Structure of the Hsp110:Hsc70 Nucleotide Exchange Machine

Schuermann, Jonathan P.; Jiang, Jianwen; Cuéllar, Jorge; Llorca, Óscar; Wang, Liping; Giménez, Luis E.; Jin, Suping; Taylor, Alexander B.; Demeler, Borries; Morano, Kevin A.; Hart, P. John; Valpuesta, José M.; Lafer, Eileen M.; Sousa, Rui

doi:10.1016/j.molcel.2008.05.006

Cited by 197 publications

(284 citation statements)

References 42 publications

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“…S1). This agrees well with previous studies that have shown Hsc70 bound to the free triskelia (14,15,22) and supports the idea previously proposed that Hsc70 acts to chaperone the released clathrin triskelia back to the plasma membrane (25), preventing formation of empty cages within the cell. It should be noted that, in a similar previous study, Schuermann et al (22) also observed a second, slower linear phase of disassembly following the initial fast exponential phase.…”

Section: Discussionsupporting

confidence: 93%

“…Using a simple perpendicular light-scattering assay we have measured the in vitro disassembly of clathrin cages by Hsc70 and auxilin, which occurs rapidly with a t 1∕2 of approximately 10 s. This is comparable to recent results from experiments that used dynamic light scattering to monitor clathrin cage disassembly (21,22) but faster than earlier centrifugation-based studies that had t 1∕2 values ranging from 2-10 min (19,20,24) but that also contained adaptor proteins such as AP180 or AP2, which are known to stabilize the cages and may consequently have slowed down disassembly. In this study, we have increased the time resolution beyond that of dynamic light scattering by monitoring perpendicular light scattering, and analysis of these data has allowed us to isolate and describe individual steps in the chaperone-mediated disassembly of cages that hitherto have remained invisible.…”

Section: Discussionsupporting

confidence: 73%

“…It was recently demonstrated that dynamic light scattering can be used effectively to monitor clathrin cage disassembly (21,22), thus providing better time resolution than previous studies that were predominantly based on centrifugation and densitometry of SDS-PAGE (19,20). We have further increased the time resolution by which disassembly kinetics can be measured, by monitoring simple perpendicular light scattering using stopped-flow methods to capture events on the milliseconds-to-seconds timescale.…”

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confidence: 99%

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A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

Rothnie

Clarke

Kuzmič³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.clathrin-mediated endocytosis | kinetic mechanism | macromolecular assemblies | vesicle uncoating | mathematical model

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 73%

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confidence: 99%

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A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

Rothnie

Clarke

Kuzmič³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous RDC analysis demonstrated that in solution the ADP-bound NBD exists in the "closed" conformation (10). X-ray structures of Hsp70 NBDs bound to their respective NEFs (12)(13)(14)(15)24), which enhance the rate of nucleotide release and binding, show subdomain IIB to be rotated about 10 to 30°with respect to the rest of the NBD and thus opening of the nucleotide-binding cleft ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…Some insight into the nature of the conformational landscape of Hsp70 NBDs comes from X-ray structures of their complexes with nucleotide exchange factors (NEFs), which show an opening of the nucleotide-binding pocket relative to other NBD structures conformation (12)(13)(14)(15). Additionally, the recently determined X-ray structure of ATP-bound yeast Hsp110, Sse1, a distant relative of the Hsp70s, emerges as a plausible model for the Hsp70 ATP-bound conformation.…”

mentioning

confidence: 99%

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

Zhuravleva

Gierasch

2011

Proc. Natl. Acad. Sci. U.S.A.

