The association of small heat shock protein Hsp16.3 with the plasma membrane of Mycobacterium tuberculosis: dissociation of oligomers is a prerequisite

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis (MTB), was originally identified as an immuno-dominant antigen and later found to be a major membrane protein. In vitro studies show that Hsp16.3 exists as nonamers and undergoes dynamic dissociation/re-association equilibrium in solutions. Nevertheless, neither the details nor the physiological implications of the presence of Hsp16.3 in the plasma membrane have been studied. In this study, we demonstrated that the purified Hsp16.3 proteins were able to interact with the MTB plasma membrane in a specific and reversible manner, suggesting that there might be subunit exchange between membrane-bound Hsp16.3 and soluble Hsp16.3 oligomers. The dissociation of Hsp16.3 oligomers appears to be a prerequisite for its membrane binding, which is interesting in view that the dissociation of small heat shock protein oligomers was also found to be necessary for it to bind denaturing substrate proteins. Furthermore, the oligomeric structure of Hsp16.3 seems to be more dynamic and flexible when incubating with the mycobacterium lipids. The physiological implications of these observations for Hsp16.3, and small heat shock proteins in general, are discussed.     

Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis

Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.     

Identification of bis-ANS binding sites in Mycobacterium tuberculosis small heat shock protein Hsp16.3: evidences for a two-step substrate-binding mechanism

Small heat shock proteins (sHSPs), as one important subclass of molecular chaperones, are able to specifically bind to denatured substrate proteins rather than to native proteins, of which their substrate-binding sites are far from clear. Our previous study showed an overlapping nature of the sites for both hydrophobic probe 1,1'-Bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding and substrate binding in Mycobacterium tuberculosis Hsp16.3 [X. Fu, H. Zhang, X. Zhang, Y. Cao, W. Jiao, C. Liu, Y. Song, A. Abulimiti, Z. Chang, A dual role for the N-terminal region of M. tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins, J. Biol. Chem. 280 (2005) 6337-6348]. In this work, two bis-ANS binding sites in Hsp16.3 were identified by a combined use of reverse phase HPLC, mass spectroscopy and N-terminal protein sequencing. One site is in the N-terminal region and the other one in the N-terminus of alpha-crystallin domain, both of which are similar to those identified so far in sHSPs. However, accumulating data suggest that these two sites differentially function in binding substrate proteins. With regard to this difference, we proposed a two-step mechanism by which Hsp16.3 binds substrate proteins, i.e., substrate proteins are recognized and initially captured by the N-terminal region that is exposed in the dissociated Hsp16.3 oligomers, and then the captured substrate proteins are further stabilized in the complex by the subsequent binding of the N-terminus of alpha-crystallin domain.     

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Inter-subunit Cross-linking Suppressed the Dynamic Oligomeric Dissociation of Mycobacterium tuberculosis Hsp16.3 and Reduced Its Chaperone Activity

Small heat shock proteins (sHsps) usually exist as dynamic oligomers and oligomeric dissociation was believed to be a prerequisite for their chaperone activities. The truth of this hypothesis was verified in our present study on Hsp16.3, one member of sHsps from Mycobacterium tuberculosis, mainly by utilizing chemical cross-linking. Analysis using size exclusion chromatography demonstrated that the heat-induced oligomeric dissociation of Hsp16.3 was severely blocked due to highly efficient inter-subunit cross-linkages generated by chemical cross-linking, as well as its chaperone activity being reduced. Further analysis by non-denaturing pore gradient polyacrylamide gel electrophoresis and fluorescence spectrometry revealed that the dynamic oligomeric dissociation/reassociation process of Hsp16.3 at room temperature was suppressed by inter-subunit cross-linkages, accompanied by significantly decreased exposure of hydrophobic surfaces that are usually hidden in oligomers. These findings supported the hypothesis that substrate-binding sites of sHsps are exposed presumably by dissociation of larger oligomers into smaller active oligomers, and therefore such a dissociation process could be adjusted to modulate chaperone activities.     

