High-resolution cryo-EM Structures Research Articles

A high resolution cryo-EM structure of Mrp from Bacillus pseudofirmus offers insights into ion transfer pathways in Mrp-type antiporters

A high resolution cryo-EM structure of Mrp from Bacillus pseudofirmus offers insights into ion transfer pathways in Mrp-type antiporters

Cryo-EM structure of human hexameric MCM2-7 complex

SummaryThe central step in the initiation of eukaryotic DNA replication is the loading of the minichromosome maintenance 2–7 (MCM2-7) complex, the core of the replicative DNA helicase, onto chromatin at replication origin. Here, we reported the cryo-EM structure of endogenous human single hexameric MCM2-7 complex with a resolution at 4.4 Å, typically an open-ring hexamer with a gap between Mcm2 and Mcm5. Strikingly, further analysis revealed that human MCM2-7 can self-associate to form a loose double hexamer which potentially implies a novel mechanism underlying the MCM2-7 loading in eukaryote. The high-resolution cryo-EM structure of human MCM2-7 is critical for understanding the molecular mechanisms governing human DNA replication, especially the MCM2-7 chromatin loading and pre-replicative complex assembly.

Opportunities and Challenges for In Silico Drug Discovery at Delta Opioid Receptors.

The delta opioid receptor is a Gi-protein-coupled receptor (GPCR) with a broad expression pattern both in the central nervous system and the body. The receptor has been investigated as a potential target for a multitude of significant diseases including migraine, alcohol use disorder, ischemia, and neurodegenerative diseases. Despite multiple attempts, delta opioid receptor-selective molecules have not been translated into the clinic. Yet, the therapeutic promise of the delta opioid receptor remains and thus there is a need to identify novel delta opioid receptor ligands to be optimized and selected for clinical trials. Here, we highlight recent developments involving the delta opioid receptor, the closely related mu and kappa opioid receptors, and in the broader area of the GPCR drug discovery research. We focus on the validity and utility of the available delta opioid receptor structures. We also discuss the increased ability to perform ultra-large-scale docking studies on GPCRs, the rise in high-resolution cryo-EM structures, and the increased prevalence of machine learning and artificial intelligence in drug discovery. Overall, we pose that there are multiple opportunities to enable in silico drug discovery at the delta opioid receptor to identify novel delta opioid modulators potentially with unique pharmacological properties, such as biased signaling.

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 channel reveal a novel binding site for positive modulators.

Kv3 ion-channels constitute a class of functionally distinct voltage-gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6Å resolution using full-length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well-resolved Zinc site and displaying a rotation by 35°. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator LuAG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage-gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite

Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the ‘ancient’ actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin ‘cofactors’, which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.

Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast α-Factor Receptor.

The Saccharomyces cerevisiae α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures.

Cryo-EM structure of the human somatostatin receptor 2 complex with its agonist somatostatin delineates the ligand-binding specificity.

Somatostatin is a peptide hormone that regulates endocrine systems by binding to G-protein-coupled somatostatin receptors. Somatostatin receptor 2 (SSTR2) is a human somatostatin receptor and is highly implicated in hormone disorders, cancers, and neurological diseases. Here, we report the high-resolution cryo-EM structure of full-length human SSTR2 bound to the agonist somatostatin (SST-14) in complex with inhibitory G (Gi) proteins. Our structural and mutagenesis analyses show that seven transmembrane helices form a deep pocket for ligand binding and that SSTR2 recognizes the highly conserved Trp-Lys motif of SST-14 at the bottom of the pocket. Furthermore, our sequence analysis combined with AlphaFold modeled structures of other SSTR isoforms provide a structural basis for the mechanism by which SSTR family proteins specifically interact with their cognate ligands. This work provides the first glimpse into the molecular recognition mechanism of somatostatin receptors and a crucial resource to develop therapeutics targeting somatostatin receptors.

Compositional complementarity between genomic RNA and coat proteins in positive-sense single-stranded RNA viruses.

During packaging in positive-sense single-stranded RNA (+ssRNA) viruses, coat proteins (CPs) interact directly with multiple regions in genomic RNA (gRNA), but the underlying physicochemical principles remain unclear. Here we analyze the high-resolution cryo-EM structure of bacteriophage MS2 and show that the gRNA/CP binding sites, including the known packaging signal, overlap significantly with regions where gRNA nucleobase-density profiles match the corresponding CP nucleobase-affinity profiles. Moreover, we show that the MS2 packaging signal corresponds to the global minimum in gRNA/CP interaction energy in the unstructured state as derived using a linearly additive model and knowledge-based nucleobase/amino-acid affinities. Motivated by this, we predict gRNA/CP interaction sites for a comprehensive set of 1082 +ssRNA viruses. We validate our predictions by comparing them with site-resolved information on gRNA/CP interactions derived in SELEX and CLIP experiments for 10 different viruses. Finally, we show that in experimentally studied systems CPs frequently interact with autologous coding regions in gRNA, in agreement with both predicted interaction energies and a recent proposal that proteins in general tend to interact with own mRNAs, if unstructured. Our results define a self-consistent framework for understanding packaging in +ssRNA viruses and implicate interactions between unstructured gRNA and CPs in the process.

