Cryo-EM structure of human mitochondrial HSPD1

Klebl, David P.; Feasey, Matthew C.; Hesketh, Emma L.; Ranson, Neil A.; Wurdak, Heiko; Sobott, Frank; Bon, Robin S.; Muench, Stephen P.

doi:10.1016/j.isci.2020.102022

Cited by 17 publications

(16 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our structural analysis with the reported single-ring mHsp60-mHsp10 shows that the mHsp60 subunit interface is increased as a result of mHsp10 binding. Note that a cryo-EM structure of mHsp60 28 was reported during our manuscript submission, however, without structural analysis on inter-subunit interactions and discussion on their roles on the structural dynamics of mHsp60.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for the structural dynamics of human mitochondrial chaperonin mHsp60

Wang

2021

Sci Rep

View full text Add to dashboard Cite

Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded β sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the β sheet and indirectly shorten β strands by disengaging the backbones of the flanking residues from hydrogen bonding in the β strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit β sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association.

show abstract

Section: Introductionmentioning

confidence: 99%

Structural basis for the structural dynamics of human mitochondrial chaperonin mHsp60

Wang

2021

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Our previous structural studies of the mitochondrial chaperonin HSPD1 (Hsp60) (Klebl et al, 2021) included cryo-EM data collection in a range of buffers and using different gridpreparation approaches. As a part of this, we prepared cryo-EM grids of HSPD1 in 25 mM Tris, 150 mM NaCl, and 5 mM MgCl 2 pH 7.7 and obtained a reconstruction at 3.4Å resolution by standard single-particle data processing methods.…”

Section: Resultsmentioning

confidence: 99%

“…HSPD1 was expressed and purified as previously described (Klebl et al, 2021). Briefly, 4 h after induction [250 µM IPTG (final concentration)], bacterial cells were harvested by centrifugation (10 min, 4,000 rpm).…”

Section: Methodsmentioning

confidence: 99%

It started with a Cys: Spontaneous cysteine modification during cryo-EM grid preparation

Klebl

Wang

Sobott

et al. 2022

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

Advances in single particle cryo-EM data collection and processing have seen a significant rise in its use. However, the influences of the environment generated through grid preparation, by for example interactions of proteins with the air-water interface are poorly understood and can be a major hurdle in structure determination by cryo-EM. Initial interactions of proteins with the air-water interface occur quickly and proteins can adopt preferred orientation or partially unfold within hundreds of milliseconds. It has also been shown previously that thin-film layers create hydroxyl radicals. To investigate the potential this might have in cryo-EM sample preparation, we studied two proteins, HSPD1, and beta-galactosidase, and show that cysteine residues are modified in a time-dependent manner. In the case of both HSPD1 and beta-galactosidase, this putative oxidation is linked to partial protein unfolding, as well as more subtle structural changes. We show these modifications can be alleviated through increasing the speed of grid preparation, the addition of DTT, or by sequestering away from the AWI using continuous support films. We speculate that the modification is oxidation by reactive oxygen species which are formed and act at the air-water interface. Finally, we show grid preparation on a millisecond timescale outruns cysteine modification, showing that the reaction timescale is in the range of 100s to 1,000s milliseconds and offering an alternative approach to prevent spontaneous cysteine modification and its consequences during cryo-EM grid preparation.

show abstract

“…The mHsp60-mHsp10 complex assists in the refolding of denatured proteins [ 25 , 26 ]. Although various conformations including the football-type, bullet-type, and single-ring HSP60-HSP10 complex were observed with the crystallographic structural analysis or cryo-electron microscopy, there is still a lack of information on HSP60 structures during macrophage activation [ 10 , 14 , 15 ]. The LiP-MS results suggest that HSP60 undergoes conformational changes during macrophage activation.…”

Section: Discussionmentioning

confidence: 99%

“…The HSP60 heptamer forms a complex with HSP10 in the presence of ATP to refold the misfolded proteins [ 11 , 12 , 13 ]. Models of the HSP60 polymorphic form have been proposed in previous studies, e.g., the football-type complex, the bullet-type complex, and the single-ring complex [ 10 , 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Structural Alternation in Heat Shock Proteins of Activated Macrophages

Zhang

Wei

Zhang

et al. 2021

Cells

View full text Add to dashboard Cite

The inflammatory response of macrophages is an orderly and complex process under strict regulation accompanied by drastic changes in morphology and functions. It is predicted that proteins will undergo structural changes during these finely regulated processes. However, changes in structural proteome in macrophages during the inflammatory response remain poorly characterized. In the present study, we applied limited proteolysis coupled mass spectrometry (LiP-MS) to identify proteome-wide structural changes in lipopolysaccharide (LPS)-activated macrophages. We identified 386 structure-specific proteolytic fingerprints from 230 proteins. Using the Gene Ontology (GO) biological process enrichment, we discovered that proteins with altered structures were enriched into protein folding-related terms, in which HSP60 was ranked as the most changed protein. We verified the structural changes in HSP60 by using cellular thermal shift assay (CETSA) and native CETSA. Our results showed that the thermal stability of HSP60 was enhanced in activated macrophages and formed an HSP10-less complex. In conclusion, we demonstrate that in situ structural systems biology is an effective method to characterize proteomic structural changes and reveal that the structures of chaperone proteins vary significantly during macrophage activation.

show abstract

Cryo-EM structure of human mitochondrial HSPD1

Cited by 17 publications

References 29 publications

Structural basis for the structural dynamics of human mitochondrial chaperonin mHsp60

Structural basis for the structural dynamics of human mitochondrial chaperonin mHsp60

It started with a Cys: Spontaneous cysteine modification during cryo-EM grid preparation

Structural Alternation in Heat Shock Proteins of Activated Macrophages

Contact Info

Product

Resources

About