Revealing biomolecular structure and motion with neural ab initio cryo-EM reconstruction.

Proteins and other biomolecules form dynamic macromolecular machines that are tightly orchestrated to move, bind, and perform chemistry. Cryo-electron microscopy (cryo-EM) can access the intrinsic heterogeneity of these complexes and is therefore a key tool for understanding mechanism and function. However, 3D reconstruction of the resulting imaging data presents a challenging computational problem, especially without any starting information, a setting termed ab initio reconstruction. Here, we introduce a method, DRGN-AI, for ab initio heterogeneous cryo-EM reconstruction. With a two-step hybrid approach combining search and gradient-based optimization, DRGN-AI can reconstruct dynamic protein complexes from scratch without input poses or initial models. Using DRGN-AI, we reconstruct the compositional and conformational variability contained in a variety of benchmark datasets, process an unfiltered dataset of the DSL1/SNARE complex fully ab initio, and reveal a new "supercomplex" state of the human erythrocyte ankyrin-1 complex. With this expressive and scalable model for structure determination, we hope to unlock the full potential of cryo-EM as a high-throughput tool for structural biology and discovery.

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite- as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 Å resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

Structural features of thyroglobulin linked to protein trafficking.

Thyroglobulin must pass endoplasmic reticulum (ER) quality control to become secreted for thyroid hormone synthesis. Defective thyroglobulin, blocked in trafficking, can cause hypothyroidism. Thyroglobulin is a large protein (~2750 residues) spanning regions I-II-III plus a C-terminal cholinesterase-like domain. The cholinesterase-like domain functions as an intramolecular chaperone for regions I-II-III, but the folding pathway leading to successful thyroglobulin trafficking remains largely unknown. Here, informed by the recent three-dimensional structure of thyroglobulin as determined by cryo-electron microscopy, we have bioengineered three novel classes of mutants yielding three entirely distinct quality control phenotypes. Specifically, upon expressing recombinant thyroglobulin, we find that first, mutations eliminating a disulfide bond enclosing a 200-amino acid loop in region I have surprisingly little impact on the ability of thyroglobulin to fold to a secretion-competent state. Next, we have identified a mutation on the surface of the cholinesterase-like domain that has no discernible effect on regional folding yet affects contact between distinct regions and thereby triggers impairment in the trafficking of full-length thyroglobulin. Finally, we have probed a conserved disulfide in the cholinesterase-like domain that interferes dramatically with local folding, and this defect then impacts on global folding, blocking the entire thyroglobulin in the ER. These data highlight variants with distinct effects on ER quality control, inhibiting domain-specific folding; folding via regional contact; neither; or both.

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

Corrigendum: Validation of the binding stoichiometry between HCN channels and their neuronal regulator TRIP8b by single molecule measurements.

[This corrects the article DOI: 10.3389/fphys.2022.998176.].

Perturbation of the host cell Ca2+ homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M.

Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO). As of February 2023, almost 670 million cases and 6,8 million deaths have been confirmed worldwide. Coronaviruses, including SARS-CoV-2, contain a single-stranded RNA genome enclosed in a viral capsid consisting of four structural proteins: the nucleocapsid (N) protein, in the ribonucleoprotein core, the spike (S) protein, the envelope (E) protein, and the membrane (M) protein, embedded in the surface envelope. In particular, the E protein is a poorly characterized viroporin with high identity amongst all the ß-coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43) and a low mutation rate. Here, we focused our attention on the study of SARS-CoV-2 E and M proteins, and we found a general perturbation of the host cell calcium (Ca2+) homeostasis and a selective rearrangement of the interorganelle contact sites. In vitro and in vivo biochemical analyses revealed that the binding of specific nanobodies to soluble regions of SARS-CoV-2 E protein reversed the observed phenotypes, suggesting that the E protein might be an important therapeutic candidate not only for vaccine development, but also for the clinical management of COVID designing drug regimens that, so far, are very limited.

Crystal structure of LGR ligand α2/ß5 from Caenorhabditis elegans with implications for the evolution of glycoprotein hormones.

