RESUMO
The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.
Assuntos
Microscopia Crioeletrônica , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.
Assuntos
Microscopia Crioeletrônica , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Animais , Humanos , Ratos , Ligantes , Endocitose , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/genética , Erros Inatos do Transporte Tubular Renal , Miopia , Hérnias Diafragmáticas Congênitas , Proteinúria , Perda Auditiva NeurossensorialRESUMO
Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca2+ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca2+ state, revealing a new conformation for Ca2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca2+ -bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+ -ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca2+ . We propose a novel mechanism of ATP binding to SERCA2b.
Assuntos
Trifosfato de Adenosina/química , Microscopia Crioeletrônica , Modelos Moleculares , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Hidrólise , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Relação Estrutura-AtividadeRESUMO
The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 ß-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1-3. Each Tom40 ß-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.
Assuntos
Microscopia Crioeletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Mitocôndrias/química , Proteínas de Transporte da Membrana Mitocondrial/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Fosfolipídeos/metabolismo , Multimerização Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Mitochondrial ß-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic ß-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last ß-strand (ß-signal) of the substrate-the 19-ß-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first ß-segment (ß1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces ß1 to promote its pairing with Tom40's last ß-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial ß-barrel folding.
Assuntos
Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Microscopia Crioeletrônica , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.2-3.1 Å-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in Zn2+-bound and unbound forms. Cryo-EM analyses show that hZnT7 exists as a dimer via tight interactions in both the cytosolic and transmembrane (TM) domains of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing conformation and widens the luminal cavity for Zn2+ release in the outward-facing conformation. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 binds two Zn2+ ions, seemingly facilitating Zn2+ recruitment to the TM metal transport pathway. These structures permit mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi to be proposed.
Assuntos
Proteínas de Transporte , Complexo de Golgi , Humanos , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Complexo de Golgi/metabolismo , Zinco/metabolismoRESUMO
Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ into the endoplasmic reticulum (ER). Herein, we present cryo-electron microscopy (EM) structures of three intermediates of SERCA2b: Ca2+-bound phosphorylated (E1P·2Ca2+) and Ca2+-unbound dephosphorylated (E2·Pi) intermediates and another between the E2P and E2·Pi states. Our cryo-EM analysis demonstrates that the E1P·2Ca2+ state exists in low abundance and preferentially transitions to an E2P-like structure by releasing Ca2+ and that the Ca2+ release gate subsequently undergoes stepwise closure during the dephosphorylation processes. Importantly, each intermediate adopts multiple sub-state structures including those like the next one in the catalytic series, indicating conformational overlap at transition steps, as further substantiated by atomistic molecular dynamic simulations of SERCA2b in a lipid bilayer. The present findings provide insight into how enzymes accelerate catalytic cycles.
Assuntos
Microscopia CrioeletrônicaRESUMO
Cardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations are implicated in severe arrhythmogenic diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remains unknown. Here, we address the gating mechanism of RyR2 by combining high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated two fundamental mechanisms for channel gating: interactions close to the channel pore stabilize the channel to prevent hyperactivity and a series of interactions in the surrounding regions is necessary for channel opening upon Ca2+ binding. Mutations at the residues involved in the former and the latter mechanisms cause gain-of-function and loss-of-function, respectively. Our results reveal gating mechanisms of the RyR2 channel and alterations by pathogenic mutations at the atomic level.
Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Microscopia Crioeletrônica , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Supramolecular protein nanoparticles and nanocages have potential in a broad range of applications. Recently, we developed a uniform supramolecular protein nanoparticle, TIP60, symmmetrically self-assembled from fusion proteins of a pentameric Sm-like protein and a dimeric MyoX-coil domain. Herein, we report the icosahedral 60-meric structure of TIP60 solved using single-particle cryo-electron microscopy. Interestingly, the structure revealed 20 regular-triangle-like pores on the surface. TIP60 and its mutants have many modifiable sites on their exterior and interior surfaces. The TIP60 architecture will be useful in the development of biomedical and biochemical nanoparticles/nanocages for future applications.
Assuntos
Nanopartículas/química , Proteínas/química , Substâncias Macromoleculares/química , Modelos Moleculares , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.
