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1.
Cell ; 184(21): 5448-5464.e22, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34624221

RESUMO

Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Conformação de Ácido Nucleico , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformação Proteica , Coesinas
2.
EMBO J ; 38(9)2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877095

RESUMO

SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptídeos/metabolismo , Proteínas SecA/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo , Proteínas SecA/química
3.
J Mol Biol ; 427(14): 2348-59, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25982945

RESUMO

Post-translational protein translocation across the bacterial plasma membrane is mediated by the interplay of the SecA ATPase and the protein-conducting SecY channel. SecA consists of several domains, including two nucleotide-binding domains (NBD1 and NBD2), a polypeptide cross-linking domain (PPXD), a helical scaffold domain (HSD), and a helical wing domain (HWD). PPXD, HSD, and NBD2 form a clamp that positions the polypeptide substrate above the channel so that it can be pushed into the channel by a two-helix finger of the HSD. How the substrate is accommodated in the clamp during translocation is unclear. Here, we report a crystal structure of Thermotoga maritima SecA at 1.9 Å resolution. Structural analysis and free-energy calculations indicate that the new structure represents an intermediate state during the transition of the clamp from an open to a closed conformation. Molecular dynamics simulations show that closure of the clamp occurs in two phases, an initial movement of PPXD, HSD, and HWD as a unit, followed by a movement of PPXD alone toward NBD2. Simulations in the presence of a polypeptide chain show that the substrate associates with the back of the clamp by dynamic hydrogen bonding and that the clamp is laterally closed by a conserved loop of the PPXD. Mutational disruption of clamp opening or closure abolishes protein translocation. These results suggest how conformational changes of SecA allow substrate binding and movement during protein translocation.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Movimento , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Thermotoga maritima/enzimologia
4.
J Cell Sci ; 128(6): 1217-29, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25616894

RESUMO

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.


Assuntos
Produtos Biológicos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Ascomicetos/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HCT116 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Canais de Translocação SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
5.
J Biol Chem ; 289(35): 24611-6, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25016015

RESUMO

While engaged in protein transport, the bacterial translocon SecYEG must maintain the membrane barrier to small ions. The preservation of the proton motif force was attributed to (i) cation exclusion, (ii) engulfment of the nascent chain by the hydrophobic pore ring, and (iii) a half-helix partly plugging the channel. In contrast, we show here that preservation of the proton motif force is due to a voltage-driven conformational change. Preprotein or signal peptide binding to the purified and reconstituted SecYEG results in large cation and anion conductivities only when the membrane potential is small. Physiological values of membrane potential close the activated channel. This voltage-dependent closure is not dependent on the presence of the plug domain and is not affected by mutation of 3 of the 6 constriction residues to glycines. Cellular ion homeostasis is not challenged by the small remaining leak conductance.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas , Transporte Proteico , Canais de Translocação SEC
6.
Cell ; 157(6): 1416-1429, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24906156

RESUMO

In bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here we show that SecA moves polypeptides through the SecY channel by a "push and slide" mechanism. In its ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state or when SecA encounters a poorly interacting amino acid in its ATP-bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed push and slide mechanism is supported by a mathematical model and explains how SecA allows translocation of a wide range of polypeptides. This mechanism may also apply to hexameric polypeptide-translocating ATPases.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA
7.
Proc Natl Acad Sci U S A ; 106(49): 20800-5, 2009 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-19933328

RESUMO

Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a "clamp" of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Adenosina Trifosfatases/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Reagentes de Ligações Cruzadas/farmacologia , Dissulfetos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Ligação Proteica/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC , Proteínas SecA , Tetra-Hidrofolato Desidrogenase/metabolismo
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