Oxidation-Induced Conformational Change of a Prokaryotic Molecular Chaperone, Hsp33, Monitored by Selective Isotope Labeling (original) (raw)

Oxidation-induced conformational change of Hsp33, monitored by NMR

Journal of the Korean Magnetic Resonance Society, 2015

Hsp33 is a prokaryotic molecular chaperon that exerts a holdase activity upon response to an oxidative stress at raised temperature. In particular, intramolecular disulfide bond formation between the four conserved cysteines that bind a zinc ion in reduced state is known to be critically associated with the redox sensing. Here we report the backbone NMR assignment results of the half-oxidized Hsp33, where only two of the four cysteines form an intramolecular disulfide bond. Almost all of the resolved peaks could be unambiguously assigned, although the total assignments extent reached just about 50%. Majority of the missing assignments could be attributed to a significant spectral collapse, largely due to the oxidation-induced unfolding of the C-terminal redox-switch domain. These results support two previous suggestions: conformational change in the first oxidation step is localized mainly in the C-terminal zinc-binding domain, and the half-oxidized form would be still inactive. However, some additional regions appeared to be potentially changed from the reduced state, which suggest that the half-oxidized conformation would be an intermediate state that is more labile to heat and/or further oxidation.

Triple isotope-[13C,15N,2H] labeling and NMR measurements of the inactive, reduced monomer form of Escherichia coli Hsp33

Journal of the Korean Magnetic Resonance Society, 2010

Hsp33 is a molecular chaperone achieving a holdase activity upon response to a dual stress by heat and oxidation. Despite several crystal structures available, the activation process is not clearly understood, because the structure inactive Hsp33 as its reduced, zinc-bound, monomeric form has not been solved yet. Thus, we initiated structural investigation of the reduced Hsp33 monomer by NMR. In this study, to overcome the high molecular weight (33 kDa), the protein was triply isotope-[ 13 C, 15 N, 2 H]labeled and its inactive, monomeric state was ensured. 2D-[ 1 H, 15 N]-TROSY and a series of triple resonance spectra could be successfully obtained on a high-field (900 MHz) NMR machine with a cryoprobe. However, under all of the different conditions tested, the number of resonances observed was significantly less than that expected from the amino acid sequence. Thus, a possible contribution of dynamic conformational exchange leading to a line broadening is suggested that might be important for activation process of Hsp33.

A NMR Investigation of Structure and Allostery in the Hsp70 Chaperone System

The Hsp70 Chaperone system is a complex molecular machine composed of proteins. It is responsible for assisting in protein folding/refolding/processing and transport. Hsp70 proteins have been historically associated with the ‘stress’ or ‘heatshock’ response of cells. They function in association with several co-chaperones in a delicately balanced and regulated two stage functional cycle. This system and its component molecules has been the subject of intensive efforts in structural biophysics for the last two decades. In this dissertation we present an analysis of the allosteric machinery, which operates in this molecular system. Using residual dipolar coupling analysis, a state-of-the-art method in solution nuclear magnetic resonance (NMR) spectroscopy, we have been able to detect distinct changes in the T.th-DnaK (the thermophilic Hsp70 protein) as the protein switches between the different stages of its ‘two-stroke’ functional cycle. Specifically, we have seen an opening of the n...

Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data

Protein Science, 2017

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes.