A family of leucine-rich-repeat-containing G-protein-coupled receptors (LGRs) mediate diverse physiological responses when complexed with their cognate ligands. LGRs are present in all metazoan animals. In humans, the LGR ligands include glycoprotein hormones (GPHs) chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (hFSH), and thyroid-stimulating hormone (hTSH). These hormones are αß heterodimers of cystine-knot protein chains. LGRs and their ligand chains have coevolved. Ancestral hormone homologs, present in both bilaterian animals and chordates, are identified as α2ß5. We have used single-wavelength anomalous diffraction and molecular replacement to determine structures of the α2ß5 hormone from Caenorhabditis elegans (Ceα2ß5). Ceα2ß5 is unglycosylated, as are many other α2ß5 hormones. Both Hsα2ß5, the human homolog of Ceα2ß5, and hTSH activate the same receptor (hTSHR). Despite having little sequence similarity to vertebrate GPHs, apart from the cysteine patterns from core disulfide bridges, Ceα2ß5 is generally similar in structure to these counterparts; however, its α2 and ß5 subunits are more symmetric as compared with α and ß of hCG and hFSH. This quasisymmetry suggests a hypothetical homodimeric antecedent of the α2ß5 and αß heterodimers. Known structures together with AlphaFold models from the sequences for other LGR ligands provide representatives for the molecular evolution of LGR ligands from early metazoans through the present-day GPHs. The experimental Ceα2ß5 structure validates its AlphaFold model, and thus also that for Hsα2ß5; and interfacial characteristics in a model for the Hsα2ß5:hTSHR complex are similar to those found in an experimental hTSH:hTSHR structure.

Defective Thyroglobulin: Cell Biology of Disease.

The primary functional units of the thyroid gland are follicles of various sizes comprised of a monolayer of epithelial cells (thyrocytes) surrounding an apical extracellular cavity known as the follicle lumen. In the normal thyroid gland, the follicle lumen is filled with secreted protein (referred to as colloid), comprised nearly exclusively of thyroglobulin with a half-life ranging from days to weeks. At the cellular boundary of the follicle lumen, secreted thyroglobulin becomes iodinated, resulting from the coordinated activities of enzymes localized to the thyrocyte apical plasma membrane. Thyroglobulin appearance in evolution is essentially synchronous with the appearance of the follicular architecture of the vertebrate thyroid gland. Thyroglobulin is the most highly expressed thyroid gene and represents the most abundantly expressed thyroid protein. Wildtype thyroglobulin protein is a large and complex glycoprotein that folds in the endoplasmic reticulum, leading to homodimerization and export via the classical secretory pathway to the follicle lumen. However, of the hundreds of human thyroglobulin genetic variants, most exhibit increased susceptibility to misfolding with defective export from the endoplasmic reticulum, triggering hypothyroidism as well as thyroidal endoplasmic reticulum stress. The human disease of hypothyroidism with defective thyroglobulin (either homozygous, or compound heterozygous) can be experimentally modeled in thyrocyte cell culture, or in whole animals, such as mice that are readily amenable to genetic manipulation. From a combination of approaches, it can be demonstrated that in the setting of thyroglobulin misfolding, thyrocytes under chronic continuous ER stress exhibit increased susceptibility to cell death, with interesting cell biological and pathophysiological consequences.

Validation of the binding stoichiometry between HCN channels and their neuronal regulator TRIP8b by single molecule measurements.

Tetratricopeptide repeat-containing Rab8b-interacting (TRIP8b) protein is a brain-specific subunit of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, a class of voltage-gated channels modulated by cyclic nucleotides. While the interaction between TRIP8b and the cytosolic C terminus of the channel has been structurally described, the HCN:TRIP8b stoichiometry is less characterized. We employed single molecule mass photometry (MP) to image HCN4 particles purified in complex with TRIP8b. Our data show that four TRIP8b subunits are bound to the tetrameric HCN4 particle, confirming a 1:1 stoichiometry.

Oligomeric interactions maintain active-site structure in a noncooperative enzyme family.

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo-EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active-site structure in one such class of proteins, the short-chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active-site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure-stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity-enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy.

Architecture of the human erythrocyte ankyrin-1 complex.

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

A drug and ATP binding site in type 1 ryanodine receptor.

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for excitation-contraction coupling in skeletal and cardiac muscle. Inherited mutations and stress-induced post-translational modifications result in an SR Ca2+ leak that causes skeletal myopathies, heart failure, and exercise-induced sudden death. A class of therapeutics known as Rycals prevent the RyR-mediated leak, are effective in preventing disease progression and restoring function in animal models, and are in clinical trials for patients with muscle and heart disorders. Using cryogenic-electron microscopy, we present a model of RyR1 with a 2.45-Å resolution before local refinement, revealing a binding site in the RY1&2 domain (3.10 Å local resolution), where the Rycal ARM210 binds cooperatively with ATP and stabilizes the closed state of RyR1.

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

Ion currents through Kir potassium channels are gated by anionic lipids.

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor.

The type 1 ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, composed of four 565-kDa protomers. Cryogenic electron microscopy (cryo-EM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryo-EM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously. This allowed us to resolve the structure of the channel in the primed and open states at 3.4 and 4.0 Å, respectively, with a single dataset. This method offers validation for detergent-based structures of the RyR and offers a starting point for utilizing a chemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.

Symmetric activation and modulation of the human calcium-sensing receptor.

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.