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.     

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.     

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.     

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.     

Stepwise disassembly and apparent nonstepwise reassembly for the oligomeric RbsD protein

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.     

Analysis of the regulation of the molecular chaperone Hsp26 by temperature-induced dissociation: the N-terminal domail is important for oligomer assembly and the binding of unfolding proteins

Small heat shock proteins (sHsps) are molecular chaperones that efficiently bind non-native proteins. All members of this family investigated so far are oligomeric complexes. For Hsp26, an sHsp from the cytosol of Saccharomyces cerevisiae, it has been shown that at elevated temperatures the 24-subunit complex dissociates into dimers. This dissociation seems to be required for the efficient interaction with unfolding proteins that results in the formation of large, regular complexes comprising Hsp26 and the non-native proteins. To gain insight into the molecular mechanism of this chaperone, we analyzed the dynamics and stability of the two oligomeric forms of Hsp 26 (i.e. the 24-mer and the dimer) in comparison to a construct lacking the N-terminal domain (Hsp26DeltaN). Furthermore, we determined the stabilities of complexes between Hsp26 and non-native proteins. We show that the temperature-induced dissociation of Hsp26 into dimers is a completely reversible process that involves only a small change in energy. The unfolding of the dissociated Hsp26 dimer or Hsp26DeltaN, which is a dimer, requires a much higher energy. Because Hsp26DeltaN was inactive as a chaperone, these results imply that the N-terminal domain is of critical importance for both the association of Hsp26 with non-native proteins and the formation of large oligomeric complexes. Interestingly, complexes of Hsp26 with non-native proteins are significantly stabilized against dissociation compared with Hsp26 complexes. Taken together, our findings suggest that the quaternary structure of Hsp26 is determined by two elements, (i) weak, regulatory interactions required to form the shell of 24 subunits and (ii) a strong and stable dimerization of the C-terminal domain.     

The determinants of the oligomeric structure in Hsp16.5 are encoded in the alpha-crystallin domain

The determinants of the oligomeric assembly of Hsp16.5, a small heat-shock protein (sHSP) from Methanococcus jannaschii, were explored via site-directed truncation and site-directed spin labeling. For this purpose, subunit contacts around the two-, three- and four-fold symmetry axes were fingerprinted using patterns of proximities between nitroxide spin labels introduced at selected sites. The lack of change in this fingerprint in an N-terminal truncation of the protein demonstrates that the interactions are encoded in the alpha-crystallin domain. In contrast, the truncation of the N-terminal domain of Mycobacterium tuberculosis Hsp16.3, a bacterial sHSP with an equally short N-terminal region, results in the dissociation of the oligomer to a trimer. These results, in conjunction with those from previous truncation studies in mammalian sHSP, suggest that as the alpha-crystallin domain evolved to encode a smaller basic unit than the overall oligomer, the control of the assembly and dynamics of the oligomeric structure became encoded in the N-terminal domain.     

The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.     

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.     

The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest

The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat-shock protein 90 (Hsp90) chaperone complex. We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast. A cdc37 mutant strain is defective in Ste11-mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11DeltaN. Moreover, Cdc37, Ste11DeltaN and Hsp90 coprecipitate pairwise. Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors. Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast.     

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.     

Role of the IXI/V motif in oligomer assembly and function of StHsp14.0, a small heat shock protein from the acidothermophilic archaeon, Sulfolobus tokodaii strain 7