Assuntos
Proteínas Reguladoras de Apoptose/química , Basigina/química , Membrana Celular/metabolismo , Proteínas de Membrana/química , Fosfolipídeos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Basigina/metabolismo , Microscopia Crioeletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/química , Estrutura Terciária de Proteína , Triptofano/químicaRESUMO
Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo-electron microscopy (cryo-EM) structures of human SERCA2b in E1â2Ca2+-adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3 - states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
Assuntos
Autofagossomos/química , Proteínas Relacionadas à Autofagia/química , Proteínas de Membrana/química , Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Sítios de Ligação , Transporte Biológico , Microscopia Crioeletrônica , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Vermelha FluorescenteRESUMO
Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. These compounds protect mice against lethal autoimmunity. Combining crystallography and cryo-electron microscopy, we identify the open conformation of the receptor and reveal the structural equilibrium between open and closed conformations that underlies TLR7 antagonism, as well as the detailed mechanism by which TLR7-specific antagonists bind to their binding pocket in TLR7. Our work provides small-molecule TLR7-specific antagonists and suggests the TLR7-targeting strategy for treating autoimmune diseases.
Assuntos
Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/química , Animais , Doenças Autoimunes , Autoimunidade , Sítios de Ligação , Microscopia Crioeletrônica , Feminino , Ligantes , Lúpus Eritematoso Sistêmico , Camundongos , Camundongos Endogâmicos NZB , Modelos Moleculares , Conformação Proteica , Taxa de SobrevidaRESUMO
Soluble immunoglobulin M (IgM) forms a pentamer containing a joining (J) chain polypeptide. While IgM pentamer has various immune functions, it also behaves as a carrier of circulating apoptosis inhibitor of macrophage (AIM; also called CD5L) protein that facilitates repair during different diseases. AIM binds to the IgM pentamer solely in the presence of the J chain. Here, using a single-particle negative-stain electron microscopy, we found that the IgM pentamer exhibits an asymmetric pentagon containing one large gap, which is markedly different from the textbook symmetric pentagon model. A single AIM molecule specifically fits into the gap, cross-bridging two IgM-Fc that form the edges of the gap through a disulfide bond at one side and a charge-based interaction at the other side. The discovery of the bona fide shape of the IgM pentamer advances our structural understanding of the pentameric IgM and its binding mode with AIM.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Membrana Celular/metabolismo , Imunoglobulina M/química , Imunoglobulina M/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Animais , Membrana Celular/química , Conformação Proteica , Receptores DepuradoresRESUMO
Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.
Assuntos
Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Sequência de Aminoácidos , Humanos , Ativação do Canal Iônico , Proteínas de Membrana/fisiologia , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Constitutive NF-κB activation has causative roles in adult T cell leukemia (ATL) caused by HTLV-1 and other cancers. Here, we report a pathway involving Polycomb-mediated miRNA silencing and NF-κB activation. We determine the miRNA signatures and reveal miR-31 loss in primary ATL cells. MiR-31 negatively regulates the noncanonical NF-κB pathway by targeting NF-κB inducing kinase (NIK). Loss of miR-31 therefore triggers oncogenic signaling. In ATL cells, miR-31 level is epigenetically regulated, and aberrant upregulation of Polycomb proteins contribute to miR-31 downregulation in an epigenetic fashion, leading to activation of NF-κB and apoptosis resistance. Furthermore, this emerging circuit operates in other cancers and receptor-initiated NF-κB cascade. Our findings provide a perspective involving the epigenetic program, inflammatory responses, and oncogenic signaling.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma de Células T do Adulto/genética , MicroRNAs/genética , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/genética , Linfócitos T/patologia , Epigênese Genética , Perfilação da Expressão Gênica , Humanos , Leucemia-Linfoma de Células T do Adulto/metabolismo , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas do Grupo Polycomb , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Transdução de Sinais , Linfócitos T/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Drosophila melanogaster has two Dicer proteins with specialized functions. Dicer-1 liberates miRNA-miRNA* duplexes from precursor miRNAs (pre-miRNAs), whereas Dicer-2 processes long double-stranded RNAs into small interfering RNA duplexes. It was recently demonstrated that Dicer-2 is rendered highly specific for long double-stranded RNA substrates by inorganic phosphate and a partner protein R2D2. However, it remains unclear how Dicer-1 exclusively recognize pre-miRNAs. Here we show that fly Dicer-1 recognizes the single-stranded terminal loop structure of pre-miRNAs through its N-terminal helicase domain, checks the loop size and measures the distance between the 3' overhang and the terminal loop. This unique mechanism allows fly Dicer-1 to strictly inspect the authenticity of pre-miRNA structures.