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.     

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.     

Reversible methionine sulfoxidation of Mycobacterium tuberculosis small heat shock protein Hsp16.3 and its possible role in scavenging oxidants

Mycobacterium tuberculosis (TB) small heat shock protein Hsp16.3 was found to be a major membrane protein that is most predominantly expressed under oxidative stress and is localized to the thickened cell envelope. Gene knock-out studies indicate that the Hsp16.3 protein is required for TB to grow in its host macrophage cells. The physiological function of Hsp16.3 has not yet revealed. Our analyses via mass spectrometry, conformation-dependent trypsin digestion, nondenaturing pore gradient electrophoresis, ANS-binding fluorescence measurements, and circular dichroism demonstrate that the three and only the three methionine residues (cysteine and tryptophan residues, which can also be readily oxidized by such oxidant as H(2)O(2), are absent in Hsp16.3) can be readily sulfoxidized with H(2)O(2) treatment in vitro, and the methionine sulfoxide can be effectively reduced back to the methionine form. Interconversion between the methionine and methioninesulfoxide has been confirmed by selective oxidation and reduction. The sulfoxidation leads to a small degree of conformational change, which in turn results in a significant decrease of the chaperone-like activity. Data presented in this report strongly implicate that reversible sulfoxidation/desulfoxidation of methionine residues may occur in Hsp16.3, which serves as a way to scavenger reactive oxygen or nitrogen species abundantly present in macrophage cells, thus protecting the plasma membrane and other components of M. tuberculosis allowing their survival in such bacteriocidal hosts.     

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.     

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.     

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.     

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.     

重组结核分枝杆菌Hsp16.3的纳米金免疫传感器检测Hsp16.3抗体

克隆和表达结核分枝杆菌热休克蛋白16.3(Hsp16.3),建立纳米金免疫传感器检测结核病患者血清Hsp16.3抗体.PCR扩增hsp16.3基因,构建重组表达质粒pQE30-hsp16.3,表达和纯化Hsp16.3,Western blot分析其反应原性;晶种生长法制备金纳米棒并连接Hsp16.3,建立纳米金免疫传感...     

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).     

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.     

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.     

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     

Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.     

Biochemical characterization of small heat shock protein HspB8 (Hsp22)–Bag3 interaction

Interaction of human Bag3 with small heat shock proteins HspB6, HspB8 and its K141E mutant was analyzed by different biochemical methods. The data of size-exclusion chromatography indicate that the wild type HspB8 forms tight complexes with Bag3. K141E mutant of HspB8 and especially HspB6 weaker interact with Bag3. The data of chemical crosslinking and analytical ultracentrifugation indicate that in vitro the stoichiometry of complexes formed by HspB8 and Bag3 is variable and is dependent on concentration of protein partners. Interaction of Bag3 and HspB8 is accompanied by increase of thermal stability measured by intrinsic tryptophan fluorescence and increased resistance to limited chymotrypsinolysis. The data of size-exclusion chromatography, analytical ultracentrifugation and limited proteolysis indicate that Bag3 belongs to the group of intrinsically disordered proteins. It is supposed that having unordered structure Bag3 might weakly interact with different small heat shock proteins which recognize unfolded proteins and this interaction is especially strong with intrinsically disordered HspB8. The complexes formed by Bag3 and HspB8 might have variable stoichiometry and can participate in different processes including clearing of the cell from improperly folded proteins.     

Self-association of a small heat shock protein

Human Hsp27 oligomerizes in vivo in a phosphorylation-dependent manner that regulates the functional activity of the protein. We have studied the self-association of wild-type Hsp27 by both sedimentation velocity and sedimentation equilibrium analysis and established that the protein forms an equilibrium mixture of monomers/dimers, tetramers, 12-mers and 16-mers (20 mM Tris-HCl (pH 8.4), 100 mM NaCl, 20 degrees C). Corresponding analysis of the S15D/S78D/S82D triple variant, which is believed to mimic the behavior of phosphorylated Hsp27, establishes that this form of the protein forms primarily monomers and dimers but also forms a small fraction of very large oligomers. Variants in which critical N-terminal sequences have been deleted exhibit oligomerization behavior that is intermediate between that of the triple variant and the wild-type protein. On the other hand a C-terminal sequence deletion variant forms larger oligomers than does the wild-type protein, but also exhibits a greater fraction of smaller oligomers. Notably, the presence of an N-terminal His6-tag induces formation of much larger oligomers than observed for any other form of the protein. The results of this work establish that the wild-type protein forms smaller oligomers than previously believed, define the roles played by various structural domains in Hsp27 oligomerization, and provide improved molecular probes with better-defined properties for the design of future experiments.     

The small heat shock protein Hsp31 cooperates with Hsp104 to modulate Sup35 prion aggregation

The yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit α-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSI⁺] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSI⁺] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSI⁺] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSI⁺] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSI⁺] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSI⁺] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSI⁺] status by the chaperone action of Hsp31. The delineation of Hsp31's role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer.     

Probing the roles of the only universally conserved leucine residue (Leu122) in the oligomerization and chaperone-like activity of Mycobacterium tuberculosis small heat shock protein Hsp16.3

To understand the role of the only universally conserved hydrophobic residue among all the members of the sHsp family, this extremely well conserved Leu122 residue in Hsp16.3 was replaced by valine, alanine, asparigine, or aspartate residues. Only very small amounts of the L122D and L122N mutant Hsp16.3 proteins were expressed in the transformed E. coli; however, both the L122V and L122A were readily expressed. The L122V and L122A mutant proteins had similar oligomeric structures to the wild-type protein at room temperature. Examination of the L122A mutant protein by native pore gradient PAGE and CD spectroscopy, however, revealed a smaller oligomeric size and different secondary structure at 37°C. Both L122V and L122A mutant proteins exhibited significantly lowered chaperone activities. Observations reported here suggest a very important role of this only universally conserved Leu residue in both the formation of specific oligomeric structures and the molecular chaperone activities of Hsp16.3.     

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.