RESUMEN
Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus nucleocapsid offers important insights into the mechanism of the helical polymerization of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design.
Asunto(s)
Virus del Sarampión/ultraestructura , Sarampión/virología , Nucleocápside/ultraestructura , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Humanos , Virus del Sarampión/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleocápside/química , Proteínas de la Nucleocápside , Nucleoproteínas/química , Nucleoproteínas/ultraestructura , Estructura Secundaria de Proteína , ARN Viral/química , ARN Viral/ultraestructura , Proteínas Virales/química , Proteínas Virales/ultraestructuraRESUMEN
Human transthyretin has an intrinsic tendency to form amyloid fibrils and is heavily implicated in senile systemic amyloidosis. Here, detailed neutron structural studies of perdeuterated transthyretin are described. The analyses, which fully exploit the enhanced visibility of isotopically replaced hydrogen atoms, yield new information on the stability of the protein and the possible mechanisms of amyloid formation. Residue Ser117 may play a pivotal role in that a single water molecule is closely associated with the γ-hydrogen atoms in one of the binding pockets, and could be important in determining which of the two sites is available to the substrate. The hydrogen-bond network at the monomer-monomer interface is more extensive than that at the dimer-dimer interface. Additionally, the edge strands of the primary dimer are seen to be favourable for continuation of the ß-sheet and the formation of an extended cross-ß structure through sequential dimer couplings. It is argued that the precursor to fibril formation is the dimeric form of the protein.
RESUMEN
Preliminary studies of perdeuterated crystals of human transthyretin (TTR) have been carried out using the LADI-III and D19 diffractometers at the Institut Laue-Langevin in Grenoble. The results demonstrate the feasibility of a full crystallographic analysis to a resolution of 2.0 Å using Laue diffraction and also illustrate the potential of using monochromatic instruments such as D19 for higher resolution studies where larger crystals having smaller unit cells are available. This study will yield important information on hydrogen bonding, amino-acid protonation states and hydration in the protein. Such information will be of general interest for an understanding of the factors that stabilize/destabilize TTR and for the design of ligands that may be used to counter TTR amyloid fibrillogenesis.
Asunto(s)
Prealbúmina/química , Humanos , Enlace de Hidrógeno , Difracción de Neutrones , Agua/químicaRESUMEN
In Bacteria and Archaea, high-affinity potassium uptake is mediated by the ATP-driven KdpFABC complex. On the basis of the biochemical properties of the ATP-hydrolyzing subunit KdpB, the transport complex is classified as type IA P-type ATPase. However, the KdpA subunit, which promotes K(+) transport, clearly resembles a potassium channel, such that the KdpFABC complex represents a chimera of ion pumps and ion channels. In the present study, we demonstrate that the blending of these two groups of transporters in KdpFABC also entails a nucleotide-binding mechanism in which the KdpC subunit acts as a catalytic chaperone. This mechanism is found neither in P-type ATPases nor in ion channels, although parallels are found in ABC transporters. In the latter, the ATP nucleotide is coordinated by the LSGGQ signature motif via double hydrogen bonds at a conserved glutamine residue, which is also present in KdpC. High-affinity nucleotide binding to the KdpFABC complex was dependent on the presence of this conserved glutamine residue in KdpC. In addition, both ATP binding to KdpC and ATP hydrolysis activity of KdpFABC were sensitive to the accessibility, presence or absence of the hydroxyl groups at the ribose moiety of the nucleotide. Furthermore, the KdpC subunit was shown to interact with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, thereby increasing the ATP-binding affinity by the formation of a transient KdpB/KdpC/ATP ternary complex.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades de Proteína/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Biocatálisis , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutamina/metabolismo , Enlace de Hidrógeno , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
Yeast RNA polymerase III is recruited upon binding of subcomplexes tauA and tauB of transcription factor IIIC (TFIIIC) to the A and B blocks of tRNA gene promoters. The tauB subcomplex consists of subunits tau60, tau91, and tau138. We determined the 3.2 A crystal structure of tau60 bound to a large C-terminal fragment of tau91 (Deltatau91). Deltatau91 protein contains a seven-bladed propeller preceded by an N-terminal extension, whereas tau60 contains a structurally homologous propeller followed by a C-terminal domain with a novel alpha/beta fold. The two propeller domains do not have any detectable DNA binding activity and mediate heterodimer formation that may serve as scaffold for tau138 assembly. We show that the C-terminal tau60 domain interacts with the TATA binding protein (TBP). Recombinant tauB recruits TBP and stimulates TFIIIB-directed transcription on a TATA box containing tRNA gene, implying a combined contribution of tauA and tauB to preinitiation complex formation.
Asunto(s)
Factores de Transcripción TFIII/química , Factores de Transcripción TFIII/fisiología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Transcripción GenéticaRESUMEN
P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe(377) and Lys(395) via a pi-pi stacking and a cation-pi interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg(317), Arg(382), Asp(399), and Glu(348)) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Potasio/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Proteínas de Transporte de Catión/metabolismo , Proteínas de Escherichia coli/metabolismo , Transporte Iónico/fisiología , Modelos Químicos , Mutación , Resonancia Magnética Nuclear Biomolecular , Nucleótidos/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Electricidad EstáticaRESUMEN
P-type ATPases are amongst the most abundant enzymes that are responsible for active transport of ions across biological membranes. Within the last 5 years a detailed picture of the structure and function of these transport ATPases has emerged. Here, we report on the recent progress in elucidating the molecular mechanism of a unique, prokaryotic member of P-type ATPases, the Kdp-ATPase. The review focuses on the catalytic parts of the central subunit, KdpB. The structure of the nucleotide-binding domain was solved by NMR spectroscopy at high resolution and a model of the nucleotide-binding mode was presented. The nucleotide turned out to be 'clipped' into the binding pocket by a pi-pi interaction to F377 on one side and a cation-pi interaction to K395 on the other. The 395KGXXD/E motif and thus the nucleotide-binding mode seems to be conserved in all P-type ATPases, except the heavy metal-transporting (class IB) ATPases. Hence, it can be concluded that KdpB is currently misgrouped as class IA. Mutational studies on two highly conserved residues (D583 and K586) in the transmembrane helix 5 of KdpB revealed that they are indispensable in coupling ATP hydrolysis to ion translocation. Based on these results, two possible pathways for the reaction cycle are discussed.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Filogenia , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico/fisiología , Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Transporte Iónico/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
P-type ATPases are involved in the active transport of ions across biological membranes. The KdpFABC complex (P-type ATPase) of Escherichia coli is a high-affinity K+ uptake system that operates only when the cell experiences osmotic stress or K+ limitation. Here, we present the solution structure of the nucleotide binding domain of KdpB (backbone RMSD 0.17 A) and a model of the AMP-PNP binding mode based on intermolecular distance restraints. The calculated AMP-PNP binding mode shows the purine ring of the nucleotide to be "clipped" into the binding pocket via a pi-pi-interaction to F377 on one side and a cation-pi-interaction to K395 on the other. This binding mechanism seems to be conserved in all P-type ATPases, except the heavy metal transporting ATPases (type IB). Thus, we conclude that the Kdp-ATPase (currently type IA) is misgrouped and has more similarities to type III ATPases. The KdpB N-domain is the smallest and simplest known for a P-type ATPase, and represents a minimal example of this functional unit. No evidence of significant conformational changes was observed within the N-domain upon nucleotide binding, thus ruling out a role for ATP-induced conformational changes in the reaction cycle.