Small heat shock proteins (sHsps) are one of the most ubiquitous molecular chaperones. They are grouped together based on a conserved domain, the alpha-crystallin domain. Generally, sHsps exist as oligomers of 9-40 subunits, and the oligomers undergo reversible temperature-dependent dissociation into smaller species as dimers, which interact with denaturing substrate proteins. Previous studies have shown that the C-terminal region, especially the consensus IXI/V motif, is responsible for oligomer assembly. In this study, we examined deletions or mutations in the C-terminal region on the oligomer assembly and function of StHsp14.0, an sHsp from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Mutated StHsp14.0 with C-terminal deletion or replacement of IIe residues in the IXI/V motif to Ala, Ser, or Phe residues could not form large oligomers and lost chaperone activity. StHsp14.0WKW, whose Ile residues in the IXI/V motif are changed to Trp, existed as an oligomer like that of the wild type. However, it dissociates to small oligomers and exhibits chaperone activity at relatively lowered temperature. Replacement of two Ile residues in the motif to relatively small residues, Ala or Ser, also resulted in the change of beta-sheet rich secondary structure and decrease of hydrophobicity. Interestingly, StHsp14.0 mutant with amino acid replacements to Phe kept almost the same secondary structure and relatively high hydrophobicity despite that it could not form an oligomeric structure. The results show that hydrophobicity and size of the amino acids in the IXI/V motif in the C-terminal region are responsible not only for assembly of the oligomer but also for the maintenance of beta-sheet rich secondary structure and hydrophobicity, which are important for the function of sHsp.     

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.     

C-terminal lysine truncation increases thermostability and enhances chaperone-like function of porcine alphaB-crystallin

The carboxyl-terminal segment of alpha-crystallin, a major lens protein of all vertebrates, has a short and flexible peptide extension of about 20 amino acid residues that are very susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role in crystallin aggregation and chaperone-like activity, we constructed a mutant of porcine alphaB-crystallin with C-terminal lysine truncated end, which unexpectedly showed better chaperone-like function than wild-type alphaB-crystallin. From circular dichroism (CD) spectra, we show that the mutant possesses similar secondary and tertiary structures to those of native purified and recombinant alphaB-crystallins. Analytical ultracentrifugation revealed that the truncated mutant was smaller than wild-type alphaB-crystallin in aggregation size and mass. The observed higher thermostability and anti-thermal aggregation propensity of the truncated alphaB-crystallin mutant than wild-type alphaB-crystallin are in contrast to the prevailing notion that mutations at the C-terminal lysines of alphaB-crystallin result in substantial loss of chaperone-like activity, despite the overall preservation of secondary structure. The detailed characterization of the C-terminal deletion mutants may provide some deeper insight into the chaperoning mechanism of the structurally related small heat-shock protein family.     

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.     

Crystal structure of an activated variant of small heat shock protein Hsp16.5

How does the sequence of a single small heat shock protein (sHSP) assemble into oligomers of different sizes? To gain insight into the underlying structural mechanism, we determined the crystal structure of an engineered variant of Methanocaldococcus jannaschii Hsp16.5 wherein a 14 amino acid peptide from human heat shock protein 27 (Hsp27) was inserted at the junction of the N-terminal region and the α-crystallin domain. In response to this insertion, the oligomer shell expands from 24 to 48 subunits while maintaining octahedral symmetry. Oligomer rearrangement does not alter the fold of the conserved α-crystallin domain nor does it disturb the interface holding the dimeric building block together. Rather, the flexible C-terminal tail of Hsp16.5 changes its orientation relative to the α-crystallin domain which enables alternative packing of dimers. This change in orientation preserves a peptide-in-groove interaction of the C-terminal tail with an adjacent β-sandwich, thereby holding the assembly together. The interior of the expanded oligomer, where substrates presumably bind, retains its predominantly nonpolar character relative to the outside surface. New large windows in the outer shell provide increased access to these substrate-binding regions, thus accounting for the higher affinity of this variant to substrates. Oligomer polydispersity regulates sHSPs chaperone activity in vitro and has been implicated in their physiological roles. The structural mechanism of Hsp16.5 oligomer flexibility revealed here, which is likely to be highly conserved across the sHSP superfamily, explains the relationship between oligomer expansion observed in disease-linked mutants and changes in chaperone activity.     

Effect of N-terminal residues on the structural stability of recombinant horse L-chain apoferritin in an acidic environment

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.     

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity

During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.     

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.