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1.
Sci Adv ; 10(15): eadk7678, 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38598631

RESUMEN

The Rpd3S complex plays a pivotal role in facilitating local histone deacetylation in the transcribed regions to suppress intragenic transcription initiation. Here, we present the cryo-electron microscopy structures of the budding yeast Rpd3S complex in both its apo and three nucleosome-bound states at atomic resolutions, revealing the exquisite architecture of Rpd3S to well accommodate a mononucleosome without linker DNA. The Rpd3S core, containing a Sin3 Lobe and two NB modules, is a rigid complex and provides three positive-charged anchors (Sin3_HCR and two Rco1_NIDs) to connect nucleosomal DNA. In three nucleosome-bound states, the Rpd3S core exhibits three distinct orientations relative to the nucleosome, assisting the sector-shaped deacetylase Rpd3 to locate above the SHL5-6, SHL0-1, or SHL2-3, respectively. Our work provides a structural framework that reveals a dynamic working model for the Rpd3S complex to engage diverse deacetylation sites.


Asunto(s)
Nucleosomas , Proteínas de Saccharomyces cerevisiae , Histonas/metabolismo , Microscopía por Crioelectrón , Metilación , Histona Desacetilasas/metabolismo , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Cell Res ; 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38658629

RESUMEN

Spliceosome is often assembled across an exon and undergoes rearrangement to span a neighboring intron. Most states of the intron-defined spliceosome have been structurally characterized. However, the structure of a fully assembled exon-defined spliceosome remains at large. During spliceosome assembly, the pre-catalytic state (B complex) is converted from its precursor (pre-B complex). Here we report atomic structures of the exon-defined human spliceosome in four sequential states: mature pre-B, late pre-B, early B, and mature B. In the previously unknown late pre-B state, U1 snRNP is already released but the remaining proteins are still in the pre-B state; unexpectedly, the RNAs are in the B state, with U6 snRNA forming a duplex with 5'-splice site and U5 snRNA recognizing the 3'-end of the exon. In the early and mature B complexes, the B-specific factors are stepwise recruited and specifically recognize the exon 3'-region. Our study reveals key insights into the assembly of the exon-defined spliceosomes and identifies mechanistic steps of the pre-B-to-B transition.

3.
Nat Commun ; 15(1): 1976, 2024 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-38438396

RESUMEN

Hemorrhagic toxin (TcsH) is a major virulence factor produced by Paeniclostridium sordellii, which is a non-negligible threat to women undergoing childbirth or abortions. Recently, Transmembrane Serine Protease 2 (TMPRSS2) was identified as a host receptor of TcsH. Here, we show the cryo-EM structures of the TcsH-TMPRSS2 complex and uncover that TcsH binds to the serine protease domain (SPD) of TMPRSS2 through the CROP unit-VI. This receptor binding mode is unique among LCTs. Five top surface loops of TMPRSS2SPD, which also determine the protease substrate specificity, constitute the structural determinants recognized by TcsH. The binding of TcsH inhibits the proteolytic activity of TMPRSS2, whereas its implication in disease manifestations remains unclear. We further show that mutations selectively disrupting TMPRSS2-binding reduce TcsH toxicity in the intestinal epithelium of the female mice. These findings together shed light on the distinct molecular basis of TcsH-TMPRSS2 interactions, which expands our knowledge of host recognition mechanisms employed by LCTs and provides novel targets for developing therapeutics against P. sordellii infections.


Asunto(s)
Serina Proteasas , Toxinas Biológicas , Embarazo , Femenino , Humanos , Animales , Ratones , Serina Proteasas/genética , Serina , Factores de Virulencia/genética , Clostridiales , Serina Endopeptidasas/genética
4.
Nat Struct Mol Biol ; 31(5): 835-845, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38196034

RESUMEN

Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.


Asunto(s)
Modelos Moleculares , Factores de Empalme de ARN , Empalmosomas , Humanos , Empalmosomas/metabolismo , Empalmosomas/química , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/genética , Microscopía por Crioelectrón , Empalme del ARN , Precursores del ARN/metabolismo , Conformación de Ácido Nucleico , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/química , Fosfoproteínas
5.
Nat Commun ; 14(1): 8426, 2023 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-38114525

RESUMEN

Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.


Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Clostridium sordellii , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridium sordellii/química , Clostridium sordellii/metabolismo , Exotoxinas/metabolismo , Metaloproteasas/metabolismo
6.
Cell Discov ; 9(1): 42, 2023 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-37076472

RESUMEN

The switch-independent 3 (SIN3)/histone deacetylase (HDAC) complexes play essential roles in regulating chromatin accessibility and gene expression. There are two major types of SIN3/HDAC complexes (named SIN3L and SIN3S) targeting different chromatin regions. Here we present the cryo-electron microscopy structures of the SIN3L and SIN3S complexes from Schizosaccharomyces pombe (S. pombe), revealing two distinct assembly modes. In the structure of SIN3L, each Sin3 isoform (Pst1 and Pst3) interacts with one histone deacetylase Clr6, and one WD40-containing protein Prw1, forming two lobes. These two lobes are bridged by two vertical coiled-coil domains from Sds3/Dep1 and Rxt2/Png2, respectively. In the structure of SIN3S, there is only one lobe organized by another Sin3 isoform Pst2; each of the Cph1 and Cph2 binds to an Eaf3 molecule, providing two modules for histone recognition and binding. Notably, the Pst1 Lobe in SIN3L and the Pst2 Lobe in SIN3S adopt similar conformation with their deacetylase active sites exposed to the space; however, the Pst3 Lobe in SIN3L is in a compact state with its active center buried inside and blocked. Our work reveals two classical organization mechanisms for the SIN3/HDAC complexes to achieve specific targeting and provides a framework for studying the histone deacetylase complexes.

7.
Nat Struct Mol Biol ; 30(6): 753-760, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37081318

RESUMEN

SIN3-HDAC (histone deacetylases) complexes have important roles in facilitating local histone deacetylation to regulate chromatin accessibility and gene expression. Here, we present the cryo-EM structure of the budding yeast SIN3-HDAC complex Rpd3L at an average resolution of 2.6 Å. The structure reveals that two distinct arms (ARM1 and ARM2) hang on a T-shaped scaffold formed by two coiled-coil domains. In each arm, Sin3 interacts with different subunits to create a different environment for the histone deacetylase Rpd3. ARM1 is in the inhibited state with the active site of Rpd3 blocked, whereas ARM2 is in an open conformation with the active site of Rpd3 exposed to the exterior space. The observed asymmetric architecture of Rpd3L is different from those of available structures of other class I HDAC complexes. Our study reveals the organization mechanism of the SIN3-HDAC complex and provides insights into the interaction pattern by which it targets histone deacetylase to chromatin.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Factores de Transcripción/metabolismo , Proteínas Represoras/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cromatina , Histona Desacetilasas/genética
8.
Mol Cell ; 83(8): 1328-1339.e4, 2023 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-37028420

RESUMEN

Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising four subunits: TSEN2, TSEN15, TSEN34, and TSEN54. Here, we report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states at average resolutions of 2.94 and 2.88 Å, respectively. Human TSEN features an extended surface groove that holds the L-shaped pre-tRNA. The mature domain of pre-tRNA is recognized by conserved structural elements of TSEN34, TSEN54, and TSEN2. Such recognition orients the anticodon stem of pre-tRNA and places the 3'-splice site and 5'-splice site into the catalytic centers of TSEN34 and TSEN2, respectively. The bulk of the intron sequences makes no direct interaction with TSEN, explaining why pre-tRNAs of varying introns can be accommodated and cleaved. Our structures reveal the molecular ruler mechanism of pre-tRNA cleavage by TSEN.


Asunto(s)
Endorribonucleasas , Precursores del ARN , Humanos , Intrones/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Endorribonucleasas/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , Conformación de Ácido Nucleico , Endonucleasas/genética
9.
Nat Commun ; 14(1): 897, 2023 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-36797247

RESUMEN

Three RNA helicases - DDX42, DDX46 and DHX15 - are found to be associated with human U2 snRNP, but their roles and mechanisms in U2 snRNP and spliceosome assembly are insufficiently understood. Here we report the cryo-electron microscopy (cryo-EM) structures of the DDX42-SF3b complex and a putative assembly precursor of 17S U2 snRNP that contains DDX42 (DDX42-U2 complex). DDX42 is anchored on SF3B1 through N-terminal sequences, with its N-plug occupying the RNA path of SF3B1. The binding mode of DDX42 to SF3B1 is in striking analogy to that of DDX46. In the DDX42-U2 complex, the N-terminus of DDX42 remains anchored on SF3B1, but the helicase domain has been displaced by U2 snRNA and TAT-SF1. Through in vitro assays, we show DDX42 and DDX46 are mutually exclusive in terms of binding to SF3b. Cancer-driving mutations of SF3B1 target the residues in the RNA path that directly interact with DDX42 and DDX46. These findings reveal the distinct roles of DDX42 and DDX46 in assembly of 17S U2 snRNP and provide insights into the mechanisms of SF3B1 cancer mutations.


