Editorial: Technical Advances in Cryo-Electron Microscopy

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Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase

Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo–electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5′-O-(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation

Coupling Lipid Nanoparticle Structure and Automated Single Particle Composition Analysis to Design Phospholipase Responsive Nanocarriers

Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here we combine Single Particle Automated Raman Trapping Analysis (SPARTA®) with small angle scattering (SAXS / SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP - phospholipase D (PLD) interactions. Our analysis demonstrates that phospholipase D, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate was an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated

Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity

Human Hsp70 Disaggregase reverses Parkinson’s-linked α-Synuclein Amyloid Fibrils

Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson’s disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases

Cryo electron microscopy to determine the structure of macromolecular complexes

Cryo-electron microscopy (cryo-EM) is a structural molecular and cellular biology technique that has experienced major advances in recent years. Technological developments in image recording as well as in processing software make it possible to obtain three-dimensional reconstructions of macromolecular assemblies at near-atomic resolution that were formerly obtained only by X-ray crystallography or NMR spectroscopy. In parallel, cryo-electron tomography has also benefitted from these technological advances, so that visualization of irregular complexes, organelles or whole cells with their molecular machines in situ has reached subnanometre resolution. Cryo-EM can therefore address a broad range of biological questions. The aim of this review is to provide a brief overview of the principles and current state of the cryo-EM field

Structural arrangement of the GINS complex

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Studio sulla struttura genetica della popolazione spagnola con l'uso di alcuni polimorfismi VNTR ed STR.

RIASSUNTO I minisatelliti e i microsatelliti appartengono ad una classe dei sequenze del DNA ripetute in tandem chiamate VNTR (variable number tandem repeats) (Nakamura e coll. 1987). Visto il loro altissimo grado di polimorfismo trovano grande applicazione in medicina forense e, sempre più frequentemente, in studi di genetica di popolazioni. Le prime applicazioni di questi marcatori allo studio dell’evoluzione umana servirono a dimostrarne l’efficace forza di risoluzione descrittiva in questo campo. La Spagna, o per meglio dire la Penisola Iberica in generale, è una della regioni d’Europa meglio studiate dal punto di vista della variabilità genetica. Sono disponibili mappe sintetiche dei marcatori classici (Cavalli-Sforza e Bertranpetit, 1991), dati con i marcatori mitocondriali (Bertranpetit e Calafell, 1996-2000) e con i polimorfismi del cromosoma Y (Carracedo, Salas, Gonzáles-Neira, 2003). Attualmente vari gruppi di ricercatori effettuano un ampio campionamento della popolazione iberica al fine di immagazzinare dati con marcatori STR (short tandem repeats, microsatelliti) (Paredes et al.2004). La ricchezza di siti archeologici nel territorio spagnolo risalenti sia al Paleolitico che al Neolitico, il suo ruolo storico di ponte fra il mondo arabo e l’occidente, fanno della peniso-la Iberica un interessante campo di indagine per la Genetica di Popolazioni. Nel presente studio sono stati tipizzati 300 individui provenienti da varie zone della Spagna con cinque marcatori autosomici VNTR: tre minisatelliti (D1S80, D4S111,D17S5) e due microsatelliti (D8S566, D8S591). L’intero campione è stato analizzato secondo tre criteri distinti: - secondo un approccio storico-politico - secondo un approccio geografico - secondo un approccio linguistico. Questa scelta è stata dettata dalla necessità di inquadrare gli studi di popolazioni secondo una prospettiva multidisciplinare. Ad un secondo livello di analisi i risultati sulla popolazione in esame sono stati confrontati con i dati presenti in letteratura su altre popolazioni del bacino del Mediterraneo. Gli obiettivi della ricerca sono stati dunque: - tracciare un profilo della popolazione spagnola secondo criteri storico-politici, geo-grafici e linguistici - inserire la popolazione spagnola nel panorama genetico del bacino del Mediterra-neo - fornire dei dati su tale popolazione con nuovi marcatori molecolari autosomici. Dall’analisi dei dati è emersa la presenza di alcune sottostrutture all’interno della popola-zione spagnola: - è stata rilevata la presenza di una probabile componente neolitica nell’attuale pool genetico ispano, dato suffragato da evidenze archeologiche - è stata identificata una condizione di particolarità genetica della popolazione Anda-lusa, probabilmente dovuta al contributo delle popolazioni arabe durante la loro oc-cupazione di quest’area geografica. Per quanto riguarda la posizione della popolazione spagnola nel complesso del bacino del Mediterraneo, è stata osservata una situazione in accordo con la tesi dell’onda di avanza-mento dal Medio Oriente al resto d’Europa (Cavalli-Sforza, 1973). ABSTRACT Minisatellites and microsatellites are a genomic DNA class known as VNTR sequences (variable number tandem repeats) (Nakamura and coll. 1987). Because of their great polymorphism, they are a very powerful tool for forensic science and genetics of populations. Early uses of these markers to reconstruct human evolution were to show their power of description resolution. The Iberian Peninsula is one of the better studied areas in genetic variability. Genetic maps made using classical markers(Cavalli-Sforza e Bertranpetit, 1991), mitochondrial markers (Bertranpetit e Calafell, 1996-2000) and Y-polymorphisms (Carracedo, Salas, Gonzáles-Neira, 2003) are available. At the moment, many research team are working hard to provide data with STR (short tandem repeats) polymorphisms. Iberian Peninsula is an interesting investigation field for genetic of population due to the presence of archeological sites related to Paleolithic and Neolithic times. Moreover, from a historical point of view, it represented a bridge between Arabic cultures and western culture. In our dissertation five VNTR markers: 3 minisatellites and 2 microsatellites were analyzed in 300 unrelated individuals from all over Spain. Data were analyzed according to three different points of view: 1) according to a historical and political approach 2) according to a geographical approach 3) according to a linguistic approach. We decided to consider all these approaches because of the importance to study population from different points of view. Another goal of our dissertation is to locate Spanish population in the Mediterranean population genetic pattern. By combining our data we find some genetic substructure within Spanish population: 1) a probable Neolithic component in the present genetic Spanish pool. This is consistent with archeological data. 2) a particular genetic pool in Andalusian population probably due to 7 centuries of Arabic occupation. Location of the Spanish population in the Mediterranean genetic pattern is consistent with Demic diffusion theory (Cavalli-Sforza, 1973)

Electron cryo-microscopy of bacteriophage PR772 reveals the elusive vertex complex and the capsid architecture

Bacteriophage PR772, a member of the Tectiviridae family, has a 70 nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 angstrom, while the resolution of the protein capsid is 2.3 angstrom. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T = 25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection

Antibacterial peptide CyclomarinA creates toxicity by deregulating the Mycobacterium tuberculosis ClpC1–ClpP1P2 protease

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1–ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity.ISSN:0021-9258ISSN:1083-351