RESUMEN
Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.
RESUMEN
Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.
Asunto(s)
Proteínas Arqueales/genética , Segregación Cromosómica , Cromosomas de Archaea/genética , ADN de Archaea/genética , Sulfolobus solfataricus/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestructura , Cristalografía por Rayos X , ADN de Archaea/química , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Sulfolobus solfataricus/metabolismoRESUMEN
In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba. sphaeroides complex shows that the PufX C-terminus, particularly the region from X49-X53, likely mediates association of core monomers. To investigate this putative interaction we analysed mutations PufX R49L, PufX R53L, PufX R49/53L and PufX G52L by measuring photosynthetic growth, fractionation of detergent-solubilised membranes, formation of 2-D crystals and electron microscopy. We show that these mutations do not affect assembly of PufX within the core or photosynthetic growth but they do prevent dimerization, consistent with predictions from the RC-LH1-PufX structure. We obtained low resolution structures of monomeric core complexes with and without PufX, using electron microscopy of negatively stained single particles and 3D reconstruction; the monomeric complex with PufX corresponds to one half of the dimer structure whereas LH1 completely encloses the RC if the gene encoding PufX is deleted. On the basis of the insights gained from these mutagenesis and structural analyses we propose a sequence for assembly of the dimeric RC-LH1-PufX complex.
Asunto(s)
Proteínas Bacterianas/fisiología , Complejos de Proteína Captadores de Luz/química , Rhodobacter sphaeroides/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Benzoquinonas/metabolismo , Cristalización , Dimerización , Hidroquinonas/metabolismo , Procesamiento de Imagen Asistido por Computador , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/fisiología , Complejos de Proteína Captadores de Luz/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Mutación Missense , Mutación Puntual , Conformación Proteica , Dominios Proteicos , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/efectos de la radiaciónRESUMEN
Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here, we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The C domain of ParB harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, which suggests the possible conservation of DNA segregation principles across the three domains of life.
Asunto(s)
Proteínas Arqueales/química , Centrómero/química , Segregación Cromosómica , Cromosomas de Archaea/genética , ADN de Archaea/genética , Sulfolobus/genética , Secuencias de Aminoácidos , Proteínas Arqueales/genética , Autoantígenos/química , Autoantígenos/genética , Bacterias/genética , Centrómero/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , ADN de Archaea/química , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , Kluyveromyces/genética , Conformación de Ácido Nucleico , Estructura Terciaria de ProteínaRESUMEN
Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αß subunits, with two BChls sandwiched between each αß pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 ß polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αß subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH2 exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc1 complex.
Asunto(s)
Proteínas Bacterianas/química , Complejos de Proteína Captadores de Luz/química , Modelos Moleculares , Rhodobacter sphaeroides/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacterioclorofila A/análisis , Bacterioclorofila A/química , Bacterioclorofila A/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Carotenoides/análisis , Carotenoides/química , Carotenoides/metabolismo , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Espectrometría de Masas , Oxidación-Reducción , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Difracción de Rayos XRESUMEN
Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A(2A) receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson's disease.
Asunto(s)
Antagonistas del Receptor de Adenosina A2/síntesis química , Antiparkinsonianos/síntesis química , Piridinas/síntesis química , Receptor de Adenosina A2A/metabolismo , Triazinas/síntesis química , Antagonistas del Receptor de Adenosina A2/farmacocinética , Antagonistas del Receptor de Adenosina A2/farmacología , Administración Oral , Animales , Antiparkinsonianos/farmacocinética , Antiparkinsonianos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Modelos Moleculares , Conformación Proteica , Piridinas/farmacocinética , Piridinas/farmacología , Ensayo de Unión Radioligante , Ratas , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Triazinas/farmacocinética , Triazinas/farmacologíaRESUMEN
Carotenoids play important roles in photosynthesis where they are involved in light-harvesting, in photo-protection and in the assembly and structural stability of light-harvesting and reaction centre complexes. In order to examine the effects of carotenoids on the oligomeric state of the reaction centre-light-harvesting 1 -PufX (RC-LH1-PufX) core complex of Rhodobacter sphaeroides two carotenoid-less mutants, TC70 and R-26, were studied. Detergent fractionation showed that in the absence of carotenoids LH2 complexes do not assemble, as expected, but also that core complexes are predominantly found as monomers, although levels of the PufX polypeptide appeared to be unaffected. Analysis of R-26 membranes by electron microscopy and atomic force microscopy reveals arrays of hexagonally packed monomeric RC-LH1-PufX complexes. Transfer of the crtB gene encoding phytoene synthase to TC70 and R-26 restores the normal synthesis of carotenoids demonstrating that the R-26 mutant of Rba. sphaeroides harbours a mutation in crtB, among its other defects. The transconjugant TC70 and R-26 strains containing crtB had regained their ability to assemble wild-type levels of dimeric RC-LH1-PufX core complexes and normal energy transfer pathways were restored, demonstrating that carotenoids are essential for the normal assembly and function of both the LH2 and RC-LH1-PufX complexes in this bacterial photosystem.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Carotenoides/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Mutación , Rhodobacter sphaeroides/metabolismo , Transferasas Alquil y Aril/genética , Western Blotting , Dimerización , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Complejos de Proteína Captadores de Luz/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Rhodobacter sphaeroides/enzimologíaRESUMEN
In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.
Asunto(s)
Citoplasma/metabolismo , Membranas Intracelulares/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Rhodobacter sphaeroides/fisiología , Secuencia de Bases , Cartilla de ADN , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismoRESUMEN
The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC-LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC-LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC-LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and ß polypeptides on the other half of the dimer.