Single-particle Electron Microscopy Research Articles

Unique structural attributes of the PSI-NDH supercomplex in Physcomitrium patens.

Cyclic electron transport around photosystem I (PSI) is essential for the protection of the photosynthetic apparatus in plants under diverse light conditions. This process is primarily mediated by Proton Gradient Regulation 5 protein/Proton Gradient Regulation 5-like photosynthetic phenotype 1 protein (PGR5/PGRL1) and NADH dehydrogenase-like complex (NDH). In angiosperms, NDH interacts with two PSI complexes through distinct monomeric antennae, LHCA5 and LHCA6, which is crucial for its higher stability under variable light conditions. This interaction represents an advanced evolutionary stage and offers limited insight into the origin of the PSI-NDH supercomplex in evolutionarily older organisms. In contrast, the moss Physcomitrium patens (Pp), which retains the lhca5 gene but lacks the lhca6, offers a glimpse into an earlier evolutionary stage of the PSI-NDH supercomplex. Here we present structural evidence of the Pp PSI-NDH supercomplex formation by single particle electron microscopy, demonstrating the unique ability of Pp to bind a single PSI in two different configurations. One configuration closely resembles the angiosperm model, whereas the other exhibits a novel PSI orientation, rotated clockwise. This structural flexibility in Pp is presumably enabled by the variable incorporation of LHCA5 within PSI and is indicative of an early evolutionary adaptation that allowed for greater diversity at the PSI-NDH interface. Our findings suggest that this variability was reduced as the structural complexity of the NDH complex increased in vascular plants, primarily angiosperms. This study not only clarifies the evolutionary development of PSI-NDH supercomplexes but also highlights the dynamic nature of the adaptive mechanisms of plant photosynthesis.

Cryo-EM analysis of Pseudomonas phage Pa193 structural components

The World Health Organization has designated Pseudomonas aeruginosa as a critical pathogen for the development of new antimicrobials. Bacterial viruses, or bacteriophages, have been used in various clinical settings, commonly called phage therapy, to address this growing public health crisis. Here, we describe a high-resolution structural atlas of a therapeutic, contractile-tailed Pseudomonas phage, Pa193. We used bioinformatics, proteomics, and cryogenic electron microscopy single particle analysis to identify, annotate, and build atomic models for 21 distinct structural polypeptide chains forming the icosahedral capsid, neck, contractile tail, and baseplate. We identified a putative scaffolding protein stabilizing the interior of the capsid 5-fold vertex. We also visualized a large portion of Pa193 ~ 500 Å long tail fibers and resolved the interface between the baseplate and tail fibers. The work presented here provides a framework to support a better understanding of phages as biomedicines for phage therapy and inform engineering opportunities.

Native mass spectrometry prescreening of G protein-coupled receptor complexes for cryo-EM structure determination.

G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-Gs complex sample prior to cryo-EM analysis, leading to a 3.51Å resolution structure from only 396 movies collected on a 200kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.

Mechanism of ASF1 Inhibition by CDAN1.

Codanin-1 (CDAN1) is an essential and ubiquitous protein named after congenital dyserythropoietic anemia type I (CDA-I), an autosomal recessive disease that manifests from mutations in the CDAN1 or CDIN1 (CDAN1 interacting nuclease 1) gene. CDAN1 interacts with CDIN1 and the paralogous histone H3-H4 chaperones ASF1A (Anti-Silencing Function 1A) and ASF1B, but its function remains unclear. Here, we biochemically and structurally analyze CDAN1 complexes. We find that CDAN1 dimerizes and assembles into cytosolic complexes with CDIN1 and multiple copies of ASF1A/B. Single-particle cryogenic electron microscopy (cryo-EM) structures of CDAN1 complexes identify interactions with ASF1 mediated by two CDAN1 B-domains commonly found in ASF1 binding partners and two helices that mimic histone H3 binding. We additionally observe that one CDAN1 can recruit two ASF1 molecules and that ASF1A and ASF1B have different requirements for CDAN1 engagement. Our findings explain how CDAN1 sequesters and inhibits the chaperone function of ASF1A/B and provide new molecular-level insights into this enigmatic complex.

Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.

Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.

Preparation of oxygen-sensitive proteins for high-resolution cryoEM structure determination using (an)aerobic blot-free vitrification.

