The force-dependent mechanism of DnaK-mediated mechanical folding

Perales-Calvo, Judit; Giganti, David; Stirnemann, Guillaume; Garcia-Manyes, Sergi

doi:10.1126/sciadv.aaq0243

Cited by 38 publications

(39 citation statements)

References 50 publications

(76 reference statements)

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“…Again, as the model substrate that we chose does not have any apparent misfolding behavior, we cannot confirm whether DnaK can suppress misfolding and/or aggregation using our model substrate. At the time of writing, another group published a study on DnaK-mediated mechanical folding using AFM-based pulling operating in force-clamp mode on ubiquitin and wild type (I27) 8 as substrate [47]. Our results are largely similar with regard to DnaK's holdase activity at different experimental conditions.…”

Section: Discussionsupporting

confidence: 59%

The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

Winardhi

Tang

You

et al. 2018

Preprint

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In Escherichia coli, the DnaK/DnaJ/GrpE system plays a critical role in mediating protein refolding and buffering against protein aggregation due to environmental stress. The underlying mechanism remains unclear. In this work, we probe the activity of DnaK/DnaJ/GrpE system with single-molecule protein refolding assay using tandem repeats of titin immunoglobulin 27 (I27)8. We provide direct evidence that DnaK in apo-and ADP-bound state is predominantly a holdase, which kinetically stabilizes the polyprotein in its unfolded form. Binding of ATP relieves DnaK's holding, allowing protein refolding. The presence of co-chaperone DnaJ and GrpE modulates this holdingrelease switching, possibly by altering DnaK's nucleotide state. Our findings thus provide important insights to the molecular mechanism of DnaK/DnaJ/GrpE system.

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Section: Discussionsupporting

confidence: 59%

The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

Winardhi

Tang

You

et al. 2018

Preprint

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“…This is modulated by the cellular response to mechanical stress, via HspB1 phosphorylation, exposure of its FLNC-binding-site, and stabilization of the reputed mechanosensitive region of FLNC. The findings add to recent reports of molecular chaperones binding to force-bearing proteins (99–101), and may provide a new avenue for understanding filaminopathies and FLNC-linked cardiac-specific diseases.…”

Section: Discussionsupporting

confidence: 65%

Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

Collier

Alderson

et al. 2018

Preprint

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Small heat-shock proteins (sHsps; HspBs) are molecular chaperones involved in the cellular stress response and a range of basal functions. Despite a multitude of targets, sHsp interactions are not well understood due their heterogeneous structures and weak binding affinities. The most widely expressed human sHsp, HspB1, is prevalent in striated muscle, where the actin cross-linker filamin C (FLNC, γ-filamin, ABP-L) is a putative binding partner. Musculoskeletal HspB1 is phosphorylated in response to a variety of cues, including mechanical stress, which promotes oligomer disassembly and association with myoarchitectural elements. Here, we report the up-regulation and interaction of both proteins in the hearts of a mouse model of heart failure, with HspB1 being phosphorylated and FLNC increasingly associated with the sarcomeric Z-disc. We used a combination of structural approaches to reveal that phosphorylation of HspB1 results in increased availability of the residues surrounding the phosphosite, facilitating their interaction with folded FLNC domains equivalent to a force-sensing region in the paralog filamin A. By employing native mass spectrometry, we show that domains 18 to 21 of FLNC are extensible under conditions mimicking force, with phosphorylated HspB1 stabilising an intermediate from further unfolding. These findings report on conformations accessible during the cycles of mechanical extension central to filamin function, and are consistent with an interaction between the chaperone and a native target that is strengthened upon the application of force. This may represent a new mode of molecular chaperone activity, allowing HspB1 to protect FLNC from over-extension during mechanical stress.

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“…Hence, titin folding contractions emerge as key transitions that not only set the stiffness of titin, but also titin's ability to produce mechanical work. Indeed, we speculate that the work produced by folding domains can be largely regulated by muscle chaperones 62,63 , molecular crowding 64 , or posttranslational modifications 28,65,66 . The HaloTag-TEV titin can now be used to test these hypotheses.…”

Section: Discussionmentioning

confidence: 99%

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

Rivas‐Pardo

Li²,

Mártonfalvi

et al. 2020

Nat Commun

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Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

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The force-dependent mechanism of DnaK-mediated mechanical folding

Abstract: Mechanical force regulates the extent of chaperone binding to the protein substrate during mechanical folding.

Cited by 38 publications

References 50 publications

The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

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