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.     

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.     

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.     

Monodisperse Hsp16.3 nonamer exhibits dynamic dissociation and reassociation,with the nonamer dissociation prerequisite for chaperone-like activity

Small heat-shock proteins (sHsps) of various origins exist commonly as oligomers and exhibit chaperone-like activities in vitro. Hsp16.3, the sHsp from Mycobacterium tuberculosis, was previously shown to exist as a monodisperse nonamer in solution when analyzed by size-exclusion chromatography and electron cryomicroscropy. This study represents part of our effort to understand the chaperone mechanism of Hsp16.3, focusing on the role of the oligomeric status of the protein. Here, we present evidence to show that the Hsp16.3 nonamer dissociates at elevated temperatures, accompanied by a greatly enhanced chaperone-like activity. Moreover, the chaperone-like activity was increased dramatically when the nonameric structure of Hsp16.3 was disturbed by chemical cross-linking, which impeded the correct reassociation of Hsp16.3 nonamer. These suggest that the dissociation of the nonameric structure is a prerequisite for Hsp16.3 to bind to denaturing substrate proteins. On the other hand, our data obtained by using radiolabeled and non-radiolabeled proteins clearly demonstrated that subunit exchange occurs readily between the Hsp16.3 oligomers, even at a temperature as low as 4 degrees C. In light of all these observations, we propose that Hsp16.3, although it appears to be homogeneous when examined at room temperature, actually undertakes rapid dynamic dissociation/reassociation, with the equilibrium, and thus the chaperone-like activities, regulated mainly by the environmental temperature.     

Hsp26: a temperature-regulated chaperone

Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.     

Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin.

Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core 'alpha-crystallin' domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human alphaB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to alphaB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mm ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPgammaS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48 degrees C. While the sequence similarity between human alphaB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core 'alpha-crystallin' domain are much closer.     

Sequence, structure, and dynamic determinants of Hsp27 (HspB1) equilibrium dissociation are encoded by the N-terminal domain

Human small heat shock protein 27 (Hsp27) undergoes concentration-dependent equilibrium dissociation from an ensemble of large oligomers to a dimer. This phenomenon plays a critical role in Hsp27 chaperone activity in vitro enabling high affinity binding to destabilized proteins. In vivo dissociation, which is regulated by phosphorylation, controls Hsp27 role in signaling pathways. In this study, we explore the sequence determinants of Hsp27 dissociation and define the structural basis underlying the increased affinity of Hsp27 dimers to client proteins. A systematic cysteine mutagenesis is carried out to identify residues in the N-terminal domain important for the equilibrium between Hsp27 oligomers and dimers. In addition, spin-labels were attached to the cysteine mutants to enable electron paramagnetic resonance (EPR) analysis of residue environment and solvent accessibility in the context of the large oligomers, upon dissociation to the dimer, and following complex formation with the model substrate T4 Lysozyme (T4L). The mutagenic analysis identifies residues that modulate the equilibrium dissociation in favor of the dimer. EPR analysis reveals that oligomer dissociation disrupts subunit contacts leading to the exposure of Hsp27 N-terminal domain to the aqueous solvent. Moreover, regions of this domain are highly dynamic with no evidence of a packed core. Interaction between T4L and sequences in this domain is inferred from transition of spin-labels to a buried environment in the substrate/Hsp27 complex. Together, the data provide the first structural analysis of sHSP dissociation and support a model of chaperone activity wherein unstructured and highly flexible regions in the N-terminal domain are critical for substrate binding.     

Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.     

Site-directed mutation on the only universally conserved residue Leu122 of small heat shock protein Hsp16.3.

Hsp16.3 from Mycobacterium tuberculosis belongs to the small heat shock protein family and has chaperone-like activity in vitro. The only universally conserved hydrophobic residue Leu122 was substituted by Val and Ala, respectively. The mutations on the Leu122 of Hsp16.3 have resulted in much lower structural stability in vivo and in vitro. Both mutant proteins exhibited much weaker chaperone-like activities than the Hsp16.3 WT under heat shock conditions. Taken together, the highly hydrophobic residue L122 of Hsp16.3 was suggested to play a very important role in maintaining not only the structural stability but also the chaperone-like activity.     

The activation mechanism of Hsp26 does not require dissociation of the oligomer

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.     

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.     

Subunit exchange of small heat shock proteins. Analysis of oligomer formation of alphaA-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations

alphaA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30-40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between alphaA-crystallin and other proteins. We found that Hsp27 and alphaB-crystallin readily exchanged with fluorescence-labeled alphaA-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of alphaA-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts alphaA-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange activity. These findings suggest residues 20-56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of alphaA-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of alphaA-crystallin, consisting of small multimers of alphaA-crystallin subunits in a dynamic equilibrium with the oligomeric complex.     

Preheat treatment for Mycobacterium tuberculosis Hsp16.3: correlation between a structural phase change at 60 degrees C and a dramatic increase in chaperone-like activity.

