Cryo-EM Structure Research Articles

Cryo-EM structures of ABCC1 revealing new conformational dynamics in the resting state

ABCC1/MRP1 in the C branch of Adenosine triphosphate binding cassette (ABC) transporters superfamily, is directly linked to multiple drug resistance in chemotherapy. Here, to further understand the conformational dynamics of ABCC1, we performed single-particle cryo-electron microscopy analysis of purified bovine ABCC1. Two conformational states were found coexisted with nearly equal population. While one state has a wider substrate transporting pathway, akin to the previously reported apo structure, the other is narrower, despite the empty substrate pocket. In addition, multiple lipid-binding interfaces were identified based on the presence of rod-shaped, unmodeled, non-protein densities in the resolved density maps, potentially contributing to the stabilization of TMD0 domain and activity regulation of ABCC1. Further, we found that three asparagine residues in bovine ABCC1 are glycosylated. Together, our study provides fresh insights into the structural features and conformational dynamics of bovine ABCC1, offering a new framework for understanding the function and regulatory mechanisms of ABCC1.

Architecture of a pentameric assembly of the tail tube protein in SPR phages

Abstract The majority of phages, viruses that infect prokaryotes, deliver their genomic material into host cells via a tail structure. Previous research has established that the central tube of these phages, composed of tail tube protein (TTP), is structurally conserved, typically forming either hexameric or trimeric rings. In this study, we revealed a novel pentameric assembly of TTP and present two cryo-EM structures at resolutions of 3.5 Å and 3.7 Å, respectively. Our detailed structural analysis demonstrates that the inner surface of the pentameric tube is highly negatively charged. Critical residues located on the loop between β3 and β4 play pivotal roles in the formation of the pentameric rings, and mismatches of the interactions between the stacked layers can induce the curvature of the tube. The cryo-EM structure of the TTP polymer at the tube’s end uncovered that the β-strands that span amino acids 27-65 move towards the central tunnel, potentially blocking the tunnel through which the phage genome is released. Our study provides new structural insights into a novel assembly of TTP, which will extend the understanding of phage assembly.

Mycobacterium tuberculosis Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for Mtb's adaptation to the human host environment. Our study focuses on one notable Mtb repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for Mtb survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of mce3R orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the yrbE3A (mce3) operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a Kd of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.

RNA sample optimization for cryo-EM analysis.

RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.

Bound by the love for cholesterol: A transporter meets a GPCR.

In a recently published article in Nature, Bayly-Jones etal. report the cryo-EM structures of a lysosomal cholesterol sensor, LYCHOS, also known as GPR155, which reveals a unique fusion of a plant auxin-transporter-like domain with a seven-transmembrane GPCR-like domain and elucidates mechanistic insights into cellular regulation of mTORC1 activity.

Binding Mode of Cyclic Chemerin-9 Peptide and ChemerinS157 Protein at CMKLR1.

The chemokine-like receptor 1 (CMKLR1) is activated by the adipokine and chemoattractant protein chemerin. Cryo-EM structures of chemerin-9-CMKLR1-Gi have been published, where chemerin-9 is the nonapeptide of the C terminus of chemerinS157. Chemerin-9 is as active as the full-length protein in Ca2+-release but shows differences in equilibrium read-outs. An equally potent cyclic chemerin-9 variant (cC9) was reported previously. Now, we have built a computational model of CMKLR1 to investigate the binding mode of cC9 and chemerinS157 in comparison to chemerin-9. Differences were investigated using CMKLR1 variants. Double-mutant cycle analysis identified CMKLR1-F2.53 as the relevant position for Phe8-binding of cC9. Energy contribution revealed slight differences in Phe8-binding to CMKLR1-F2.53 and space for larger residues. This was confirmed as the chemerin-9 variant with 1-naphthyl-L-alanine at position 8 showed a 4-fold increased potency of 2 nM (pEC50=8.6±0.15). While chemerin-9 and cC9 share their interactions at the CMKLR1, chemerinS157 tolerates most mutations of CMKLR1 in the deep binding site. The computational model of chemerinS157 suggests a β-sheet interaction between the N-terminal CMKLR1-segment I25VVL28 and the β-sheet D108KVLGRLVH116 of ChemS157, which was confirmed experimentally. Our data add to the knowledge by identifying the binding mode of chemerinS157 and cC9 at CMKLR1, facilitating the future structure-based drug design.

