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

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

Investigation and validation of a cubic turbulence model in isothermal and combusting flows

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