart, in addition to its primary role in blood circulation, functions as an endocrine organ by producing cardiac hormone natriuretic peptides. These hormones regulate blood pressure through the single-pass transmembrane receptor guanylyl cyclase A (GC-A), also known as natriuretic peptide receptor 1. The binding of the peptide hormones to the extracellular domain of the receptor activates the intracellular guanylyl cyclase domain of the receptor to produce the second messenger cyclic guanosine monophosphate. Despite their importance, the detailed architecture and domain interactions within full-length GC-A remain elusive. Here we present cryo-electron microscopy structures, functional analyses and molecular dynamics simulations of full-length human GC-A, in both the absence and the presence of atrial natriuretic peptide. The data reveal the architecture of full-length GC-A, highlighting the spatial arrangement of its various functional domains. This insight is crucial for understanding how different parts of the receptor interact and coordinate during activation. The study elucidates the molecular basis of how extracellular signals are transduced across the membrane to activate the intracellular guanylyl cyclase domain.

Architecture of the spinach plastid-encoded RNA polymerase.

The plastid-encoded RNA polymerase serves as the principal transcription machinery within chloroplasts, transcribing over 80% of all primary plastid transcripts. This polymerase consists of a prokaryotic-like core enzyme known as the plastid-encoded RNA polymerase core, and is supplemented by newly evolved associated proteins known as PAPs. However, the architecture of the plastid-encoded RNA polymerase and the possible functions of PAPs remain unknown. Here, we present the cryo-electron microscopy structure of a 19-subunit plastid-encoded RNA polymerase complex derived from spinach (Spinacia oleracea). The structure shows that the plastid-encoded RNA polymerase core resembles bacterial RNA polymerase. Twelve PAPs and two additional proteins (FLN2 and pTAC18) bind at the periphery of the plastid-encoded RNA polymerase core, forming extensive interactions that may facilitate complex assembly and stability. PAPs may also protect the complex against oxidative damage and has potential functions in transcriptional regulation. This research offers a structural basis for future investigations into the functions and regulatory mechanisms governing the transcription of plastid genes.

Inhibition mechanism of potential antituberculosis compound lansoprazole sulfide

Tuberculosis is one of the most common causes of death worldwide, with a rapid emergence of multi-drug-resistant strains underscoring the need for new antituberculosis drugs. Recent studies indicate that lansoprazole-a known gastric proton pump inhibitor and its intracellular metabolite, lansoprazole sulfide (LPZS)-are potential antituberculosis compounds. Yet, their inhibitory mechanism and site of action still remain unknown. Here, we combine biochemical, computational, and structural approaches to probe the interaction of LPZS with the respiratory chain supercomplex III2IV2 of Mycobacterium smegmatis, a close homolog of Mycobacterium tuberculosis supercomplex. We show that LPZS binds to the Qo cavity of the mycobacterial supercomplex, inhibiting the quinol substrate oxidation process and the activity of the enzyme. We solve high-resolution (2.6 Å) cryo-electron microscopy (cryo-EM) structures of the supercomplex with bound LPZS that together with microsecond molecular dynamics simulations, directed mutagenesis, and functional assays reveal key interactions that stabilize the inhibitor, but also how mutations can lead to the emergence of drug resistance. Our combined findings reveal an inhibitory mechanism of LPZS and provide a structural basis for drug development against tuberculosis.

STK19 positions TFIIH for cell-free transcription-coupled DNA repair.

In transcription-coupled nucleotide excision repair (TC-NER), stalled RNA polymerase II (RNA Pol II) binds CSB and CRL4CSA, which cooperate with UVSSA and ELOF1 to recruit TFIIH. To explore the mechanism of TC-NER, we recapitulated this reaction invitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also requires STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9-Å cryo-electron microscopy structure shows that STK19 binds the TC-NER complex through CSA and the RPB1 subunit of RNA Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of RNA Pol II for lesion verification. Our analysis of cell-free TC-NER suggests that STK19 couples RNA Pol II stalling to downstream repair events.

Structural basis of the mechanism and inhibition of a human ceramide synthase.

Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for different acyl-CoA substrates. Ceramide with a 16-carbon acyl chain (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, along with biochemical characterization, reveal that CerS catalysis proceeds through a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, yielding insights into its inhibition of CerS. These results provide a framework for understanding CerS function, selectivity and inhibition and open routes for future drug discovery.