Probing structural dynamics by mass spectrometry provides new insights into HIV's structural and antigenic diversity

High-resolution cryo-EM and crystal structures of the HIV Env glycoprotein captured in the prefusion closed conformation have revealed a high degree of structural similarity across diverse strains of HIV. Biophysical data however indicate that Env is a highly dynamic assembly, and the level of dynamics and conformational sampling embodied in the trimeric complex can vary dramatically between HIV strains. Sequence, structural, and antigenic variation in HIV Env is a major barrier to development of an effective HIV vaccine.

Heat-dependent opening of TRPV1 in the presence of capsaicin

Noxious heat (> 42°C) and capsaicin are two well-known stimuli for nociception. Our ability to sense noxious heat and spiciness is owed to the transient receptor potential vanilloid member 1 (TRPV1), a calcium-permeable ion channel expressed in primary sensory nerve terminals. TRPV1 also serves as a sensor for other vanilloid compounds and is a member of the TRP channel superfamily. Here we present six high-resolution cryo-EM structures of apo and capsaicin-bound full-length TRPV1 reconstituted into lipid nanodiscs over a range of temperatures.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics.

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.

High-resolution cryo-EM structures of the magnesium-inhibited and calcium-inactivated RyR1

RyR is an intracellular Ca2+ channel that also is gated by this ion. RyR1, the skeletal muscle isoform, is closed at submicromolar Ca2+ concentrations, maximally activated at ∼20-50 micromolar Ca2+, and begins to inactivate at ∼100 micromolar cytosolic Ca2+ concentrations. Mg2+, a divalent cation with a constant concentration in the cell, inhibits RyR1 at physiological (mM) concentrations. Functional studies using tritiated ryanodine binding of RyR1 under different conditions of Ca2+ and Mg2+ revealed a dominant effect of Mg2+ over a wide range of Ca2+ concentrations, and similar inhibiting effect of the two divalent cations at mM concentrations.

Structural basis for DNA targeting by the Tn7 transposon.

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with the viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). First we constructed atomic structure models of the initially bound and inserted state using recently resolved high-resolution cryo-EM structures for the SARS-CoV-2 RdRp.

High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

Ribosomes are complex and highly conserved ribonucleoprotein assemblies catalyzing protein biosynthesis in every organism. Here we present high-resolution cryo-EM structures of the 80S ribosome from a thermophilic fungus in two rotational states, which due to increased 80S stability provide a number of mechanistic details of eukaryotic translation. We identify a universally conserved ‘nested base-triple knot’ in the 26S rRNA at the polypeptide tunnel exit with a bulged-out nucleotide that likely serves as an adaptable element for nascent chain containment and handover. We visualize the structure and dynamics of the ribosome protective factor Stm1 upon ribosomal 40S head swiveling. We describe the structural impact of a unique and essential m1acp3 Ψ 18S rRNA hyper-modification embracing the anticodon wobble-position for eukaryotic tRNA and mRNA translocation. We complete the eEF2-GTPase switch cycle describing the GDP-bound post-hydrolysis state. Taken together, our data and their integration into the structural landscape of 80S ribosomes furthers our understanding of protein biogenesis.

Discovery and Characterization of Potent Dual P-Glycoprotein and CYP3A4 Inhibitors: Design, Synthesis, Cryo-EM Analysis, and Biological Evaluations.

Targeted concurrent inhibition of intestinal drug efflux transporter P-glycoprotein (P-gp) and drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is a promising approach to improve oral bioavailability of their common substrates such as docetaxel, while avoiding side effects arising from their pan inhibitions. Herein, we report the discovery and characterization of potent small molecule inhibitors of P-gp and CYP3A4 with encequidar (minimally absorbed P-gp inhibitor) as a starting point for optimization. To aid in the design of these dual inhibitors, we solved the high-resolution cryo-EM structure of encequidar bound to human P-gp. The structure guided us to prudently decorate the encequidar scaffold with CYP3A4 pharmacophores, leading to the identification of several analogues with dual potency against P-gp and CYP3A4. In vivo, dual P-gp and CYP3A4 inhibitor 3a improved the oral absorption of docetaxel by 3-fold as compared to vehicle, while 3a itself remained poorly absorbed.

Structure, mechanism, and inhibition of Hedgehog acyltransferase

SummaryThe Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism.

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding are only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin.

Structural basis of human ghrelin receptor signaling by ghrelin and the synthetic agonist ibutamoren

The hunger hormone ghrelin activates the ghrelin receptor GHSR to stimulate food intake and growth hormone secretion and regulate reward signaling. Acylation of ghrelin at Ser3 is required for its agonistic action on GHSR. Synthetic agonists of GHSR are under clinical evaluation for disorders related to appetite and growth hormone dysregulation. Here, we report high-resolution cryo-EM structures of the GHSR-Gi signaling complex with ghrelin and the non-peptide agonist ibutamoren as an investigational new drug. Our structures together with mutagenesis data reveal the molecular basis for the binding of ghrelin and ibutamoren. Structural comparison suggests a salt bridge and an aromatic cluster near the agonist-binding pocket as important structural motifs in receptor activation. Notable structural variations of the Gi and GHSR coupling are observed in our cryo-EM analysis. Our results provide a framework for understanding GHSR signaling and developing new GHSR agonist drugs.

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.