RESUMEN
The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer's disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity. Within the ternary Hsp90/FKBP51/Tau complex, Hsp90 serves as a scaffold that traps the PPIase and nucleates multiple conformations of Tau's proline-rich region next to the PPIase catalytic pocket in a phosphorylation-dependent manner. Our study defines a conceptual model for dynamic Hsp90/co-chaperone/client recognition.
Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/toxicidad , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/toxicidad , Proteínas tau/química , Proteínas tau/toxicidad , Biocatálisis/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Conformación Proteica , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas tau/metabolismoRESUMEN
The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome's catalytic RNA network.
Asunto(s)
Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U5/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , ARN Helicasas DEAD-box/química , Activación Enzimática , Células HeLa , Humanos , Modelos Moleculares , Factores de Elongación de Péptidos/química , Conformación Proteica , ARN Helicasas/química , Proteínas de Unión al ARN/química , Ribonucleoproteínas Nucleares Pequeñas/química , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/metabolismo , Ubiquitina Tiolesterasa/químicaRESUMEN
As an enzyme superfamily, proteases are rivaled only by kinases in terms of their abundance within the human genome. Two ratiometric quantum dot (QD) Förster resonance energy transfer-based sensors designed to monitor the activity of the proteolytic enzymes collagenase and elastase are investigated here. Given the unique material constraints of these sensing constructs, assays are realized utilizing excess enzyme and fixed substrate in progress curve format to yield enzyme specificity or k cat/K m ratios. The range of k cat/Km values derived is 0.5-1.1 mM(-1) s(-1) for the collagenase sensor and 3.7-4.2 mM(-1) s(-1) for the elastase sensor. Of greater interest is the observation that the elastase sensor can be well represented by the Michaelis-Menten model while the collagenase sensor cannot. The latter demonstrates increased specificity at higher peptide substrate/QD loading values and an apparent QD-caused reversible inhibition as the reaction progresses. Understanding the detailed kinetic mechanisms that underpin these types of sensors will be important especially for their further quantitative utilization.
Asunto(s)
Técnicas Biosensibles , Puntos Cuánticos , Cinética , Proteolisis , Espectrometría de FluorescenciaRESUMEN
The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer's disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau-microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.
Asunto(s)
Microtúbulos/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Fenómenos Biofísicos , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Porcinos , Vinblastina/metabolismo , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
Splicing of precursor messenger RNA is a hallmark of eukaryotic cells, which is carried out by the spliceosome, a multi-megadalton ribonucleoprotein machinery. The splicing reaction removes non-coding regions (introns) and ligates coding regions (exons). The spliceosome is a highly dynamic ribonucleoprotein complex that undergoes dramatic structural changes during its assembly, the catalysis and its disassembly. The transitions between the different steps during the splicing cycle are promoted by eight conserved DExD/H box ATPases. The DEAH-box protein Prp43 is responsible for the disassembly of the intron-lariat spliceosome and its helicase activity is activated by the G-patch protein Ntr1. Here, we investigate the activation of Prp43 by Ntr1 in the presence and absence of RNA substrate by functional assays and structural proteomics. Residues 51-110 of Ntr1 were identified to be the minimal fragment that induces full activation. We found protein-protein cross-links that indicate that Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Additionally, we report on functionally important RNA binding residues in both proteins and propose a model for the activation of the helicase.
Asunto(s)
ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Activación Enzimática , Proteínas Intrínsecamente Desordenadas/química , Espectrometría de Masas , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , ARN/metabolismoRESUMEN
Misfolding of the natively α-helical prion protein into a ß-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.