Cryo-EM of human rhinovirus reveals capsid-RNA duplex interactions that provide insights into virus assembly and genome uncoating.

The cryo-EM structure of the human rhinovirus B14 determined in this study reveals 13-bp RNA duplexes symmetrically bound to regions around each of the 30 two-fold axes in the icosahedral viral capsid. The RNA duplexes (~12% of the ssRNA genome) define a quasi-dodecahedral cage that line a substantial part of the capsid interior surface. The RNA duplexes establish a complex network of non-covalent interactions with pockets in the capsid inner wall, including coulombic interactions with a cluster of basic amino acid residues that surround each RNA duplex. A direct comparison was made between the cryo-EM structure of RNA-filled virions and that of RNA-free (empty) capsids that resulted from genome release from a small fraction of viruses. The comparison reveals that some specific residues involved in capsid-duplex RNA interactions in the virion undergo remarkable conformational rearrangements upon RNA release from the capsid. RNA release is also associated with the asynchronous opening of channels at the 30 two-fold axes. The results provide further insights into the molecular mechanisms leading to assembly of rhinovirus particles and their genome uncoating during infection. They may also contribute to development of novel antiviral strategies aimed at interfering with viral capsid-genome interactions during the infectious cycle.

Inhibition mechanism of potential antituberculosis compound lansoprazole sulfide

Tuberculosis is one of the most common causes of death worldwide, with a rapid emergence of multi-drug-resistant strains underscoring the need for new antituberculosis drugs. Recent studies indicate that lansoprazole-a known gastric proton pump inhibitor and its intracellular metabolite, lansoprazole sulfide (LPZS)-are potential antituberculosis compounds. Yet, their inhibitory mechanism and site of action still remain unknown. Here, we combine biochemical, computational, and structural approaches to probe the interaction of LPZS with the respiratory chain supercomplex III2IV2 of Mycobacterium smegmatis, a close homolog of Mycobacterium tuberculosis supercomplex. We show that LPZS binds to the Qo cavity of the mycobacterial supercomplex, inhibiting the quinol substrate oxidation process and the activity of the enzyme. We solve high-resolution (2.6 Å) cryo-electron microscopy (cryo-EM) structures of the supercomplex with bound LPZS that together with microsecond molecular dynamics simulations, directed mutagenesis, and functional assays reveal key interactions that stabilize the inhibitor, but also how mutations can lead to the emergence of drug resistance. Our combined findings reveal an inhibitory mechanism of LPZS and provide a structural basis for drug development against tuberculosis.

LARP1 binds ribosomes and TOP mRNAs in repressed complexes.

Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies have implicated LARP1 in 40S- or 80S-ribosome complexes that are thought to repress and stabilize TOPs. However, a molecular understanding of how LARP1 and TOPs interact with these ribosome complexes is lacking. Here, we show that LARP1 directly binds non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the mRNA channel of the 40S subunit. Increased availability of free ribosomal subunits downstream of various stresses promote 60S joining at the same interface to form LARP1-80S complexes. Simultaneously, LARP1 engages the TOP via its previously characterized La/PAM2 and DM15 domains. Contrary to expectations, ribosome binding within these complexes is not required for LARP1-mediated TOP repression or stabilization, two canonical LARP1 functions. Together, this work provides molecular insight into how LARP1 directly binds ribosomal subunits and challenges existing models describing the function of repressed LARP1-40S/80S-TOP complexes.

Structural basis of MICAL autoinhibition.

MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.

Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair.

The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist-a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems.

The N-terminal region of DNMT3A engages the nucleosome surface to aid chromatin recruitment.

DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs.

Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome.

Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being trafficked to the cell membrane at different extents and capable of binding PIP2 (phosphatidylinositol-4,5-bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K+ current, albeit through different molecular mechanisms. Coexpression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K+ flow. In addition, the cryo-EM structure of the Kir2.1-R312H mutant has been reconstructed. This study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS.

HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2

In vertebrate spindles, most microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation through enhanced localization to TPX2 condensates, which form the core of the branch site on microtubules. Lastly, we provide a high-resolution cryo-EM structure of HURP on the microtubule, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

Structural basis of 3'-tRNA maturation by the human mitochondrial RNase Z complex.

Maturation of human mitochondrial tRNA is essential for cellular energy production, yet the underlying mechanisms remain only partially understood. Here, we present several cryo-EM structures of the mitochondrial RNase Z complex (ELAC2/SDR5C1/TRMT10C) bound to different maturation states of mitochondrial tRNAHis, showing the molecular basis for tRNA-substrate selection and catalysis. Our structural insights provide a molecular rationale for the 5'-to-3' tRNA processing order in mitochondria, the 3'-CCA antideterminant effect, and the basis for sequence-independent recognition of mitochondrial tRNA substrates. Furthermore, our study links mutations in ELAC2 to clinically relevant mitochondrial diseases, offering a deeper understanding of the molecular defects contributing to these conditions.

A proteolytic AAA+ machine poised to unfold protein substrates

AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP’s ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX’s ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.

Cryo-EM structure of a novel α-synuclein filament subtype from multiple system atrophy.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by accumulation of α-synuclein cross-β amyloid filaments in the brain. Previous structural studies of these filaments by cryo-electron microscopy (cryo-EM) revealed three discrete folds distinct from α-synuclein filaments associated with other neurodegenerative diseases. Here, we use cryo-EM to identify a novel, low-populated MSA filament subtype (designated Type I2) in addition to a predominant class comprising MSA Type II2 filaments. The 3.3-Å resolution structure of the Type I2 filament reveals a fold consisting of two asymmetric protofilaments, one of which adopts a novel structure that is chimeric between two previously reported protofilaments. These results further define MSA-specific folds of α-synuclein filaments and have implications for designing MSA diagnostics and therapeutics.

Cryo-EM structures of the membrane repair protein dysferlin

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

Cryo-EM structure of a lysozyme-derived amyloid fibril from hereditary amyloidosis

Systemic ALys amyloidosis is a debilitating protein misfolding disease that arises from the formation of amyloid fibrils from C-type lysozyme. We here present a 2.8 Å cryo-electron microscopy structure of an amyloid fibril, which was isolated from the abdominal fat tissue of a patient who expressed the D87G variant of human lysozyme. We find that the fibril possesses a stable core that is formed by all 130 residues of the fibril precursor protein. There are four disulfide bonds in each fibril protein that connect the same residues as in the globularly folded protein. As the conformation of lysozyme in the fibril is otherwise fundamentally different from native lysozyme, our data provide a structural rationale for the need of protein unfolding in the development of systemic ALys amyloidosis.

An all-atom model of the human cardiac sodium channel in a lipid bilayer

Voltage-gated sodium channels (NaV) are complex macromolecular proteins that are responsible for the initial upstroke of an action potential in excitable cells. Appropriate function is necessary for many physiological processes such as heartbeat, voluntary muscle contraction, nerve conduction, and neurological function. Dysfunction can have life-threatening consequences. During the past decade, there have been significant advancements with ion channel structural characterization by CryoEM, yet descriptions of cytosolic components are often lacking. Many investigations have biophysically characterized reconstituted cytosolic components and their interactions. However, extrapolating the structural alterations and allosteric communication within an intact ion channel can be challenging. To address this, we have developed an all-atom model of the human cardiac sodium channel (NaV1.5) in a lipid bilayer with explicit salt and water. Our simulations contain descriptions of cytosolic components that are poorly predicted by AlphaFold and lacking in many CryoEM structures. Leveraging the latest advancements of the Amber force fields (ff19sb and Lipid21) and water model (OPC), our simulations improved protein backbone torsion angles and generated structural information across time (four independent one-microsecond simulations). Our analysis provided descriptions of lipid and solvent contacts and insight into the C-Terminal Domain - inactivation gate and inactivation gate - latch receptor interactions.