Asunto(s)
Neoplasias , Empalmosomas , Humanos , Empalmosomas/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Microscopía por Crioelectrón , Unión Proteica , ARN Nuclear Pequeño/metabolismo , Neoplasias/metabolismo , Empalme del ARN , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo
10.
Mol Cell ; 82(15): 2769-2778.e4, 2022 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-35705093

RESUMEN

Pre-mRNA splicing involves two sequential reactions: branching and exon ligation. The C complex after branching undergoes remodeling to become the C∗ complex, which executes exon ligation. Here, we report cryo-EM structures of two intermediate human spliceosomal complexes, pre-C∗-I and pre-C∗-II, both at 3.6 Å. In both structures, the 3' splice site is already docked into the active site, the ensuing 3' exon sequences are anchored on PRP8, and the step II factor FAM192A contacts the duplex between U2 snRNA and the branch site. In the transition of pre-C∗-I to pre-C∗-II, the step II factors Cactin, FAM32A, PRKRIP1, and SLU7 are recruited. Notably, the RNA helicase PRP22 is positioned quite differently in the pre-C∗-I, pre-C∗-II, and C∗ complexes, suggesting a role in 3' exon binding and proofreading. Together with information on human C and C∗ complexes, our studies recapitulate a molecular choreography of the C-to-C∗ transition, revealing mechanistic insights into exon ligation.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Empalmosomas , Exones/genética , Humanos , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN Nuclear Pequeño/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo
11.
Science ; 376(6598): eabl8280, 2022 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-35679404

RESUMEN

INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo-electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo-electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo-electron microscopy (cryo-EM) to image the intact NPC from the NE of Xenopus laevis oocytes. Reconstructions of the core region and the Nup358 region of the X. laevis CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the X. laevis NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the ß-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named "the clamp," hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 ß-propellers may strengthen the scaffold. CONCLUSION Our EM map-based model of the X. laevis CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. [Figure: see text].


Asunto(s)
Proteínas de Complejo Poro Nuclear , Poro Nuclear , Proteínas de Xenopus , Xenopus laevis , Animales , Microscopía por Crioelectrón , Citoplasma/metabolismo , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Conformación Proteica , Proteínas de Xenopus/química , Xenopus laevis/metabolismo
12.
Cell Res ; 32(5): 451-460, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35301439

RESUMEN

Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy structure of the inner ring (IR) subunit from the Xenopus laevis NPC at an average resolution of 4.2 Å. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin hetero-trimer of Nup62/58/54 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure reveals the molecular basis for the IR subunit assembly of a vertebrate NPC.


Asunto(s)
Poro Nuclear , Proteínas de Xenopus , Transporte Activo de Núcleo Celular , Animales , Microscopía por Crioelectrón , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
13.
Cell ; 185(6): 980-994.e15, 2022 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-35303428

RESUMEN

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.


Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Clostridioides difficile/genética , Lipoproteínas/genética
14.
Cell Res ; 32(4): 349-358, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35177819

RESUMEN

Nuclear pore complex (NPC) shuttles cargo across the nuclear envelope. Here we present single-particle cryo-EM structure of the nuclear ring (NR) subunit from Xenopus laevis NPC at an average resolution of 5.6 Å. The NR subunit comprises two 10-membered Y complexes, each with the nucleoporin ELYS closely associating with Nup160 and Nup37 of the long arm. Unlike the cytoplasmic ring (CR) or inner ring (IR), the NR subunit contains only one molecule each of Nup205 and Nup93. Nup205 binds both arms of the Y complexes and interacts with the stem of inner Y complex from the neighboring subunit. Nup93 connects the stems of inner and outer Y complexes within the same NR subunit, and places its N-terminal extended helix into the axial groove of Nup205 from the neighboring subunit. Together with other structural information, we have generated a composite atomic model of the central ring scaffold that includes the NR, IR, and CR. The IR is connected to the two outer rings mainly through Nup155. This model facilitates functional understanding of vertebrate NPC.


