Cryo-EM structure of the human Asc-1 transporter complex.

The Alanine-Serine-Cysteine transporter 1 (Asc-1 or SLC7A10) forms a crucial heterodimeric transporter complex with 4F2hc (SLC3A2) through a covalent disulfide bridge. This complex enables the sodium-independent transport of small neutral amino acids, including L-Alanine (L-Ala), Glycine (Gly), and D-Serine (D-Ser), within the central nervous system (CNS). D-Ser and Gly are two key endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors by binding to the allosteric site. Mice deficient in Asc-1 display severe symptoms such as tremors, ataxia, and seizures, leading to early postnatal death. Despite its physiological importance, the functional mechanism of the Asc-1-4F2hc complex has remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the human Asc-1-4F2hc complex in its apo state, D-Ser bound state, and L-Ala bound state, resolved at 3.6 Å, 3.5 Å, and 3.4 Å, respectively. Through detailed structural analysis and transport assays, we uncover a comprehensive alternating access mechanism that underlies conformational changes in the complex. In summary, our findings reveal the architecture of the Asc-1 and 4F2hc complex and provide valuable insights into substrate recognition and the functional cycle of this essential transporter complex.

Structures and dynamics of Rpd3S complex bound to nucleosome.

The Rpd3S complex plays a pivotal role in facilitating local histone deacetylation in the transcribed regions to suppress intragenic transcription initiation. Here, we present the cryo-electron microscopy structures of the budding yeast Rpd3S complex in both its apo and three nucleosome-bound states at atomic resolutions, revealing the exquisite architecture of Rpd3S to well accommodate a mononucleosome without linker DNA. The Rpd3S core, containing a Sin3 Lobe and two NB modules, is a rigid complex and provides three positive-charged anchors (Sin3_HCR and two Rco1_NIDs) to connect nucleosomal DNA. In three nucleosome-bound states, the Rpd3S core exhibits three distinct orientations relative to the nucleosome, assisting the sector-shaped deacetylase Rpd3 to locate above the SHL5-6, SHL0-1, or SHL2-3, respectively. Our work provides a structural framework that reveals a dynamic working model for the Rpd3S complex to engage diverse deacetylation sites.

Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

Differential Interactions of the Proteasome Inhibitor PI31 with Constitutive and Immuno-20S Proteasomes.

PI31 (Proteasome Inhibitor of 31,000 Da) is a 20S proteasome binding protein originally identified as an in vitro inhibitor of 20S proteasome proteolytic activity. Recently reported cryo-electron microscopy structures of 20S-PI31 complexes have revealed that the natively disordered proline-rich C-terminus of PI31 enters the central chamber in the interior of the 20S proteasome and interacts directly with the proteasome's multiple catalytic threonine residues in a manner predicted to inhibit their enzymatic function while evading its own proteolysis. Higher eukaryotes express an alternative form of the 20S proteasome (termed "immuno-proteasome") that features genetically and functionally distinct catalytic subunits. The effect of PI31 on immuno-proteasome function is unknown. We examine the relative inhibitory effects of PI31 on purified constitutive (20Sc) and immuno-(20Si) 20S proteasomes in vitro and show that PI31 inhibits 20Si hydrolytic activity to a significantly lesser degree than that of 20Sc. Unlike 20Sc, 20Si hydrolyzes the carboxyl-terminus of PI31 and this effect contributes to the reduced inhibitory activity of PI31 toward 20Si. Conversely, loss of 20Sc inhibition by PI31 point mutants leads to PI31 degradation by 20Sc. These results demonstrate unexpected differential interactions of PI31 with 20Sc and 20Si and document their functional consequences.

Structural analysis of ATP bound to the F1-ATPase ß-subunit monomer by solid-state NMR- insight into the hydrolysis mechanism in F1.

ATP-hydrolysis-associated conformational change of the ß-subunit during the rotation of F1-ATPase (F1) has been discussed using cryo-electron microscopy (cryo-EM). Since it is worthwhile to further investigate the conformation of ATP at the catalytic subunit through an alternative approach, the structure of ATP bound to the F1ß-subunit monomer (ß) was analyzed by solid-state NMR. The adenosine conformation of ATP-ß was similar to that of ATP analog in F1 crystal structures. 31P chemical shift analysis showed that the Pα and Pß conformations of ATP-ß are gauche-trans and trans-trans, respectively. The triphosphate chain is more extended in ATP-ß than in ATP analog in F1 crystals. This appears to be in the state just before ATP hydrolysis. Furthermore, the ATP-ß conformation is known to be more closed than the closed form in F1 crystal structures. In view of the cryo-EM results, ATP-ß would be a model of the most closed ß-subunit with ATP ready for hydrolysis in the hydrolysis stroke of the F1 rotation.

Bridging structural and cell biology with cryo-electron microscopy.

Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.

EMinsight: a tool to capture cryoEM microscope configuration and experimental outcomes for analysis and deposition.

The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the single-particle analysis (SPA) workflow into a reasonably standardized procedure. Significant investments of development time have been made, particularly in sample preparation, microscope data-collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practices developed at facilities worldwide has also begun to establish a degree of standardization to data structures coming from the SPA workflow. There is opportunity to capitalize on this moment in the maturation of the field, to capture metadata from SPA experiments and correlate the metadata with experimental outcomes, which is presented here in a set of programs called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as to define methods for metadata capture to assist with the archiving of cryoEM SPA data. It is also envisaged that this tool will be useful to microscope operators and facilities looking to rapidly generate reports on SPA data-collection and screening sessions.

Architecture and regulation of filamentous human cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

Structural and quantum chemical basis for OCP-mediated quenching of phycobilisomes.

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using cryo-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6- to 2.1-angstrom resolution, showcasing its inherent flexibility. Using multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthin's transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria.

A glimpse into Giardia lamblia unique translational machinery.

Eiler et al. used cryo-electron microscopy to determine a 2.49 Å resolution structure of Giardia lamblia 80S ribosome bound to tRNA, mRNA, and the anti-protozoal drug emetine. The structure reveals some critical aspects of translation in G. lamblia, including the lack of ribosomal protein RACK1, and how emetine blocks translation by interacting with both the ribosome and mRNA.

Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment.

GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 - 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.

A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Emergence of fractal geometries in the evolution of a metabolic enzyme.

Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2-4; however, so far, molecular assembly into fractals is restricted to synthetic systems5-12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpinski triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.

Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase.

5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product S-adenosyl-L-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.

Cryo-EM structures of adenosine receptor A3AR bound to selective agonists.

The adenosine A3 receptor (A3AR), a key member of the G protein-coupled receptor family, is a promising therapeutic target for inflammatory and cancerous conditions. The selective A3AR agonists, CF101 and CF102, are clinically significant, yet their recognition mechanisms remained elusive. Here we report the cryogenic electron microscopy structures of the full-length human A3AR bound to CF101 and CF102 with heterotrimeric Gi protein in complex at 3.3-3.2 Å resolution. These agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays reveal the critical role of extracellular loop 3 in A3AR's ligand selectivity and receptor activation. Key mutations, including His3.37, Ser5.42, and Ser6.52, in a unique sub-pocket of A3AR, significantly impact receptor activation. Comparative analysis with the inactive A2AAR structure highlights a conserved receptor activation mechanism. Our findings provide comprehensive insights into the molecular recognition and signaling of A3AR, paving the way for designing subtype-selective adenosine receptor ligands.

Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles.

Purpose: Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Patients and Methods: Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. Results: The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. Conclusion: The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.