The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: conformational flexibility and molecular chaperone activity

Hsp16.3, the alpha-crystallin-related small heat shock protein of Mycobacterium tuberculosis that is maximally expressed during the stationary phase and is a major membrane protein, has been reported to form specific trimer-of-trimers structure and to act as an effective molecular chaperone (Chang Z et al., 1996, J. Biol Chem 271:7218-7223). However, little is known about its action mechanism. In this study, Hsp16.3 conformational intermediates with dramatically increased chaperone activities were detected after treatment with very low concentrations of guanidine hydrochloride (0.05 M), urea (0.3 M), or mild heating (30 degrees C). The intermediates showed a significant increase in their capacity to bind the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS), indicating an increased exposure of hydrophobic surfaces. Interestingly, the greatest chaperone activities of Hsp16.3 were observed in the presence of 0.3 M guanidine HCl or when heated to 35 degrees C. CD spectroscopy studies revealed no significant changes in protein secondary and tertiary structures at these mild treatments. Our in vitro studies also indicate that long-time-heated Hsp16.3, heated even to temperatures as high as 85 degrees C, has almost the same, if not a slightly greater, chaperone activities as the native protein when cooled to room temperature and its secondary structures also almost recovered. Together, these results suggest that Hsp16.3 modulates its chaperone activity by exposing hydrophobic surfaces and that the protein structure is highly stable and flexible, thus highly adapted for its function.     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

Chaperone-Like Activity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Hsp16.3 Does Not Require Its Intact (Native) Structures

Small heat shock proteins (sHsps) were found to exhibit efficient chaperone-like activities under stress conditions although their native structures are severely disturbed. Here, using an alternative approach (site-directed mutagenesis), we obtained two structurally and functionally distinct Mycobacterium tuberculosis Hsp16.3 single-site mutant proteins. The G59W mutant protein (with Gly59 substituted by Trp) is capable of exhibiting efficient chaperone-like activity even under non-stress conditions although its secondary, tertiary, and quaternary structures are very different from that of the wild type protein. By contrast, the G59A mutant protein (with Gly59 substituted by Ala) resembles with the wild type protein in structure and function. These observations suggest that the Gly59 of the Hsp16.3 protein is critical for its folding and assembly. In particular, we propose that the exhibition of chaperone-like activity for Hsp16.3 does not require its intact (native) structures but requires the disturbance of its native structures (i.e., the native structure-disturbed Hsp16.3 retains its chaperone-like activity or even becomes more active). In addition, the behavior of such an active mutant protein (G59W) also strongly supports our previous suggestion that Hsp16.3 exhibits chaperone-like activity via oligomeric dissociation.     

Ursolic acid and carvacrol may be potential inhibitors of dormancy protein small heat shock protein16.3 of Mycobacterium tuberculosis

Small heat shock protein16.3 (sHSP16.3) is a crucial protein for survival of Mycobacterium tuberculosis (MTB) in its host. Besides, this protein acts as a molecular chaperone during stress and is indispensable for MTB’s growth, virulence and cell-wall thickening. sHSP16.3 is also a promising candidate for vaccine, serodiagnosis and drug design as well. In the present study, we have targeted sHSP16.3 with two phytochemicals, namely ursolic acid and carvacrol using in silico approach. Molecular docking analysis showed that both phytochemicals (ursolic acid and carvacrol) have docked with sHSP16.3 and shown tendency to inhibit the function of this vital protein of MTB. In addition, both compounds have exhibited strong compatibility with sHSP16.3 during whole 60 ns duration of molecular dynamics simulation. Further, the molecular mechanic/generalized Born/Poisson–Boltzmann surface area (MM/G/P/BSA) free energies were calculated which showed that both phytocompounds have stable and favourable binding energies causing strong binding with binding site of sHSP16.3. Taking together, the data of present study suggest that both phytocompounds may be potential inhibitor of sHSP16.3 of MTB and a best alternative to standard anti-tuberculosis drugs.     

Some properties of human small heat shock protein Hsp22 (H11 or HspB8)

Untagged recombinant human small heat shock protein with apparent molecular mass 22 kDa (Hsp22) was obtained in homogeneous state. Size exclusion chromatography and chemical crosslinking with dimethylsuberimidate indicate that Hsp22 forms stable dimers. Being highly susceptible to oxidation Hsp22 forms disulfide crosslinked dimers and poorly soluble high molecular mass oligomers. According to CD spectroscopy oxidation of Hsp22 results in disturbing of both secondary and tertiary structure. Hsp22 possesses a negligibly low autophosphorylation activity and under the conditions used is unable to phosphorylate casein or histone. Hsp22 effectively prevents heat-induced aggregation of yeast alcohol dehydrogenase and bovine liver rhodanese with chaperone activity comparable to that of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20).     