Asunto(s)
Proteínas de Complejo Poro Nuclear , Poro Nuclear , Animales , Microscopía por Crioelectrón , Citoplasma/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
15.
Nat Commun ; 12(1): 4871, 2021 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-34381056

RESUMEN

The heteromeric complex between PKD1L3, a member of the polycystic kidney disease (PKD) protein family, and PKD2L1, also known as TRPP2 or TRPP3, has been a prototype for mechanistic characterization of heterotetrametric TRP-like channels. Here we show that a truncated PKD1L3/PKD2L1 complex with the C-terminal TRP-fold fragment of PKD1L3 retains both Ca2+ and acid-induced channel activities. Cryo-EM structures of this core heterocomplex with or without supplemented Ca2+ were determined at resolutions of 3.1 Å and 3.4 Å, respectively. The heterotetramer, with a pseudo-symmetric TRP architecture of 1:3 stoichiometry, has an asymmetric selectivity filter (SF) guarded by Lys2069 from PKD1L3 and Asp523 from the three PKD2L1 subunits. Ca2+-entrance to the SF vestibule is accompanied by a swing motion of Lys2069 on PKD1L3. The S6 of PKD1L3 is pushed inward by the S4-S5 linker of the nearby PKD2L1 (PKD2L1-III), resulting in an elongated intracellular gate which seals the pore domain. Comparison of the apo and Ca2+-loaded complexes unveils an unprecedented Ca2+ binding site in the extracellular cleft of the voltage-sensing domain (VSD) of PKD2L1-III, but not the other three VSDs. Structure-guided mutagenic studies support this unconventional site to be responsible for Ca2+-induced channel activation through an allosteric mechanism.


Asunto(s)
Canales de Calcio/química , Calcio/metabolismo , Receptores de Superficie Celular/química , Canales Catiónicos TRPP/química , Aminoácidos , Animales , Sitios de Unión , Calcio/química , Canales de Calcio/genética , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Activación del Canal Iónico , Ratones , Mutagénesis , Conformación Proteica , Dominios Proteicos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo
16.
Nat Commun ; 12(1): 4541, 2021 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-34315898

RESUMEN

Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A ß-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.


Asunto(s)
Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/ultraestructura , Proteína Wnt3A/ultraestructura , Receptores Frizzled/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Conformación Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3A/química , Proteína Wnt3A/metabolismo
17.
Nat Commun ; 11(1): 5739, 2020 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-33159063

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

18.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32636384

RESUMEN

SWI/SNF remodelers play a key role in regulating chromatin architecture and gene expression. Here, we report the cryo-EM structure of the Saccharomyces cerevisiae Swi/Snf complex in a nucleosome-free state. The structure consists of a stable triangular base module and a flexible Arp module. The conserved subunits Swi1 and Swi3 form the backbone of the complex and closely interact with other components. Snf6, which is specific for yeast Swi/Snf complex, stabilizes the binding of the ATPase-containing subunit Snf2 to the base module. Comparison of the yeast Swi/Snf and RSC complexes reveals conserved structural features that govern the assembly and function of these two subfamilies of chromatin remodelers. Our findings complement those from recent structures of the yeast and human chromatin remodelers and provide further insights into the assembly and function of the SWI/SNF remodelers.


Asunto(s)
Adenosina Trifosfatasas/química , Cromatina/química , Proteínas Cromosómicas no Histona/química , Proteínas Nucleares/química , Proteínas Represoras/química , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/química , Microscopía por Crioelectrón , Proteínas de Unión al ADN/química , Humanos , Nucleosomas , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/química
19.
Proc Natl Acad Sci U S A ; 117(18): 9876-9883, 2020 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-32303654

RESUMEN

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryogenic electron microscopy (cryo-EM) structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 Å. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex.


Asunto(s)
Proteínas Relacionadas con la Autofagia/ultraestructura , Proteína C9orf72/ultraestructura , Proteínas Portadoras/ultraestructura , Complejos Multiproteicos/ultraestructura , Secuencia de Aminoácidos/genética , Esclerosis Amiotrófica Lateral/genética , Arginina/genética , Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Proteína C9orf72/genética , Proteínas Portadoras/genética , Microscopía por Crioelectrón , Filaminas/genética , Filaminas/ultraestructura , Demencia Frontotemporal/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/ultraestructura , Predisposición Genética a la Enfermedad , Humanos , Complejos Multiproteicos/genética , Alineación de Secuencia , Proteínas de Unión al GTP rab/genética
20.
Annu Rev Biochem ; 89: 333-358, 2020 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31815536

RESUMEN

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.


Asunto(s)
Factores de Empalme de ARN/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Empalmosomas/metabolismo , Dominio Catalítico , Secuencia Conservada , Exones , Humanos , Intrones , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/química , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Empalmosomas/ultraestructura
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