High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from traditional grid preparation workflows are particularly exacerbated for oxygen-sensitive proteins, including metalloproteins, whereby oxygen-induced damage and alteration of oxidation states can result in protein inactivation, denaturation, and/or aggregation. Indeed, 99% of the current structures in the EMBD were prepared aerobically and limited successes for anaerobic cryoEM grid preparation exist. Current practices for anaerobic grid preparation involve a vitrification device located in an anoxic chamber, which presents significant challenges including temperature and humidity control, optimization of freezing conditions, costs for purchase and operation, as well as accessibility. Here, we present a streamlined approach that allows for the (an)aerobic vitrification of oxygen-sensitive proteins using an automated aerobic blot-free grid vitrification device - the SPT Labtech chameleon. This robust workflow allows for high-resolution structure determination of dynamic, oxygen-sensitive proteins, of varying complexity and molecular weight.

Discovery of isoquinoline sulfonamides as allosteric gyrase inhibitors with activity against fluoroquinolone-resistant bacteria

Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of DNA gyrase with antibacterial activity against fluoroquinolone-resistant clinical isolates of Escherichia coli. Screening of a small-molecule library revealed an initial isoquinoline sulfonamide hit, which was optimized via medicinal chemistry efforts to afford the more potent antibacterial LEI-800. Target identification studies, including whole-genome sequencing of in vitro selected mutants with resistance to isoquinoline sulfonamides, unanimously pointed to the DNA gyrase complex, an essential bacterial topoisomerase and an established antibacterial target. Using single-particle cryogenic electron microscopy, we determined the structure of the gyrase–LEI-800–DNA complex. The compound occupies an allosteric, hydrophobic pocket in the GyrA subunit and has a mode of action that is distinct from the clinically used fluoroquinolones or any other gyrase inhibitor reported to date. LEI-800 provides a chemotype suitable for development to counter the increasingly widespread bacterial resistance to fluoroquinolones.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis

BackgroundHydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown.ResultsWe report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes.ConclusionsThe hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

Molecular basis for antibody recognition of multiple drug–peptide/MHC complexes

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Structural insights into thermophilic chaperonin complexes

Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.

The α-crystallin Chaperones Undergo a Quasi-ordered Co-aggregation Process in Response to Saturating Client Interaction

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of α-crystallins, carrying potential implications for a pathway toward cataract formation.

Cryo-EM structure and functional basis of prothrombin recognition by a type I antiprothrombin antiphospholipid antibody

AbstractAntiprothrombin antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered type I and type II antibodies and proposed that type I recognizes the open form. In this study, we report the discovery and structural and functional characterization in human plasma of a type I antibody, POmAb (prothrombin open monoclonal antibody). Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium toward the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2-Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russell’s viper venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a type I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.

Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding

Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.

Custom Design of a Humidifier Chamber for InMeso Crystallization.

Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community.

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain.

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.

Structural insights into intron catalysis and dynamics during splicing

The group II intron ribonucleoprotein is an archetypal splicing system with numerous mechanistic parallels to the spliceosome, including excision of lariat introns1,2. Despite the importance of branching in RNA metabolism, structural understanding of this process has remained elusive. Here we present a comprehensive analysis of three single-particle cryogenic electron microscopy structures captured along the splicing pathway. They reveal the network of molecular interactions that specifies the branchpoint adenosine and positions key functional groups to catalyse lariat formation and coordinate exon ligation. The structures also reveal conformational rearrangements of the branch helix and the mechanism of splice site exchange that facilitate the transition from branching to ligation. These findings shed light on the evolution of splicing and highlight the conservation of structural components, catalytic mechanism and dynamical strategies retained through time in premessenger RNA splicing machines.

Elucidating the Structural Dynamics of Integrin α IIbβ 3 from Native Platelet Membranes By Cryo-EM Coupled with Build and Retrieve Data Processing Methodology

