The holdase function of <i>Escherichia coli</i> Hsp70 (DnaK) chaperone

Winardhi, Ricksen S.; Tang, Qingnan; You, Huijuan; Sheetz, Michael P.; Yan, Jie

doi:10.1101/305854

Cited by 10 publications

(14 citation statements)

References 47 publications

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“…Due to structural differences, these proteins exhibit different folding-unfolding pattern with distinct force-response, however, their folding landscape are modulated exactly similar during the chaperone interactions-unfoldase shifts the dynamics towards the unfolded state, while the foldase chaperones favor the folded state. Recently, different groups have also tested the unfoldase chaperone activity (DnaK/DnaJ) under force with immunoglobulin substrates, including titin I 27 and Z1 proteins and observed similar results 34,61 , which certainly coincides with our observation. Therefore, our results, along with other similar force-spectroscopy studies with structurally diverse substrates, have evidently strengthen and generalized our hypothesis of mechanical activity of chaperones.…”

Section: Mechanical Chaperone Activity Tested On Talin Proteinsupporting

confidence: 89%

Direct observation of the mechanical role of bacterial chaperones in protein folding

Chaudhuri

Banerjee

Chakraborty

et al. 2020

Preprint

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Protein folding under force is an integral source of generating mechanical power to carry out diverse cellular functions. Though chaperones interact with proteins throughout the different stages of folding pathways, how they behave and interact with client proteins under force was not known. Here we introduce the ‘mechanical role’ of chaperone and explained it with seven independent chaperones using single molecule based real-time microfluidics-magnetic-tweezers. We showed and quantified how chaperones increase or decrease mechanical work output by shifting the folding energy landscape of the client proteins towards the folded or unfolded state. Notably, we found chaperones could behave differently under force. For instance: trigger factor, ribosomal-tunnel associated chaperone, working as a holdase in absence of force, but assist folding under force. This phenomenon generates extra mechanical energy to pull the polyprotein from the stalled ribosome. This is also relevant for SecYEG tunnel associated oxidoreductase DsbA, which act similarly like TF and increases the mechanical energy up to ~59 zJ, to facilitate membrane translocation in an energy efficient manner. However cytoplasmic oxidoreductases such as PDI and Thioredoxin, unlike DsbA, do not have the mechanical folding ability. Interestingly, we observed a highly potential foldase- DnaKJE chaperone complex, only restores the folding ability of the client protein and fails to act like TF or DsbA under force. However, the individual components of this complex, DnaK or DnaJ, act as a mechanical holdase and inhibits folding; similar to that of SecB. Together our study provides an emerging insight of mechanical chaperone behavior, where tunnel associated chaperones generate extra mechanical work whereas the cytoplasmic chaperones are unable to generate that, which might have evolved to minimize the energy consumption in biological processes.

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Section: Mechanical Chaperone Activity Tested On Talin Proteinsupporting

confidence: 89%

Direct observation of the mechanical role of bacterial chaperones in protein folding

Chaudhuri

Banerjee

Chakraborty

et al. 2020

Preprint

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“…Moreover, new native species were formed and rapidly accumulated during 12 minutes, reaching a maximal activity level of 82% of native MLucV, against their natural tendency to denature at 37.7 o C. Yet, indicating that ATP was soon consumed, the luciferase activity dropped again and the FRET signal for aggregation concomitantly increased to a higher level than previously. This suggests that in the presence of ADP, none of the chaperones, although in 8 fold excess for DnaK alone, could passively "hold" the misfolded MLucV monomers and prevent their aggregation, contrary to the so-called "holdase" function generally ascribed to these chaperones (Winardhi, Tang et al 2018, Hall 2020) Addition, after 68 minutes, of a second dose of 800 µM ATP further accumulated up to 46% native monomers in 32 minutes. Again, the eventual consumption of ATP led to a loss of the activity and to an increase in the FRET efficiency.…”

Section: Non-equilibrium Activity Of Hsp70mentioning

confidence: 98%

A novel fluorescent multi-domain protein construct reveals the individual steps of the unfoldase action of Hsp70

Tiwari

Fauvet

Assenza

et al. 2022

Preprint

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A detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the detailed characterization of the aggregates, which are typically heterogeneous. To tackle this problem, we designed here a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase core, flanked by stress-resistant fluorescent mTFP and Venus domains, which upon denaturation formed a discrete stable population of small aggregates. Combining Förster Resonance Energy Transfer and enzymatic activity measurements provided unprecedent details on MLucV states, including native, aggregated, unfolded and chaperone-bound conformations. Using MLucV, we probed the various steps undertaken by bacterial Hsp70 to convert stable discrete aggregates into native proteins. The mechanism first involved an ATP-fuelled disaggregation and unfolding step of the stable pre-aggregated substrate, with a consequent stretching of MLucV beyond simply-unfolded conformations, followed, upon release, by native refolding. Furthermore, the ATP-fuelled unfolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures, highly adverse to the native state. These results unambiguously excluded binding and preventing aggregation from the non-equilibirum mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

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“…Surprisingly, the complex had transcribed over the T-less cassette that should stall further elongation. The ADP (Sigma-Aldrich) used in the formation of ADP•BeF 3 is reported to be contaminated with up to 2.76 % ATP 50 , possibly providing the required substrate to transcribe past the end of the T-less cassette. Identification of DNA-RNA register was aided by map B.…”

Section: Reconstitution Of Transcribing Pol Ii-nucleosome Com-mentioning

confidence: 99%

Structural basis of nucleosome transcription mediated by Chd1 and FACT

Farnung

Ochmann

Engeholm

et al. 2020

Preprint

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Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through nucleosomes. Efficient Pol II passage through nucleosomes depends on the chromatin remodelling factor Chd11 and the histone chaperone FACT2. How Chd1 and FACT mediate Pol II passage through nucleosomes remains unclear. Here we first show that Chd1 and FACT cooperate with the elongation factors Spt4/5 and TFIIS to facilitate Pol II transcription through a nucleosome in a defined biochemical system. We then determine cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT at 2.9 Å and 3.1 Å resolution, respectively. In the first structure, transcribing Pol II has partially unwrapped nucleosomal DNA and exposed the proximal histone H2A/H2B dimer, which is bound by the acidic N-terminal region of Spt5 (Spt5N). The inhibitory DNA-binding region of Chd1 is released3 and the Chd1 translocase adopts an activated state that is poised to pump DNA towards Pol II. In the second structure, transcribing Pol II has generated a partially unravelled nucleosome that binds FACT in a manner that excludes Chd1 and Spt5N. These results suggest a dynamic model of Pol II passage through a nucleosome. In the model, Pol II enters the nucleosome4, activates Chd1 by releasing its DNA-binding region, and thereby stimulates its own progression. Pol II progression then enables FACT binding, liberates Chd1 and Spt5N, and eventually displaces a complex of FACT with histones that is transferred to upstream DNA.

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The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

Cited by 10 publications

References 47 publications

Direct observation of the mechanical role of bacterial chaperones in protein folding

Direct observation of the mechanical role of bacterial chaperones in protein folding

A novel fluorescent multi-domain protein construct reveals the individual steps of the unfoldase action of Hsp70

Structural basis of nucleosome transcription mediated by Chd1 and FACT

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