A conserved mechanism couples cytosolic domain movements to pore gating in the TRPM2 channel

Transient Receptor Potential Melastatin 2 (TRPM2) cation channels contribute to immunocyte activation, insulin secretion, and central thermoregulation. TRPM2 opens upon binding cytosolic Ca2+ and ADP ribose (ADPR). We present here the 2.5 Å cryo-electronmicroscopy structure of TRPM2 from Nematostella vectensis (nvTRPM2) in a lipid nanodisc, complexed with Ca2+ and ADPR-2'-phosphate. Comparison with nvTRPM2 without nucleotide reveals that nucleotide binding-induced movements in the protein's three "core" layers deconvolve into a set of rigid-body rotations conserved from cnidarians to man. By covalently crosslinking engineered cysteine pairs we systematically trap the cytosolic layers in specific conformations and study effects on gate opening/closure. The data show that nucleotide binding in Layer 3 disrupts inhibitory intersubunit interactions, allowing rotation of Layer 2 which in turn expands the gate located in Layer 1. Channels trapped in that "activated" state are no longer nucleotide dependent, but are opened by binding of Ca2+ alone.

Cryo-EM structure of a lysozyme-derived amyloid fibril from hereditary amyloidosis

Systemic ALys amyloidosis is a debilitating protein misfolding disease that arises from the formation of amyloid fibrils from C-type lysozyme. We here present a 2.8 Å cryo-electron microscopy structure of an amyloid fibril, which was isolated from the abdominal fat tissue of a patient who expressed the D87G variant of human lysozyme. We find that the fibril possesses a stable core that is formed by all 130 residues of the fibril precursor protein. There are four disulfide bonds in each fibril protein that connect the same residues as in the globularly folded protein. As the conformation of lysozyme in the fibril is otherwise fundamentally different from native lysozyme, our data provide a structural rationale for the need of protein unfolding in the development of systemic ALys amyloidosis.

Structural mechanisms of potent lysophosphatidic acid receptor 1 activation by nonlipid basic agonists.

Lysophosphatidic acid receptor 1 (LPA1) is one of the G protein-coupled receptors activated by the lipid mediator, lysophosphatidic acid (LPA). LPA1 is associated with a variety of diseases, and LPA1 agonists have potential therapeutic value for treating obesity and depression. Although potent nonlipid LPA1 agonists have recently been identified, the mechanisms of nonlipid molecule-mediated LPA1 activation remain unclear. Here, we report a cryo-electron microscopy structure of the human LPA1-Gi complex bound to a nonlipid basic agonist, CpY, which has 30-fold higher agonistic activity as compared with LPA. Structural comparisons of LPA1 with other lipid GPCRs revealed that the negative charge in the characteristic binding pocket of LPA1 allows the selective recognition of CpY, which lacks a polar head. In addition, our structure show that the ethyl group of CpY directly pushes W2716.48 to fix the active conformation. Endogenous LPA lacks these chemical features, which thus represent the crucial elements of nonlipid agonists that potently activate LPA1. This study provides detailed mechanistic insights into the ligand recognition and activation of LPA1 by nonlipid agonists, expanding the scope for drug development targeting the LPA receptors.

Crystal structure of the alternative complex III from the phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) is a multi-subunit quinol:electron acceptor oxidoreductase that couples quinol oxidation with transmembrane proton translocation in bacterial respiratory and photosynthetic electron transport chains. Four ACIII cryoelectron microscopy (cryo-EM) structures are known. However, the effects of cryo-EM versus X-ray crystallography structure determination on ACIII structure are unclear. Here, we report a 3.25Å crystal structure of photosynthetic ACIII from Chloroflexus aurantiacus (CaACIIIp), revealing eight subunits (ActA-G and I) with four iron-sulfur clusters and six c-type hemes, a menaquinol-binding site, and two proton translocation passages. Structural comparisons with the previously reported cryo-EM structures reveal slight local conformational changes in the solvent-exposed regions of ActB, ActD, ActG, and the transmembrane (TM) helix of subunit I. The regions conferring structural flexibility possess low sequence conservation across species. However, the core functional modules containing the menaquinol-binding pocket, redox centers, and proton translocation passages remain unchanged, preserving the enzyme's activity.

Isolation and escape mapping of broadly neutralizing antibodies against emerging delta-coronaviruses.