Background: Platelets play a critical role in physiological processes by sensing the extracellular environment through their membrane proteins. Changing the quantity or quality of these proteins can profoundly affect platelet physiology and impact health. The native membrane environment provides essential additional regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins and large macromolecules to be solved from highly purified, homogeneous samples. Our recent breakthroughs in data processing make it feasible to obtain high-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the novel “Build and Retrieve” (BaR) data processing methodology. By performing in silico purification, image sorting, and model building from large heterogenous datasets, this iterative bottom-up methodology opens new avenues for an in-depth systems biology approach with structural biology that will uncover how the native environment, including lipid and metal binding, post-translational modifications, and co-factor associations regulate platelet function at the molecular level. Method: Whole blood was collected, and the platelet membrane proteins were solubilized by 1% n-Dodecyl-β-D-Maltoside (DDM) and incorporated into lipidic nanodisc MSP1E3D1 directly from the endogenous source. After being enriched by size exclusion chromatography, the mixture of proteins at ~200 kDa was directly applied on cryo-EM grids. Data were collected on Titan Krios G3i cryogenic transmission electron microscope equipped with a BioQuantum K3 camera. All data processing was done in cryoSPARC using the BaR protocol. The near-atomic-resolution cryo-EM maps were used for protein identification using the program phenix.sequence_from_map in the PHENIX suite. The final protein models were built by Coot and refined by PHENIX. Results: We have used using cryo-EM followed by the BaR method to solve the first unmodified integrin α IIbβ 3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.76Å and 2.49Å, respectively. From these two structures, we observed the ion binding sites at the β1-domain and β-propeller domain in agreement with previous “classical” α IIbβ 3 structural studies using a purified sample. More importantly, we were able to assign endogenous N-linked glycan sites, which reflected how glycosylation naturally regulates α IIbβ 3 dynamic and function on resting human platelets. Finally, we also solved a novel third unique dimer conformation of α IIbβ 3 at 2.61Å formed by two intermediate-states of α IIbβ 3. This may indicate a previously unknown self-regulatory mechanism of α IIbβ 3 in its native environment. Conclusion: In conclusion, our data show the power of using cryo-EM with the BaR method to uncover novel structures directly from natural sources to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. A distinct advantage of the BaR method is that it is an unbiased approach for solving multiple structures from raw samples, which highlights the potential of building a protein structural atlas from platelets. These data have the potential to enrich our understanding of platelet signaling circuitry and foster the development of higher-quality diagnoses and treatments for patients with platelet disorders.

Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.

Biophysical characterization of the cetacean morbillivirus haemagglutinin glycoprotein

Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal disease outbreaks worldwide. Among the eight proteins encoded by the CeMV genome, the haemagglutinin (H) glycoprotein is responsible for the virus attachment to host cell receptors. CeMV H represents an attractive target for antiviral and diagnostic research, yet the elucidation of the molecular mechanisms underlying its role in infection and inter-species transmission was hampered thus far due to the unavailability of recombinant versions of the protein. Here we present the cloning, expression and purification of a recombinant CeMV H ectodomain (rH-ecto), providing an initial characterization of its biophysical and structural properties. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) combined to Western blot analysis and periodic acid Schiff assay showed that CeMV rH-ecto is purifiable at homogeneity from insect cells as a secreted, soluble and glycosylated protein. Miniaturized differential scanning fluorimetry, Blue Native PAGE and size exclusion chromatography coupled to multiangle light scattering revealed that CeMV rH-ecto is globularly folded, thermally stable and exists in solution in the oligomeric states of dimer and multiple of dimers. Furthermore, negative stain electron microscopy single particle analysis allowed us to delineate a low-resolution molecular architecture of the CeMV rH-ecto dimer, which recapitulates native assemblies from other morbilliviral H proteins, such as those from measles virus and canine distemper virus. This set of experiments by orthogonal techniques validates the CeMV rH-ecto as an experimental model for future biochemical studies on its structure and functions.

MyoD-family inhibitor proteins act as auxiliary subunits of Piezo channels.

Piezo channels are critical cellular sensors of mechanical forces. Despite their large size, ubiquitous expression, and irreplaceable roles in an ever-growing list of physiological processes, few Piezo channel-binding proteins have emerged. In this work, we found that MyoD (myoblast determination)-family inhibitor proteins (MDFIC and MDFI) are PIEZO1/2 interacting partners. These transcriptional regulators bind to PIEZO1/2 channels, regulating channel inactivation. Using single-particle cryogenic electron microscopy, we mapped the interaction site in MDFIC to a lipidated, C-terminal helix that inserts laterally into the PIEZO1 pore module. These Piezo-interacting proteins fit all the criteria for auxiliary subunits, contribute to explaining the vastly different gating kinetics of endogenous Piezo channels observed in many cell types, and elucidate mechanisms potentially involved in human lymphatic vascular disease.