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

The gene encoding a small heat shock protein (sHSP) from Pyrococcus furiosus was redesigned and chemically synthesized by using bacteria-preferred codons. The gene product was over-expressed in Escherichia coli BL21(DE)₃ and purified to homogeneity. In the presence of this protein, the activities of Taq DNA polymerase, DNA restriction endonuclease HindIII and lysozyme were protected at elevated temperature, and also, thermal aggregation of lysozyme was prevented by this purified recombinant sHSP.Huayou Chen, Zhongmei Chu, Contributed equally to this work     

Electrostatic interactions play a critical role in Mycobacterium tuberculosis Hsp16.3 binding of substrate proteins

Mycobacterium tuberculosis Hsp16.3, a member of a small heat shock protein family, has chaperone-like activity in vitro and suppresses thermally or chemically induced aggregation of proteins. The nature of the interactions between Hsp16.3 and the denatured substrate proteins was investigated. A dramatic enhancement of chaperone-like activity of Hsp16.3 upon increasing temperature was accompanied by decreased ANS-detectable surface hydrophobicity. Hsp16.3 exhibited significantly enhanced chaperone-like activity after preincubation at 100°C with almost unchanged surface hydrophobicity. The interaction between Hsp16.3 and dithiothreitol-treated insulin B chains was markedly weakened in the presence of NaCl but greatly enhanced by the addition of a low-polarity alcohol, accompanied by significantly increased and decreased surface hydrophobicity, respectively. A working model for Hsp16.3 binding to its substrate proteins is proposed.     

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (alpha-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.     

15-Deoxyspergualin modulates Plasmodium falciparum heat shock protein function

Heat shock proteins are essential for the survival of all cells. The C-terminal EEVD motif of Hsp70 has previously been implicated in binding 15-deoxyspergualin (DSG), an immunosuppressant with antimalarial activity whose mechanism of action is uncertain. We report the cloning, overexpression, and characterization of three members of the heat shock family, PfHsp70-1 (an Hsp70 protein with a C-terminal EEVD motif), PfHsp70-2 (an Hsp70 protein without the EEVD motif), and PfHsp70 interacting protein. The chaperone activity of PfHsp70-1, and PfHsp70-2 was enhanced by ATP and by PfHip. Interestingly, while binding of protein substrates to PfHsp70-1, PfHsp70-2 and PfHip was unaffected in the presence of DSG, the ATP enhanced chaperone activity of PfHsp70-1 but not PfHsp70-2 was stimulated further by DSG. Our finding suggests that the binding partner of DSG in the parasite cellular milieu is PfHsp70-1 and paves the way for the elucidation of the mechanism of antimalarial action of DSG.     

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.     

Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine-rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.     

Babesia divergens: identification and characterization of BdHSP-20, a small heat shock protein

This study describes the identification and characterization of the Babesia divergens α-crystallin/small heat shock protein 20 (BdHSP-20). BdHSP-20 was recognized by the DG7 monoclonal antibody (DG7 mAb) originally produced by Precigout et al. [Precigout, E., Valentin, A., Carcy, B., Gorenflot, A., Nakamura, K., Aikawa, M., Schrevel, J. 1993. Babesia divergens: characterization of a 17-kDa merozoite membrane protein. Experimental Parasitology 77, 425-434] against B. divergens merozoites. We used DG7 mAb to immunoscreen a B. divergens cDNA library to clone the gene encoding the small heat shock protein. Bdhsp-20 is a single copy gene interrupted by one intron. The deduced gene product (BdHSP-20) clearly belongs to the α-crystallin family and shows significant homology to Babesia bovis, Plasmodiumfalciparum and Toxoplasma gondii sHSPs, with the highest degree of sequence identity around the catalytic domain. Nutritient stress (serum depletion) treatment of the parasites induced the upregulation of BdHSP-20 gene expression observed by semi-quantitative PCR and immunoprecipitation. This regulation pattern suggests that BdHSP-20 could probably be of importance for parasite survival in the case of environmental stress. BdHSP-20 has previously been shown to be highly conserved among different strains and antibodies against the protein drastically reduce parasitemia in vitro.     

Structural dynamics of archaeal small heat shock proteins

Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.     

A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein

The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR‐like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast‐localized and highly expressed in actively photosynthesizing tissue ( Sadanandom A et al., 2000 ). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast‐localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR‐catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time‐scale, was ultimately DTT‐dependent and led to recovery of Hsp21 conformation and chaperone‐like activity, both of which are lost upon methionine sulfoxidation ( Härndahl et al., 2001 ). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress.