RESUMO
The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.
Assuntos
Proteínas Cromossômicas não Histona/química , Cromossomos/ultraestrutura , Microscopia Crioeletrônica/métodos , DNA/química , Proteínas de Escherichia coli/química , Proteínas Repressoras/química , Adenosina Trifosfatases/química , Proteínas de Ciclo Celular/química , Cromossomos Bacterianos , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Dimerização , Escherichia coli/metabolismo , Técnicas Genéticas , Genoma Bacteriano , Complexos Multiproteicos/química , Photorhabdus , Ligação Proteica , Conformação Proteica , Domínios Proteicos , CoesinasRESUMO
SMC proteins support vital cellular processes in all domains of life by organizing chromosomal DNA. They are composed of ATPase "head" and "hinge" dimerization domains and a connecting coiled-coil "arm." Binding to a kleisin subunit creates a closed tripartite ring, whose â¼47-nm-long SMC arms act as barrier for DNA entrapment. Here, we uncover another, more active function of the bacterial Smc arm. Using high-throughput genetic engineering, we resized the arm in the range of 6-60 nm and found that it was functional only in specific length regimes following a periodic pattern. Natural SMC sequences reflect these length constraints. Mutants with improper arm length or peptide insertions in the arm efficiently target chromosomal loading sites and hydrolyze ATP but fail to use ATP hydrolysis for relocation onto flanking DNA. We propose that SMC arms implement force transmission upon nucleotide hydrolysis to mediate DNA capture or loop extrusion.
Assuntos
Adenosina Trifosfatases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Bacterianos/enzimologia , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Engenharia Genética/métodos , Ensaios de Triagem em Larga Escala , Hidrólise , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Relação Estrutura-AtividadeRESUMO
Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase "head" and a "hinge" dimerization domain connected by a 49 nm coiled-coil "arm." The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Cisteína , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Relação Estrutura-AtividadeRESUMO
SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive "hinge" dimerization domain with an ATP-regulated "head" dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB's association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc's dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.
Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Ciclo Celular/ultraestrutura , DNA Bacteriano/química , Pyrococcus furiosus , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin-condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast.
Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Structural maintenance of chromosomes (SMC) complexes guard and organize the three-dimensional structure of chromosomal DNA across the tree of life. Many SMC functions can be explained by an inherent motor activity that extrudes large DNA loops while the complexes move along their substrate. Here, we review recent structural insights into the architecture and conservation of these molecular machines, their interaction with DNA, and the conformational changes that are linked to their ATP hydrolysis cycle.
Assuntos
Cromossomos , Complexos Multiproteicos , Complexos Multiproteicos/química , DNA/química , Proteínas de Ligação a DNA , Biologia , Proteínas de Ciclo Celular/genéticaRESUMO
Dynamins are a family of large GTPases that are involved in key cellular processes, where they mediate events of membrane fission and fusion. The dynamin superfamily is not restricted to eukaryotes but might have a bacterial origin, with many species containing an operon of two genes related to mitofusins. However, it is not clear whether bacterial dynamins promote membrane fission or fusion. The dynamin-like protein DynA of Bacillus subtilis is remarkable in that it arose from a gene fusion of two dynamins and contains two separate dynamin-like subunits and GTPase domains. We found that DynA exhibits strictly auto-regulated GTP hydrolysis, and that progress through the GTPase cycle is concerted within DynA oligomers. Furthermore, we show that DynA can tether membranes and mediates nucleotide-independent membrane fusion in vitro. This process merely requires magnesium as a cofactor. Our results provide a set of minimal requirements for membrane fusion by dynamin-like proteins and have mechanistic implications in particular for the fusion of mitochondria.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Dinaminas/metabolismo , Fusão de Membrana , Nucleotídeos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dinaminas/química , Dinaminas/genética , Guanosina Trifosfato/metabolismo , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estrutura Terciária de ProteínaRESUMO
During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA-FtsN interaction site in the IA-IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Actinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipídeos , Lipossomos , Proteínas de Membrana/metabolismo , Peptidoglicano/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Site-directed mutagenesis is a key tool in the analysis of biological mechanisms. We have established an efficient and systematic gene targeting strategy for Bacillus subtilis based on the Golden Gate cloning methodology. Our approach permits the introduction of single or multiple point mutations or of heavily engineered alleles into the endogenous gene locus in a single step using a 96-well microtiter plate format. We have successfully applied this system for high-throughput functional screening of resized variants of the Structural Maintenance of Chromosome (Smc) protein and for exhaustive cysteine cross-linking mutagenesis. Here we describe, in detail, the experimental setup for high-throughput introduction of modifications into the B. subtilis chromosome. With minor modifications, the approach should be applicable to other bacteria and yeast.
Assuntos
Bacillus subtilis/genética , Ensaios de Triagem em Larga Escala/métodos , Alelos , Cromossomos/genética , Mutagênese/genética , Mutagênese Sítio-Dirigida/métodos , Mutação Puntual/genéticaRESUMO
Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head-kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Escherichia coli/metabolismo , Dobramento de Proteína , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Escherichia coli , Conformação Proteica , Saccharomyces cerevisiae , CoesinasRESUMO
Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Bacterianos/química , DNA Primase/metabolismo , DNA Bacteriano/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Segregação de Cromossomos , Cromossomos Bacterianos/ultraestrutura , DNA Primase/genética , DNA Bacteriano/genética , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Alinhamento de SequênciaRESUMO
Smc-ScpAB forms elongated, annular structures that promote chromosome segregation, presumably by compacting and resolving sister DNA molecules. The mechanistic basis for its action, however, is only poorly understood. Here, we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc-ScpAB ring. To do so, we have chemically cross-linked the three ring interfaces in Smc-ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions. Exclusively species of Smc-ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA. DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc-ScpAB is loaded onto the chromosome at parS sites near the replication origin. We furthermore report a physical interaction between native Smc-ScpAB and chromosomal DNA fragments.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Bacterianos/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Bacillus subtilis , HidróliseRESUMO
The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Actinas/genética , Actinas/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Western Blotting , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Membrana Celular/química , Proteínas do Citoesqueleto/genética , Citoesqueleto/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Oxazinas/química , Transporte Proteico/efeitos dos fármacos , Ionóforos de Próton/farmacologia , Imagem com Lapso de TempoRESUMO
Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3's adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin's association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin's dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA/química , Mutação , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Eukaryotic structural maintenance of chromosomes (SMC)-kleisin complexes form large, ring-shaped assemblies that promote accurate chromosome segregation. Their asymmetric structural core comprises SMC heterodimers that associate with both ends of a kleisin subunit. However, prokaryotic condensin Smc-ScpAB is composed of symmetric Smc homodimers associated with the kleisin ScpA in a postulated symmetrical manner. Here, we demonstrate that Smc molecules have two distinct binding sites for ScpA. The N terminus of ScpA binds the Smc coiled coil, whereas the C terminus binds the Smc ATPase domain. We show that in Bacillus subtilis cells, an Smc dimer is bridged by a single ScpAB to generate asymmetric tripartite rings analogous to eukaryotic SMC complexes. We define a molecular mechanism that ensures asymmetric assembly, and we conclude that the basic architecture of SMC-kleisin rings evolved before the emergence of eukaryotes.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Complexos Multiproteicos/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Streptococcus pneumoniae/químicaRESUMO
Membrane dynamics are involved in crucial processes in eukaryotic and prokaryotic cells. Membrane fusion and fission events are often catalyzed by proteins that belong to the dynamin family of large GTPases. It has recently been shown that members of the dynamin superfamily are also present in many bacterial species. Although structural information about full length bacterial dynamin-like proteins is available, their molecular role remains unclear. We have shown previously that DynA, a dynamin-like protein found in the firmicute Bacillus subtilis is able to fuse membranes in vitro. In contrast to other members of the dynamin family this membrane remodeling activity was not dependent on guanosine nucleotides, but required magnesium. DynA assemblies localize in foci that are often enriched at sites of septation and hence a potential role during bacterial cytokinesis was discussed. In order to identify potential interaction partners we constructed a bacterial-two hybrid (B2H) library and screened for DynA interacting proteins. Three potential interaction partner have been identified, YneK, RNaseY (YmdA), and YwpG. Localization of these proteins phenocopies that of DynA, supporting the potential interaction in vivo.