Activation of the Redox-Regulated Molecular Chaperone Hsp33—A Two-Step Mechanism

Structure, 2001

5 reduced, thiolate anion state, coordinate one zinc atom, and constitute a novel zinc binding motif [5]. Upon exposure to oxidizing conditions such as H 2 O 2 , two intramolecular disulfide bonds form in Hsp33 [2, 6]. These disulof Hsp33's disulfide bonds is accompanied by the release of zinc and leads to the activation of Hsp33's University of Halle Halle, Germany chaperone function. In its oxidized and activated state, Hsp33 is able to recognize and bind aggregation-sensitive folding intermediates and can prevent nonspecific side reactions such as aggregation. In vivo thiol-trapping experiments of Hsp33's cysteines as well as ge-Summary netic studies using Hsp33 deletion mutants suggest that the redox regulation of Hsp33's chaperone function Background: Hsp33 is a novel redox-regulated molecular chaperone. Hsp33 is present in the reducing envi-plays an important role in protecting cells against the deleterious effects of reactive oxygen species [2]. Under ronment of the cytosol and is, under normal conditions, inactive. The four highly conserved cysteines found in nonstress conditions, the cytoplasmic Hsp33 is predominantly in its reduced, inactive state. Oxidizing condi-Hsp33 constitute a novel zinc binding motif. Upon exposure to oxidative stress, Hsp33's chaperone activity is tions lead to the accumulation of disulfide-bonded, activated Hsp33 in the cytoplasm of Escherichia coli. turned on. This activation process is initiated by the formation of two intramolecular disulfide bonds. Re-Deletion mutants in Hsp33 show an increased sensitivity toward oxidative stress treatment [2]. cently, the 2.2 Å crystal structure of Hsp33 has been solved, revealing that Hsp33 is present as a dimer in the Over the past few years, an increasing number of redox-regulated proteins have been identified. These structure Vijayalakshmi et al., this issue, 367-375 [1]). proteins include the prokaryotic oxidative stress transcription factor OxyR [7]; the antisigma factor RsrA [8]; Results: We show here that oxidized, highly active Hsp33 is a dimer in solution. In contrast, reduced and the oxidative stress transcription factor of yeast, Yap1 [9-11]; several zinc finger transcription factors; and pro-inactive Hsp33 is monomeric. The incubation of reduced Hsp33 in H 2 O 2 leads to the simultaneous formation of tein kinase C [12]

NMR-monitored titration of acid-stress bacterial chaperone HdeA reveals that Asp and Glu charge neutralization produces a loosened dimer structure in preparation for protein unfolding and chaperone activation

Protein Science, 2013

HdeA is a periplasmic chaperone found in several gram-negative pathogenic bacteria that are linked to millions of cases of dysentery per year worldwide. After the protein becomes activated at low pH, it can bind to other periplasmic proteins, protecting them from aggregation when the bacteria travel through the stomach on their way to colonize the intestines. It has been argued that one of the major driving forces for HdeA activation is the protonation of aspartate and glutamate side chains. The goal for this study, therefore, was to investigate, at the atomic level, the structural impact of this charge neutralization on HdeA during the transition from near-neutral conditions to pH 3.0, in preparation for unfolding and activation of its chaperone capabilities. NMR spectroscopy was used to measure pK a values of Asp and Glu residues and monitor chemical shift changes. Measurements of R 2 /R 1 ratios from relaxation experiments confirm that the protein maintains its dimer structure between pH 6.0 and 3.0. However, calculated correlation times and changes in amide protection from hydrogen/deuterium exchange experiments provide evidence for a loosening of the tertiary and quaternary structures of HdeA; in particular, the data indicate that the dimer structure becomes progressively weakened as the pH decreases. Taken together, these results provide insight into the process by which HdeA is primed to unfold and carry out its chaperone duties below pH 3.0, and it also demonstrates that neutralization of aspartate and glutamate residues is not likely to be the sole trigger for HdeA dissociation and unfolding.

Per-deuteration and NMR experiments for the backbone assignment of 62 kDa protein, Hsp31

Journal of the Korean Magnetic Resonance Society, 2015

Hsp31 protein is one of the members of DJ-1 superfamily proteins and has a dimeric structure of which molecular weight (MW) is 62 kDa. The mutation of DJ-1 is closely related to early onset of Parkinson's disease. Hsp31 displays Zn +2-binding activity and was first reported to be a holding chaperone in E. coli. Its additional glyoxalase III active has recently been characterized. Moreover, an incubation at 60°C induces Hsp31 protein to form a high MW oligomer (HMW) in vitro, which accomplishes an elevated holding chaperone activity. The NMR technique is elegant method to probe any local or global structural change of a protein in responses to environmental stresses (heat, pH, and metal). Although the presence of the backbone chemical shifts (bbCSs) is a prerequisite for detailed NMR analyses of the structural changes, general HSQC-based triple resonance experiments could not be used for 62 kDa Hsp31 protein. Here, we prepared the per-deuterated Hsp31 and performed the TROSY-based triple resonance experiments for the bbCSs assignment. Here, detailed processes of per-deuteration and the NMR experiments are described for other similar NMR approaches.

Oligomeric Hsp33 with Enhanced Chaperone Activity: GEL FILTRATION, CROSS-LINKING, AND SMALL ANGLE X-RAY SCATTERING (SAXS) ANALYSIS

Journal of Biological Chemistry, 2004

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration-and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.

Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions

eLife, 2018

Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecul...

Differences in conformational dynamics within the Hsp90 chaperone family reveal mechanistic insights

Frontiers in Molecular Biosciences, 2014

The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90β (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre-or posthydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.