Cryo-electron Microscopy Structure Research Articles

Filament structures unveil the dynamic organization of human acetyl-CoA carboxylase.

Human acetyl-coenzyme A (CoA) carboxylases (ACCs) catalyze the carboxylation of acetyl-CoA, which is the rate-limiting step in fatty acid synthesis. The molecular mechanism underlying the dynamic organization of ACCs is largely unknown. Here, we determined the cryo-electron microscopy (EM) structure of human ACC1 in its inactive state, which forms a unique filament structure and is in complex with acetyl-CoA. We also determined the cryo-EM structure of human ACC1 activated by dephosphorylation and citrate treatment, at a resolution of 2.55 Å. Notably, the covalently linked biotin binds to a site that is distant from the acetyl-CoA binding site when acetyl-CoA is absent, suggesting a potential coordination between biotin binding and acetyl-CoA binding. These findings provide insights into the structural dynamics and regulatory mechanisms of human ACCs.

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.

Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168.

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5c~Ub-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses.

Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre- and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors.

Structural insights into human zinc transporter ZnT1 mediated Zn2+ efflux

Zinc transporter 1 (ZnT1), the principal carrier of cytosolic zinc to the extracellular milieu, is important for cellular zinc homeostasis and resistance to zinc toxicity. Despite recent advancements in the structural characterization of various zinc transporters, the mechanism by which ZnTs-mediated Zn2+ translocation is coupled with H+ or Ca2+ remains unclear. To visualize the transport dynamics, we determined the cryo-electron microscopy (cryo-EM) structures of human ZnT1 at different functional states. ZnT1 dimerizes via extensive interactions between the cytosolic (CTD), the transmembrane (TMD), and the unique cysteine-rich extracellular (ECD) domains. At pH 7.5, both protomers adopt an outward-facing (OF) conformation, with Zn2+ ions coordinated at the TMD binding site by distinct compositions. At pH 6.0, ZnT1 complexed with Zn2+ exhibits various conformations [OF/OF, OF/IF (inward-facing), and IF/IF]. These conformational snapshots, together with biochemical investigation and molecular dynamic simulations, shed light on the mechanism underlying the proton-dependence of ZnT1 transport.

Structural insights into inhibitor mechanisms on immature HIV-1 Gag lattice revealed by high-resolution in situ single-particle cryo-EM.

HIV-1 inhibitors, such as Bevirimat (BVM) and Lenacapavir (LEN), block the production and maturation of infectious virions. However, their mechanisms remain unclear due to the absence of high-resolution structures for BVM complexes and LEN's structural data being limited to the mature capsid. Utilizing perforated virus-like particles (VLPs) produced from mammalian cells, we developed an approach to determine in situ cryo-electron microscopy (cryo-EM) structures of HIV-1 with inhibitors. This allowed for the first structural determination of the native immature HIV-1 particle with BVM and LEN bound inside the VLPs at high resolutions. Our findings offer a more accurate model of BVM engaging the Gag lattice and, importantly, demonstrate that LEN not only binds the mature capsid but also targets the immature lattice in a distinct manner. The binding of LEN induces a conformational change in the capsid protein (CA) region and alters the architecture of the Gag lattice, which may affect the maturation process. These insights expand our understanding of the inhibitory mechanisms of BVM and LEN on HIV-1 and provide valuable clues for the design of future inhibitors.

Structural mechanism of HP1⍺-dependent transcriptional repression and chromatin compaction

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.Z-nucleosome, revealing two distinct HP1α-nucleosome interfaces. The primary HP1α binding site is located at the N terminus of histone H3, specifically at the trimethylated lysine 9(K9me3) region, while a secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. Our study offers a model for HP1α-mediated heterochromatin maintenance and gene silencing. It also sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

MreC-MreD structure reveals a multifaceted interface that controls MreC conformation.

The peptidoglycan (PG) cell wall is critical for bacterial growth and survival and is a primary antibiotic target. MreD is an essential accessory factor of the Rod complex, which carries out PG synthesis during elongation, yet little is known about how MreD facilitates this process. Here, we present the cryo-electron microscopy structure of Thermus thermophilus MreD in complex with another essential Rod complex component, MreC. The structure reveals that a periplasmic-facing pocket of MreD interacts with multiple membrane-proximal regions of MreC. We use single-molecule FRET to show that MreD controls the conformation of MreC through these contacts, inducing a state primed for Rod complex activation. Using E. coli as a model, we demonstrate that disrupting these interactions abolishes Rod complex activity in vivo . Our findings reveal the role of MreD in bacterial cell shape determination and highlight its potential as an antibiotic target.

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.

Ribosomes hibernate on mitochondria during cellular stress.

Cell survival under nutrient-deprived conditions relies on cells' ability to adapt their organelles and rewire their metabolic pathways. In yeast, glucose depletion induces a stress response mediated by mitochondrial fragmentation and sequestration of cytosolic ribosomes on mitochondria. This cellular adaptation promotes survival under harsh environmental conditions; however, the underlying mechanism of this response remains unknown. Here, we demonstrate that upon glucose depletion protein synthesis is halted. Cryo-electron microscopy structure of the ribosomes show that they are devoid of both tRNA and mRNA, and a subset of the particles depicted a conformational change in rRNA H69 that could prevent tRNA binding. Our in situ structural analyses reveal that the hibernating ribosomes tether to fragmented mitochondria and establish eukaryotic-specific, higher-order storage structures by assembling into oligomeric arrays on the mitochondrial surface. Notably, we show that hibernating ribosomes exclusively bind to the outer mitochondrial membrane via the small ribosomal subunit during cellular stress. We identify the ribosomal protein Cpc2/RACK1 as the molecule mediating ribosomal tethering to mitochondria. This study unveils the molecular mechanism connecting mitochondrial stress with the shutdown of protein synthesis and broadens our understanding of cellular responses to nutrient scarcity and cell quiescence.

