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1.
Nucleic Acids Res ; 48(18): 10045-10075, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32894288

RESUMO

ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time we provide a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. We present a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Adenosina Trifosfatases , Evolução Molecular , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/classificação , Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/classificação , Adenosina Trifosfatases/fisiologia , Bactérias/enzimologia , Eucariotos/enzimologia , Nucleoproteínas/metabolismo
2.
Nat Struct Mol Biol ; 27(8): 743-751, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661420

RESUMO

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.


Assuntos
Proteínas de Transporte/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
3.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636384

RESUMO

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.


Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Humanos , Nucleossomos , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química
4.
Nucleic Acids Res ; 48(14): 7991-8005, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32621607

RESUMO

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Geobacillus stearothermophilus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , DNA , DNA Helicases/química , DNA Helicases/isolamento & purificação , Desoxirribonuclease I/química , Desoxirribonuclease I/isolamento & purificação
5.
Nat Commun ; 11(1): 2728, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483114

RESUMO

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Conformação Proteica , Multimerização Proteica , Transativadores/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenol/metabolismo , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(26): 14970-14977, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541053

RESUMO

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Humanos , Peroxissomos/genética , Peroxissomos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Transporte Proteico
7.
Mol Cell ; 79(1): 99-114.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32445620

RESUMO

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodos
8.
Science ; 368(6498): 1454-1459, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32409525

RESUMO

As a ring-shaped adenosine triphosphatase (ATPase) machine, cohesin organizes the eukaryotic genome by extruding DNA loops and mediates sister chromatid cohesion by topologically entrapping DNA. How cohesin executes these fundamental DNA transactions is not understood. Using cryo-electron microscopy (cryo-EM), we determined the structure of human cohesin bound to its loader NIPBL and DNA at medium resolution. Cohesin and NIPBL interact extensively and together form a central tunnel to entrap a 72-base pair DNA. NIPBL and DNA promote the engagement of cohesin's ATPase head domains and ATP binding. The hinge domains of cohesin adopt an "open washer" conformation and dock onto the STAG1 subunit. Our structure explains the synergistic activation of cohesin by NIPBL and DNA and provides insight into DNA entrapment by cohesin.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , DNA/química , Microscopia Crioeletrônica , Humanos , Domínios Proteicos , Multimerização Proteica
9.
Nucleic Acids Res ; 48(12): 6980-6995, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453425

RESUMO

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one ß-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/ultraestrutura , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Conformação Proteica
10.
Nucleic Acids Res ; 48(11): 6326-6339, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374860

RESUMO

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Archaeoglobus fulgidus/química , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Soluções , Eletricidade Estática , Sulfolobus/química
11.
Nucleic Acids Res ; 48(9): 4769-4779, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232335

RESUMO

The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Caulobacter crescentus/enzimologia , Proteínas de Ligação a DNA/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Difusão , Espectrometria de Massa com Troca Hidrogênio-Deutério , Mutação , Ligação Proteica
12.
Biochem Biophys Res Commun ; 525(3): 668-674, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32139119

RESUMO

Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the internal carotid arteries. Genetic studies originally identified RNF213 as an MMD susceptibility gene that encodes a large 591 kDa protein with a functional RING domain and dual AAA+ ATPase domains. As the functions of RNF213 and its relationship to MMD onset are unknown, we set out to characterize the ubiquitin ligase activity of RNF213, and the effects of MMD patient mutations on these activities and on other cellular processes. In vitro ubiquitination assays, using the RNF213 RING domain, identified Ubc13/Uev1A as a key ubiquitin conjugating enzyme that together generate K63-linked polyubiquitin chains. However, nearly all MMD patient mutations in the RING domain greatly reduced this activity. When full-length proteins were overexpressed in HEK293T cells, patient mutations that abolished the ubiquitin ligase activities conversely enhanced nuclear factor κB (NFκB) activation and induced apoptosis accompanied with Caspase-3 activation. These induced activities were dependent on the RNF213 AAA+ domain. Our results suggest that the NFκB- and apoptosis-inducing functions of RNF213 may be negatively regulated by its ubiquitin ligase activity and that disruption of this regulation could contribute towards MMD onset.


Assuntos
Domínio AAA , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Apoptose , Doença de Moyamoya/genética , Mutação/genética , NF-kappa B/metabolismo , Domínios RING Finger , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Células HEK293 , Humanos , Lisina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Poliubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
13.
Nature ; 579(7799): 438-442, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132705

RESUMO

Condensin, a key component of the structure maintenance of chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA1. It remains unclear, however, how condensin complexes work together to collectively package DNA into chromosomes. Here we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these motor proteins, which, individually, extrude DNA in one direction only are able to dynamically change each other's DNA loop sizes, even when far apart. When they are in close proximity, condensin complexes are able to traverse each other and form a loop structure, which we term a Z-loop-three double-stranded DNA helices aligned in parallel with one condensin at each edge. Z-loops can fill gaps left by single loops and can form symmetric dimer motors that pull in DNA from both sides. These findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae , Adenosina Trifosfatases/química , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/química , Cromossomos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Motores Moleculares/química , Complexos Multiproteicos/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Imagem com Lapso de Tempo
14.
Mol Cell Biol ; 40(12)2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32205407

RESUMO

Maintenance of protein homeostasis in eukaryotes under normal growth and stress conditions requires the functions of Hsp70 chaperones and associated cochaperones. Here, we investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phosphomimetic substitutions at this site (S151D) dramatically downregulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phosphomimetic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 cochaperones Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phosphomimetic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [PSI+ ] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Sítios de Ligação , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSP70/química , Humanos , Metais Pesados/metabolismo , Fosforilação , Agregados Proteicos , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteostase , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Estresse Fisiológico
15.
Proc Natl Acad Sci U S A ; 117(14): 7814-7823, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198203

RESUMO

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an "ADP-like" undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70-Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Conformação Proteica , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Transferência Ressonante de Energia de Fluorescência , Heterogeneidade Genética , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/química , Humanos , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Ligação Proteica/genética , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica/genética
16.
Arch Biochem Biophys ; 685: 108350, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32220566

RESUMO

Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Potássio/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
17.
Nature ; 579(7799): 448-451, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188943

RESUMO

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and -1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity5. The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13.


Assuntos
Microscopia Crioeletrônica , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Drosophila melanogaster , Humanos , Camundongos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Xenopus laevis
18.
Nucleic Acids Res ; 48(5): 2762-2776, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009148

RESUMO

OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC-Escherichia coli hosts, indicating that both the ATPase and nuclease activities are required for OLD function in vivo.


Assuntos
Trifosfato de Adenosina/metabolismo , Biocatálise , Endonucleases/química , Endonucleases/metabolismo , Thermus/enzimologia , Adenosina Trifosfatases/química , Sequência Conservada , Hidrólise , Metais/metabolismo , Modelos Moleculares , Mutação/genética , Domínios Proteicos
19.
Nat Struct Mol Biol ; 27(3): 233-239, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066964

RESUMO

Genome regulation requires control of chromosome organization by SMC-kleisin complexes. The cohesin complex contains the Smc1 and Smc3 subunits that associate with the kleisin Scc1 to form a ring-shaped complex that can topologically engage chromatin to regulate chromatin structure. Release from chromatin involves opening of the ring at the Smc3-Scc1 interface in a reaction that is controlled by acetylation and engagement of the Smc ATPase head domains. To understand the underlying molecular mechanisms, we have determined the 3.2-Šresolution cryo-electron microscopy structure of the ATPγS-bound, heterotrimeric cohesin ATPase head module and the 2.1-Šresolution crystal structure of a nucleotide-free Smc1-Scc1 subcomplex from Saccharomyces cerevisiae and Chaetomium thermophilium. We found that ATP-binding and Smc1-Smc3 heterodimerization promote conformational changes within the ATPase that are transmitted to the Smc coiled-coil domains. Remodeling of the coiled-coil domain of Smc3 abrogates the binding surface for Scc1, thus leading to ring opening at the Smc3-Scc1 interface.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaetomium/química , Chaetomium/genética , Chaetomium/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
20.
Science ; 367(6481): 1039-1042, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32108112

RESUMO

The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.


Assuntos
Actinas/química , Adenosina Trifosfatases/química , Glucoquinase/química , Hexoquinase/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Glucoquinase/genética , Hexoquinase/genética , Polimerização , Proteínas de Saccharomyces cerevisiae/genética
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