Porcine delta-coronavirus (PDCoV) spillovers were recently detected in febrile children, underscoring the recurrent zoonoses of divergent CoVs. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated PDCoV spike (S)-directed monoclonal antibodies (mAbs) from humanized mice and found that two, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryoelectron microscopy (cryo-EM) structures of PD33 and PD41 in complex with the S receptor-binding domain (RBD) and ectodomain trimer revealed the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs competitively interfere with host aminopeptidase N binding to neutralize PDCoV and used deep-mutational scanning epitope mapping to associate RBD antigenic sites with mAb-mediated neutralization potency. Our results indicate a PD33-PD41 mAb cocktail may heighten the barrier to escape. PD33 and PD41 are candidates for clinical advancement against future PDCoV outbreaks.

Amyloid fibril structures and ferroptosis activation induced by ALS-causing SOD1 mutations.

Over 200 genetic mutations in copper-zinc superoxide dismutase (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). Among these, two ALS-causing mutants, histidine-46→arginine (H46R) and glycine-85→arginine (G85R), exhibit a decreased capacity to bind metal ions. Here, we report two cryo-electron microscopy structures of amyloid fibrils formed by H46R and G85R. These mutations lead to the formation of amyloid fibrils with unique structures distinct from those of the native fibril. The core of these fibrils features a serpentine arrangement with seven or eight β strands, secured by a hydrophobic cavity and a salt bridge between arginine-85 and aspartic acid-101 in the G85R fibril. We demonstrate that these mutant fibrils are notably more toxic and capable of promoting the aggregation of wild-type SOD1 more effectively, causing mitochondrial impairment and activating ferroptosis in cell cultures, compared to wild-type SOD1 fibrils. Our study provides insights into the structural mechanisms by which SOD1 mutants aggregate and induce cytotoxicity in ALS.

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein–protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex.

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein-protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Cryo-EM structure of the heteromeric TRPC1/TRPC4 channel.

Transient receptor potential (TRP) ion channels have a crucial role as cellular sensors, mediating diverse physical and chemical stimuli. The formation of heteromeric structures expands the functionality of TRP channels; however, their molecular architecture remains largely unknown. Here we present the cryo-electron microscopy structures of the human TRPC1/TRPC4 heteromer in the apo and antagonist-bound states, both consisting of one TRPC1 subunit and three TRPC4 subunits. The heteromer structure reveals a distinct ion-conduction pathway, including an asymmetrically constricted selectivity filter and an asymmetric lower gate, primarily attributed to the incorporation of TRPC1. Through a structure-guided electrophysiological assay, we show that both the selectivity filter and the lower part of the S6 helix participate in deciding overall preference for permeating monovalent cations. Moreover, we reveal that the introduction of one lysine residue of TRPC1 into the tetrameric central cavity is enough to render one of the most important functional consequences of TRPC heteromerization: reduced calcium permeability. Our results establish a framework for addressing the structure-function relationship of the heteromeric TRP channels.

A structural snapshot of the multiple working states of the Mpox virus helicase-primase D5.

The Mpox virus (or Monkeypox virus, MPXV) uses its own encoded proteins to form a replication machine that replicates the viral genome in the host cell cytoplasm, making this machinery a key target for antiviral drug design. The D5 (also known as the OPG117 or E5) protein, a bi-functional helicase-primase enzyme, is crucial in the MPXV replication machinery and genome uncoating process. Recently, cryo-electron microscopy (cryo-EM) structures of D5 in multiple states have been determined. These structures have elucidated the full trajectory of the MPXV D5 helicase-primase as it moves along single-stranded DNA, providing unprecedented advancements in the molecular dynamics and unwinding mechanism. This structural snapshot describes the structural features of the D5 protein and dissects the broader implications of its pivotal role in MPXV replication.

Cryo-EM reveals transition states of the Acinetobacter baumannii F1-ATPase rotary subunits γ and ε, unveiling novel compound targets.

Priority 1: critical WHO pathogen Acinetobacter baumannii depends on ATP synthesis and ATP:ADP homeostasis and its bifunctional F1FO-ATP synthase. While synthesizing ATP, it regulates ATP cleavage by its inhibitory ε subunit to prevent wasteful ATP consumption. We determined cryo-electron microscopy structures of the ATPase active A. baumannii F1-αßγεΔ134-139 mutant in four distinct conformational states, revealing four transition states and structural transformation of the ε's C-terminal domain, forming the switch of an ATP hydrolysis off- and an ATP synthesis on-state based. These alterations go in concert with altered motions and interactions in the catalytic- and rotary subunits of this engine. These A. baumannii interacting sites provide novel pathogen-specific targets for inhibitors, with the aim of ATP depletion and/or ATP synthesis and growth inhibition. Furthermore, the presented diversity to other bacterial F-ATP synthases extends the view of structural elements regulating such a catalyst.