Structure of the Nipah virus polymerase phosphoprotein complex

The Nipah virus (NiV), a member of the Paramyxoviridae family, is notorious for its high fatality rate in humans. The RNA polymerase machinery of NiV, comprising the large protein L and the phosphoprotein P, is essential for viral replication. This study presents the 2.9-Å cryo-electron microscopy structure of the NiV L-P complex, shedding light on its assembly and functionality. The structure not only demonstrates the molecular details of the conserved N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP polyribonucleotidyltransferase of the L protein, but also the intact central oligomerization domain and the C-terminal X domain of the P protein. The P protein interacts extensively with the L protein, forming an antiparallel β-sheet among the P protomers and with the fingers subdomain of RdRp. The flexible linker domain of one P promoter extends its contact with the fingers subdomain to reach near the nascent RNA exit, highlighting the distinct characteristic of the NiV L-P interface. This distinctive tetrameric organization of the P protein and its interaction with the L protein provide crucial molecular insights into the replication and transcription mechanisms of NiV polymerase, ultimately contributing to the development of effective treatments and preventive measures against this Paramyxoviridae family deadly pathogen.

Engineering substrate channeling in a bifunctional terpene synthase

Fusicoccadiene synthase from Phomopsis amygdala (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate, and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are connected by a flexible 69-residue linker. The PT domain mediates oligomerization to form predominantly octamers, with cyclase domains randomly splayed out around the PT core. Surprisingly, despite the random positioning of cyclase domains, substrate channeling is operative in catalysis since most of the GGPP generated by the PT remains on the enzyme for cyclization. Here, we demonstrate that covalent linkage of the PT and cyclase domains is not required for GGPP channeling, although covalent linkage may improve channeling efficiency. Moreover, GGPP competition experiments with other diterpene cyclases indicate that the PaFS PT and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryoelectron microscopy structure of the 600-kD "linkerless" construct, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the PT octamer and exhibit fascinating quaternary structural flexibility. These results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the PT octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.

P2X7 receptors exhibit at least three modes of allosteric antagonism.

P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A number of small-molecule antagonists have been identified for these important pharmaceutical targets. However, the molecular pharmacology of P2X receptors is poorly understood because of the chemically disparate nature of antagonists and their differential actions on the seven constituent subtypes. Here, we report high-resolution cryo-electron microscopy structures of the homomeric rat P2X7 receptor bound to five previously known small-molecule allosteric antagonists and a sixth antagonist that we identify. Our structural, biophysical, and electrophysiological data define the molecular determinants of allosteric antagonism in this pharmacologically relevant receptor, revealing three distinct classes of antagonists that we call shallow, deep, and starfish. Starfish binders, exemplified by the previously unidentified antagonist methyl blue, represent a unique class of inhibitors with distinct functional properties that could be exploited to develop potent P2X7 ligands with substantial clinical impact.

Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants

Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).

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.

Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] ∼100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50]<50μg/mL) and total lineage-concentrations estimates of 50-200μg/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide invivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

Structure of TnsABCD transpososome reveals mechanisms of targeted DNA transposition

Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the transpososome and facilitate transposition. Although this pathway is well established, atomic-level structural insights of this process remain largely elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of the TnsC-TnsD-att DNA complex and the TnsABCD transpososome from the Tn7-like transposon in Peltigera membranacea cyanobiont 210A, a type I-B CRISPR-associated transposon. Our structures reveal a striking bending of the att DNA, featured by the intercalation of an arginine side chain of TnsD into a CC/GG dinucleotide step. The TnsABCD transpososome structure reveals TnsA-TnsB interactions and demonstrates that TnsC not only recruits TnsAB but also directly participates in the transpososome assembly. These findings provide mechanistic insights into targeted DNA insertion by Tn7-like transposons, with implications for improving the precision and efficiency of their genome-editing applications.

Integrated all-atom and coarse-grained simulations uncover structural, dynamics and energetic shifts in SARS-CoV-2 JN.1 and BA.2.86 variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, is an enveloped, positive-stranded RNA virus that enters human cells by using its spike protein to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Since its emergence, the virus has mutated, producing variants with increased transmissibility, immune evasion, and infectivity. The JN.1 variant, detected in January 2024, features a single substitution mutation (Leu455Ser) in the receptor-binding domain (RBD) of its spike protein, setting it apart from its parent lineage, BA.2.86. This variant has rapidly become globally predominant due to its enhanced transmission and significant epidemiological impact. To understand the causes behind the dominance of the JN.1 variant, we conducted a comprehensive study using all-atom molecular dynamics (MD) and coarse-grained MD simulations. This allowed us to examine the structural, dynamic, energetics and binding properties of the wild-type (Wuhan strain), BA.2.86, and JN.1 variants. Principal component and free energy landscape analyses revealed enhanced structural stability in the JN.1 variant. Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) assessments indicated lower binding affinity for JN.1 as compared to BA.2.86. Intermolecular interaction analyses further confirmed BA.2.86′s superior binding affinity over JN.1 and wild-type. Additionally, we compared and validated our findings against experimentally determined cryo-electron microscopy (cryo-EM) structures of JN.1 and BA.2.86 variants, confirming the reliability of our simulation results. Overall, this study provides crucial insights into the structural-dynamics-energetics features and physicochemical properties that have contributed to the global prevalence of the JN.1 variant and sheds light on its potential to generate future subvariants.