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198

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The 70-kDa heat shock protein (Hsp70) chaperones perform a wide array of cellular functions that all derive from the ability of their N-terminal nucleotide-binding domains (NBDs) to allosterically regulate the substrate affinity of their C-terminal substrate-binding domains in a nucleotide-dependent mechanism. To explore the structural origins of Hsp70 allostery, we performed NMR analysis on the NBD of DnaK, the Escherichia coli Hsp70, in six different states (ligand-bound or apo) and in two constructs, one that retains the conserved and functionally crucial portion of the interdomain linker (residues 389 VLLL 392 ) and another that lacks the linker. Chemical-shift perturbation patterns identify residues at subdomain interfaces that constitute allosteric networks and enable the NBD to act as a nucleotide-modulated switch. Nucleotide binding results in changes in subdomain orientations and long-range perturbations along subdomain interfaces. In particular, our findings provide structural details for a key mechanism of Hsp70 allostery, by which information is conveyed from the nucleotide-binding site to the interdomain linker. In the presence of ATP, the linker binds to the edge of the IIA β-sheet, which structurally connects the linker and the nucleotide-binding site. Thus, a pathway of allosteric communication leads from the NBD nucleotide-binding site to the substrate-binding domain via the interdomain linker. T he 70-kDa heat shock proteins (Hsp70s) compose one of the most well studied and ubiquitously distributed families of allosteric proteins (1). Hsp70s assist in an extraordinarily broad spectrum of cellular processes, including protein folding, disaggregation, and translocation. All chaperone activities of Hsp70s are based on their ability to interact with short hydrophobic peptide segments of protein substrate in an ATP-dependent fashion. Hsp70s contain two domains-a 44-kDa N-terminal nucleotidebinding domain (NBD) and a 15-kDa C-terminal substrate-binding domain (SBD)-connected by a highly conserved hydrophobic linker. The allosteric cycle of Hsp70s involves an alternation between the ATP-bound state with low affinity and fast exchange rates for substrates, and the ADP-bound state with high affinity and low exchange rates for substrates. In turn, substrate binding to the SBD results in about 10-fold stimulation of ATPase activity of the NBD. However, the same ATPase stimulation can be achieved for the isolated NBD in the presence of the conserved interdomain linker sequence motif ( 389 VLLL 392 ) (2-4), indicating that the linker plays a key role in NBD function and allostery.The Hsp70 NBD belongs to the Actin/Hexokinase/Hsp70 superfamily, members of which share a number of common features (5, 6). The NBD is composed of two lobes, I and II; each lobe consists, in turn, of two subdomains: IA and IB for lobe I, and IIA and IIB for lobe II. Nucleotide binds at the bottom of the deep central cleft at the interface between subdomains IB and IIB, and all four subdomains are involved in nucleotide coordination...

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Hsp110 Chaperones

Sousa

2016

Encyclopedia of Life Sciences

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Hsp70s are a diverse family of chaperones that execute a variety of proteostatic and protein processing reactions. They include the eukaryotic Hsp110s, which function as nucleotide exchange factors for other Hsp70s and thereby regulate association of Hsp70s with their protein substrates. Hsp110s also display protein substrate‐binding domains that shield misfolded proteins from aggregation. Through these activities, Hsp110s contribute to a range of protein processing reactions including recovery from stresses such as heat or ischemic shocks, protein aggregation inhibition and aggregate solubilisation, prion propagation and assembly/disassembly of polymeric protein complexes. These functions result in Hsp110s, contributing both positively and negatively to multiple disease etiologies. Positively, because they inhibit degenerative diseases associated with protein aggregation and negatively because these same activities stimulate cancer proliferation and allow cancer cells to survive the stress of radiologic and chemotherapies. Consequently, Hsp110s are targets of therapeutic approaches that aim to both stimulate and inhibit their activities. Key Concepts Hsp70s are proteins that bind other, often misfolded, proteins so as to either inhibit interactions made by those proteins, break up protein:protein associations or move proteins between cellular compartments. ATP binding causes Hsp70s to release their bound protein substrates, while ADP causes them to hold tightly to those substrates. Hsp110s cause Hsp70s to release ADP so that they can then bind ATP, which then causes the Hsp70 to release its bound protein substrate. In this way, Hsp110s regulate Hsp70:substrate associations. The crystal structure of an Hsp110:Hsp70 complex reveals how Hsp110 induces nucleotide exchange and why it is so effective at doing so. Hsp110s are also able to bind directly to misfolded proteins to block their aggregation, but cannot unfold and refold these proteins as canonical Hsp70s can. Hsp110s strongly stimulate the ability of canonical Hsp70s to solubilise protein aggregates in vitro . Hsp110s are induced by stresses such as heat or ischaemic shocks and assist cellular recovery from such traumas. Hsp110s inhibit protein aggregation and help solubilise protein aggregates in vivo . Hsp110 roles in aggregate solubilisation and aggregation inhibition makes stimulation of their activities a goal for treatment of degenerative diseases associated with protein aggregation. Hsp110 activities support cancer cell growth and therapeutic resistance and make Hsp110 inhibition a goal in cancer treatment.

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Structure of the Hsp110:Hsc70 Nucleotide Exchange Machine

Cited by 197 publications

References 42 publications

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

Hsp110 Chaperones

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