The in vitro chaperone-like activity of Mycobacterium tuberculosis small heat shock protein Hsp16.3 was found to be dramatically enhanced to the same extent after preheat treatment at or over 60 degrees C. Structural analysis using gel filtration, native pore-gradient PAGE, nondenaturing PAGE, and far-UV CD spectroscopy consistently revealed no significant difference between the native and the preheated Hsp16.3 proteins. However, near-UV CD spectroscopy clearly demonstrated that the tertiary structure of preheated Hsp16.3 is quite similar to its native conformation, with a minor but significant difference. Further analysis using differential scanning calorimetry indicated that Hsp16.3 exhibited a structural transition near 60 degrees C. All these results together indicate that Hsp16.3 suffers a phase change at approximately 60 degrees C, which seem to remove a structural energy barrier for the protein to refold to a conformational status with increased chaperone-like activity.     

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.     

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.     

The dynamics of Hsp25 quaternary structure. Structure and function of different oligomeric species.

Small heat shock proteins (sHsps), including alpha-crystallin, represent a conserved and ubiquitous family of proteins. They form large oligomers, ranging in size from 140 to more than 800 kDa, which seem to be important for the interaction with non-native proteins as molecular chaperones. Here we analyzed the stability and oligomeric structure of murine Hsp25 and its correlation with function. Upon unfolding, the tertiary and quaternary structure of Hsp25 is rapidly lost, whereas the secondary structure remains remarkably stable. Unfolding is completely reversible, leading to native hexadecameric structures. These oligomers are in a concentration-dependent equilibrium with tetramers and dimers, indicating that tetramers assembled from dimers represent the basic building blocks of Hsp25 oligomers. At high temperatures, the Hsp25 complexes increase in molecular mass, consistent with the appearance of "heat shock granules" in vivo after heat treatment. This high molecular mass "heat shock form" of Hsp25 is in a slow equilibrium with hexadecameric Hsp25. Thus, it does not represent an off-pathway reaction. Interestingly, the heat shock form exhibits unchanged chaperone activity even after incubation at 80 degrees C. We conclude that Hsp25 is a dynamic tetramer of tetramers with a unique ability to refold and reassemble into its active quaternary structure after denaturation. So-called heat shock granules, which have been reported to appear in response to stress, seem to represent a novel functional species of Hsp25.     

Small heat shock protein Hsp17.8 functions as an AKR2A cofactor in the targeting of chloroplast outer membrane proteins in Arabidopsis

Plastid proteins that are encoded by the nuclear genome and synthesized in the cytosol undergo posttranslational targeting to plastids. Ankyrin repeat protein 2A (AKR2A) and AKR2B were recently shown to be involved in the targeting of proteins to the plastid outer envelope. However, it remains unknown whether other factors are involved in this process. In this study, we investigated a factor involved in AKR2A-mediated protein targeting to chloroplasts in Arabidopsis (Arabidopsis thaliana). Hsp17.8, a member of the class I (CI) cytosolic small heat shock proteins (sHsps), was identified in interactions with AKR2A. The interaction between Hsp17.8 and AKR2A was further confirmed by coimmunoprecipitation experiments. The carboxyl-terminal ankyrin repeat domain of AKR2A was responsible for AKR2A binding to Hsp17.8. Other CI cytosolic sHsps also interact with AKR2A to varying degrees. Additionally, Hsp17.8 binds to chloroplasts in vitro and enhances AKR2A binding to chloroplasts. HSP17.8 was expressed under normal growth conditions, and its expression increased after heat shock. Hsp17.8 exists as a dimer under normal physiological conditions, and it is converted to high oligomeric complexes, ranging from 240 kD to greater than 480 kD, after heat shock. High levels of Hsp17.8 together with AKR2A resulted in increased plastid targeting of Outer Envelope Protein7 (OEP7), a plastid outer envelope protein expressed as a green fluorescent protein fusion protein. In contrast, artificial microRNA suppression of HSP17.8 and closely related CI cytosolic sHSPs in protoplasts resulted in a reduction of OEP7:green fluorescent protein targeting to plastids. Based on these data, we propose that Hsp17.8 functions as an AKR2A cofactor in targeting membrane proteins to plastid outer membranes under normal physiological conditions.     

Self-association and chaperone activity of Hsp27 are thermally activated

The small heat shock protein 27 (Hsp27) is an oligomeric, molecular chaperone in vitro. This chaperone activity and other physiological roles attributed to Hsp27 have been reported to depend on the state of self-association. In the present work, we have used sedimentation velocity experiments to demonstrate that the self-association of Hsp27 is independent of pH and ionic strength but increases significantly as the temperature is increased from 10 to 40 degrees C. The largest oligomers formed at 10 degrees C are approximately 8-12 mer, whereas at 40 degrees C oligomers as large as 22-30 mer are observed. Similarly, the chaperone activity of Hsp27 as indicated by its ability to inhibit dithiothreitol-induced insulin aggregation also increases with increased temperature, with a particularly sharp increase in activity as temperature is increased from 34 to 43 degrees C. Similar studies of an Hsp27 triple variant that mimics the behavior of the phosphorylated protein establish that this protein has greatly diminished chaperone activity that responds minimally to increased temperature. We conclude that Hsp27 can exploit a large number of oligomerization states and that the range of oligomer size and the magnitude of chaperone activity increase significantly as temperature is increased over the range that is relevant to the physiological heat shock response.     

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.