Cryo-EM Structure Research Articles

Recommended Tool Compounds for the Melanocortin Receptor (MCR) G Protein-Coupled Receptors (GPCRs).

The melanocortin receptors are a centrally and peripherally expressed family of Class A GPCRs with physiological roles, including pigmentation, steroidogenesis, energy homeostasis, and others yet to be fully characterized. There are five melanocortin receptor subtypes that, apart from the melanocortin-2 receptor (MC2R), are stimulated by a shared set of endogenous agonists. Until 2020, X-ray crystallographic and cryo-electron microscopic (cryo-EM) structures of these receptors were unavailable, and the investigation of their mechanisms of action and putative ligand-receptor interactions was driven by site-directed mutagenesis studies of the receptors and targeted structure-activity relationship (SAR) studies of the endogenous and derivative synthetic ligands. Synthetic derivatives of the endogenous agonist ligand α-MSH have evolved into a suite of powerful ligands such as NDP-MSH (melanotan I), melanotan II (MTII), and SHU9119. This suite of tool compounds now enables the study of the melanocortin receptors and serves as scaffolds for FDA-approved drugs, means of validating stably expressing melanocortin receptor cell lines, core ligands in assessing cryo-EM structures of active and inactive receptor complexes, and essential references for high-throughput discovery and mechanism of action studies. Herein, we review the history and significance of a finite set of these essential tool compounds and discuss how they are being utilized to further the field's understanding of melanocortin receptor physiology and greater druggability.

Differences in oxidoreductase activity of cytochromes bd-I and bd-II oxidases from Mycobacterium smegmatis and Cryo-EM structure of cyt. bd-II

Differences in oxidoreductase activity of cytochromes bd-I and bd-II oxidases from Mycobacterium smegmatis and Cryo-EM structure of cyt. bd-II

High resolution cryoEM structures of terminal oxidases

High resolution cryoEM structures of terminal oxidases

Channelrhodopsins with distinct chromophores and binding patterns

Channelrhodopsins are popular optogenetic tools in neuroscience, but remain poorly understood mechanistically. Here we report the cryo-EM structures of channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii and H. catenoides kalium channelrhodopsin (KCR1). We show that ChR2 recruits an endogenous N-retinylidene-PE-like molecule to a previously unidentified lateral retinal binding pocket, exhibiting a reduced light response in HEK293 cells. In contrast, H. catenoides kalium channelrhodopsin (KCR1) binds an endogenous retinal in its canonical retinal binding pocket under identical condition. However, exogenous ATR reduces the photocurrent magnitude of wild type KCR1 and also inhibits its leaky mutant C110T. Our results uncover diverse retinal chromophores with distinct binding patterns for channelrhodopsins in mammalian cells, which may further inspire next generation optogenetics for complex tasks such as cell fate control.

High-resolution structures of the UapA purine transporter reveal unprecedented aspects of the elevator-type transport mechanism.

UapA is an extensively studied elevator-type purine transporter from the model fungus Aspergillus nidulans . Determination of a 3.6Å inward-facing crystal structure lacking the cytoplasmic N-and C-tails, molecular dynamics (MD), and functional studies have led to speculative models of its transport mechanism and determination of substrate specificity. Here, we report full-length cryo-EM structures of UapA in new inward-facing apo- and substrate-loaded conformations at 2.05-3.5 Å in detergent and lipid nanodiscs. The structures reveal in an unprecedented level of detail the role of water molecules and lipids in substrate binding, specificity, dimerization, and activity, rationalizing accumulated functional data. Unexpectedly, the N-tail is structured and interacts with both the core and scaffold domains. This finding, combined with mutational and functional studies and MD, points out how N-tail interactions couple proper subcellular trafficking and transport activity by wrapping UapA in a conformation necessary for ER-exit and but also critical for elevator-type conformational changes associated with substrate translocation once UapA has integrated into the plasma membrane. Our study provides detailed insights into important aspects of the elevator-type transport mechanism and opens novel issues on how the evolution of extended cytosolic tails in eukaryotic transporters, apparently needed for subcellular trafficking, might have been integrated into the transport mechanism.

Cryo-EM structure of the SPFH-NfeD family protein complex QmcA-YbbJ

The SPFH (stomatin, prohibitin, flotillin, and HflK/C) protein family is universally present and encompasses the evolutionarily conserved SPFH domain. These proteins are predominantly localized in lipid raft and implicated in various biological processes. The NfeD (nodulation formation efficiency D) protein family is often encoded in tandem with SPFH proteins, suggesting a close functional relationship. Here, we elucidate the cryoelectron microscopy (cryo-EM) structure of the Escherichia coli QmcA-YbbJ complex belonging to the SPFH and NfeD families, respectively. Our findings reveal that the QmcA-YbbJ complex forms an intricate cage-like structure composed of 26 copies of QmcA-YbbJ heterodimers. The transmembrane helices of YbbJ act as adhesive elements bridging adjacent QmcA molecules, while the oligosaccharide-binding domain of YbbJ encapsulates the SPFH domain of QmcA. Our structural study significantly contributes to understanding the functional role of the NfeD protein family and sheds light on the interplay between SPFH and NfeD family proteins.

Scorpion α-toxin LqhαIT specifically interacts with a glycan at the pore domain of voltage-gated sodium channels

Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an "S4 down" conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.

Structural insights into Frizzled3 through nanobody modulators

The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.

Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19)

The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.

Structure of a truncated human GlcNAc-1-phosphotransferase variant reveals the basis for its hyperactivity

Mutations that cause loss of function of GlcNAc-1-phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) targeting system that is responsible for tagging lysosomal hydrolases with the M6P moiety for their delivery to the lysosome. We had previously generated a truncated hyperactive form of PTase termed S1S3 which was shown to notably increase the phosphorylation level of secreted lysosomal enzymes and enhance their uptake by cells. Here, we report the 3.4Å cryo-EM structure of soluble S1S3 lacking both transmembrane domains and cytosolic tails. The structure reveals a high degree of conservation of the catalytic core to full-length PTase. In this dimeric structure, the EF-hand of one protomer is observed interacting with the conserved region four of the other. In addition, we present a high-quality EM 3D map of the UDP-GlcNAc bound form of the full-length soluble protein showing the key molecular interactions between the nucleotide sugar donor and side chain amino acids of the protein. Finally, although the domain organization of S1S3 is very similar to that of the Drosophila melanogaster (fruit fly) PTase homolog, we establish that the latter does not act on lysosomal hydrolases.

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

Cryo-EM structures of the human band 3 transporter indicate a transport mechanism involving the coupled movement of chloride and bicarbonate ions.

The band 3 transporter is a critical integral membrane protein of the red blood cell (RBC), as it is responsible for catalyzing the exchange of bicarbonate and chloride anions across the plasma membrane. To elucidate the structural mechanism of the band 3 transporter, detergent solubilized human ghost membrane reconstituted in nanodiscs was applied to a cryo-EM holey carbon grid to define its composition. With this approach, we identified and determined structural information of the human band 3 transporter. Here, we present 5 different cryo-EM structures of the transmembrane domain of dimeric band 3, either alone or bound with chloride or bicarbonate. Interestingly, we observed that human band 3 can form both symmetric and asymmetric dimers with a different combination of outward-facing (OF) and inward-facing (IF) states. These structures also allow us to obtain the first model of a human band 3 molecule at the IF conformation. Based on the structural data of these dimers, we propose a model of ion transport that is in favor of the elevator-type mechanism.

Structural basis for substrate binding and selection by human mitochondrial RNA polymerase

The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.

Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42–62 and 74–96 relieves autoinhibition of ACA8 activity.Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition.We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.

Structural prediction of GluN3 NMDA receptors.

N-methyl-D-aspartate (NMDA) receptors are heterotetrametric ion channels composed of two obligatory GluN1 subunits and two alternative GluN2 or GluN3 subunits, forming GluN1-N2, GluN1-N3, and GluN1-N2-N3 type of NMDA receptors. Extensive research has focused on the functional and structural properties of conventional GluN1-GluN2 NMDA receptors due to their early discovery and high expression levels. However, the knowledge of unconventional GluN1-N3 NMDA receptors remains limited. In this study, we modeled the GluN1-N3A, GluN1-N3B, and GluN1-N3A-N3B NMDA receptors using deep-learned protein-language predication algorithms AlphaFold and RoseTTAFold All-Atom. We then compared these structures with GluN1-N2 and GluN1-N3A receptor cryo-EM structures and found that GluN1-N3 receptors have distinct properties in subunit arrangement, domain swap, and domain interaction. Furthermore, we predicted the agonist- or antagonist-bound structures, highlighting the key molecular-residue interactions. Our findings shed new light on the structural and functional diversity of NMDA receptors and provide a new direction for drug development. This study uses advanced AI algorithms to model GluN1-N3 NMDA receptors, revealing unique structural properties and interactions compared to conventional GluN1-N2 receptors. By highlighting key molecular-residue interactions and predicting ligand-bound structures, our research enhances the understanding of NMDA receptor diversity and offers new insights for targeted drug development.

Structural determinants of spike infectivity in bat SARS-like coronaviruses RsSHC014 and WIV1.

The bat SARS-like CoVs RsSHC014 and WIV1 can use hACE2 for cell entry without further adaptation, indicating their potential risk of emergence in human populations. The S glycoprotein, responsible for receptor recognition and membrane fusion, plays a crucial role in cross-species transmission and infection. In this study, we determined the cryo-EM structures of the S glycoproteins of RsSHC014 and WIV1. Detailed comparisons revealed dynamic structural variations within spike proteins. We also elucidated the complex structure of WIV1 S-hACE2, providing structural evidence for the potential emergence of WIV1 in humans. Although RsSHC014 and WIV1 had similar hACE2-binding affinities, they exhibited distinct pseudovirus cell entry behavior. Through mutagenesis and cryo-EM analysis, we revealed that besides the structural variations, the 623 site in the SD2 region is another important structural determinant of spike infectivity.

Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

Biased receptor signalling and intracellular trafficking profiles of structurally distinct formylpeptide receptor 2 agonists.

There is increasing interest in developing FPR2 agonists (compound 43, ACT-389949 and BMS-986235) as potential pro-resolving therapeutics, with ACT-389949 and BMS-986235 having entered phase I clinical development. FPR2 activation leads to diverse downstream outputs. ACT-389949 was observed to cause rapid tachyphylaxis, while BMS-986235 and compound 43 induced cardioprotective effects in preclinical models. We aim to characterise the differences in ligand-receptor engagement and downstream signalling and trafficking bias profile. Concentration-response curves to G protein dissociation, β-arrestin recruitment, receptor trafficking and second messenger signalling were generated using FPR2 ligands (BMS-986235, ACT-389949, compound 43 and WKYMVm), in HEK293A cells. Log(τ/KA) was obtained from the operational model for bias analysis using WKYMVm as a reference ligand. Docking of FPR2 ligands into the active FPR2 cryoEM structure (PDBID: 7T6S) was performed using ICM pro software. Bias analysis revealed that WKYMVm and ACT-389949 shared a very similar bias profile. In comparison, BMS-986235 and compound 43 displayed approximately 5- to 50-fold bias away from β-arrestin recruitment and trafficking pathways, while being 35- to 60-fold biased towards cAMP inhibition and pERK1/2. Molecular docking predicted key amino acid interactions at the FPR2 shared between WKYMVm and ACT-389949, but not with BMS-986235 and compound 43. In vitro characterisation demonstrated that WKYMVm and ACT-389949 differ from BMS-986235 and compound 43 in their signalling and protein coupling profile. This observation may be explained by differences in the ligand-receptor interactions. In vitro characterisation provided significant insights into identifying the desired bias profile for FPR2-based pharmacotherapy.

Cryo-EM structure of ACE2-SIT1 in complex with tiagabine

The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.

Mechanistic insights into lanthipeptide modification by a distinct subclass of LanKC enzyme that forms dimers

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a “negative cooperativity” mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.