Structural basis for hepatitis B virus restriction by a viral receptor homologue

Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV.

Mefloquine-induced conformational shift in Cx36 N-terminal helix leading to channel closure mediated by lipid bilayer

Connexin 36 (Cx36) forms interneuronal gap junctions, establishing electrical synapses for rapid synaptic transmission. In disease conditions, inhibiting Cx36 gap junction channels (GJCs) is beneficial, as it prevents abnormal synchronous neuronal firing and apoptotic signal propagation, mitigating seizures and progressive cell death. Here, we present cryo-electron microscopy structures of human Cx36 GJC in complex with known channel inhibitors, such as mefloquine, arachidonic acid, and 1-hexanol. Notably, these inhibitors competitively bind to the binding pocket of the N-terminal helices (NTH), inducing a conformational shift from the pore-lining NTH (PLN) state to the flexible NTH (FN) state. This leads to the obstruction of the channel pore by flat double-layer densities of lipids. These studies elucidate the molecular mechanisms of how Cx36 GJC can be modulated by inhibitors, providing valuable insights into potential therapeutic applications.

Molecular Dynamics (MD) Simulations Provide Insights into the Activation Mechanisms of 5-HT2A Receptors

Recent breakthroughs in the determination of atomic resolution 3-D cryo-electron microscopy structures of membrane proteins present an unprecedented opportunity for drug discovery. Structure-based drug discovery utilizing in silico methods enables the study of dynamic connectivity of stable conformations induced by the drug in achieving its effect. With the ever-expanding computational power, simulations of this type reveal protein dynamics in the nano-, micro-, and even millisecond time scales. In the present study, aiming to characterize the protein dynamics of the 5HT2A receptor stimulated by ligands (agonist/antagonist), we performed 1 µs MD simulations on 5HT2A/DOI (agonist), 5HT2A/GSK215083 (antagonist), and 5HT2A (APO, no ligand) systems. The crystal structure of 5HT2A/zotepine (antagonist) (PDB: 6A94) was used to set up the simulation systems in a lipid bilayer environment. We found the monitoring of the ionic lock residue pair (R3.50-E6.30) of 5HT2A in MD simulations to be a good approximation of the effects of agonists (ionic lock breakage) or antagonists (ionic lock formation) on receptor activation. We further performed analyses of the MD trajectories, including Principal Component Analysis (PCA), hydrogen bond, salt bridge, and hydrophobic interaction network analyses, and correlation between residues to identify key elements of receptor activation. Our results suggest that in order to trigger receptor activation, DOI must interact with 5HT2A through residues V5.39, G5.42, S5.43, and S5.46 on TM5, inducing significant conformational changes in the backbone angles of G5.42 and S5.43. DOI also interacted with residues W6.48 (toggle switch) and F6.51 on TM6, causing major conformational shifts in the backbone angles of F6.44 and V6.45. These structural changes were transmitted to the intracellular ends of TM5, TM6, and ICL3, resulting in the breaking of the ionic lock and subsequent G protein activation. The studies could be helpful in future design of selective agonists/antagonists for various serotonin receptors (5HT1A, 5HT2A, 5HT2B, 5HT2C, and 5HT7) involved in detrimental disorders, such as addiction and schizophrenia.

Membrane structure-responsive lipid scrambling by TMEM63B to control plasma membrane lipid distribution.

Phospholipids are asymmetrically distributed in the plasma membrane (PM), with phosphatidylcholine and sphingomyelin abundant in the outer leaflet. However, the mechanisms by which their distribution is regulated remain unclear. Here, we show that transmembrane protein 63B (TMEM63B) functions as a membrane structure-responsive lipid scramblase localized at the PM and lysosomes, activating bidirectional lipid translocation upon changes in membrane curvature and thickness. TMEM63B contains two intracellular loops with palmitoylated cysteine residue clusters essential for its scrambling function. TMEM63B deficiency alters phosphatidylcholine and sphingomyelin distributions in the PM. Persons with heterozygous mutations in TMEM63B are known to develop neurodevelopmental disorders. We show that V44M, the most frequent substitution, confers constitutive scramblase activity on TMEM63B, disrupting PM phospholipid asymmetry. We determined the cryo-electron microscopy structures of TMEM63B in its open and closed conformations, uncovering a lipid translocation pathway formed in response to changes in the membrane environment. Together, our results identify TMEM63B as a membrane structure-responsive scramblase that controls PM lipid distribution and we reveal the molecular basis for lipid scrambling and its biological importance.