Disassembly intermediates of RbsD protein remain oligomeric despite the loss of an intact secondary structure

Many proteins exist as homo-oligomers in living organisms wherein the change of oligomeric status apparently serves as an effective means for modulating their biological activities. We have previously reported that the homo-decameric RbsD from Escherichia coli undergoes stepwise disassembly and non-stepwise reassembly. Here the structural status of the urea-induced RbsD disassembly intermediates was examined, mainly using urea-containing polyacrylamide gel electrophoresis and chemical cross-linking. Such intermediates were found to remain oligomeric while losing their intact secondary structures. Such disassembly intermediates were able to effectively refold when the concentration of the urea denaturant was reduced to a lower level, or to refold/reassemble into the native decamers when urea was completely removed, as detected by non-denaturing polyacrylamide gel electrophoresis. These novel observations strongly suggest that the assembly of oligomeric proteins may occur before the completion of subunit folding.     

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 disassembly and reassembly of mutants of Escherichia coli heat-labile enterotoxin: replacement of proline 93 does not abolish the reassembly-competent and reassembly-incompetent states

The carrier moiety of heat-labile enterotoxin of Escherichia coli (EtxB) is formed by the noncovalent association of identical monomeric subunits, which assemble, in vivo and in vitro, into exceptionally stable pentameric complexes. In vitro, acid disassembly followed by neutralization results in reassembly yields of between 20% and 60% depending on the identity of the salts present during the acid denaturation process. Loss of reassembly competence has been attributed to isomerization of the native cis-proline residue at position 93. To characterize this phenomenon further, two mutants of EtxB at proline 93 (P93G and P93A) were generated and purified. The proline variants reveal only minor differences in their biophysical and biochemical properties relative to wild-type protein, but major changes were observed in the kinetics of pentamer disassembly and reassembly. Additionally, a loss of assembly competence was observed following longer term acid treatment, which was even more marked than that of the wild-type protein. We present evidence that the loss of assembly competence of these mutants is best explained by a cis/trans peptidyl isomerization of the unfolded mutant subunits in acid conditions; this limited reassembly competence and the biophysical properties of the native P93 mutant pentamers imply the retention of the native cis conformation in the nonproline peptide bond between residues 92 and 93 in the mutated proteins.     

A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(12) approximately 8 min) and to migrate as SDS-stable pentamers (t(12) approximately 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of approximately 8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.     

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.     

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.     

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.     

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.     

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.     

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.     

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.     

A dual role for the N-terminal region of Mycobacterium tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins

The N-terminal regions, which are highly variable in small heat-shock proteins, were found to be structurally disordered in all the 24 subunits of Methanococcus jannaschii Hsp16.5 oligomer and half of the 12 subunits of wheat Hsp16.9 oligomer. The structural and functional roles of the corresponding region (potentially disordered) in Mycobacterium tuberculosis Hsp16.3, existing as nonamers, were investigated in this work. The data demonstrate that the mutant Hsp16.3 protein with 35 N-terminal residues removed (DeltaN35) existed as trimers/dimers rather than as nonamers, failing to bind the hydrophobic probe (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid) and exhibiting no chaperone-like activity. Nevertheless, another mutant protein with the C-terminal extension (of nine residues) removed, although existing predominantly as dimers, exhibited efficient chaperone-like activity even at room temperatures, indicating that pre-existence as nonamers is not a prerequisite for its chaperone-like activity. Meanwhile, the mutant protein with both the N- and C-terminal ends removed fully exists as a dimer lacking any chaperone-like activity. Furthermore, the N-terminal region alone, either as a synthesized peptide or in fusion protein with glutathione S-transferase, was capable of interacting with denaturing proteins. These observations strongly suggest that the N-terminal region of Hsp16.3 is not only involved in self-oligomerization but also contains the critical site for substrate binding. Such a dual role for the N-terminal region would provide an effective mechanism for the small heat-shock protein to modulate its chaperone-like activity through oligomeric dissociation/reassociation. In addition, this study demonstrated that the wild-type protein was able to form heterononamers with DeltaN35 via subunit exchange at a subunit ratio of 2:1. This implies that the 35 N-terminal residues in three of the nine subunits in the wild-type nonamer are not needed for the assembly of nonamers from trimers and are thus probably structurally disordered.     

Sequential Depletion and Acquisition of Proteins during Golgi Stack Disassembly and Reformation

Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP‐locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi‐resident N‐glycan processing enzymes and matrix proteins (golgins) with specific cis–trans‐Golgi sub‐locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans‐Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis‐Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno‐blotting. The sequential redistribution of Golgi components in a trans–cis sequence may highlight a novel retrograde trafficking pathway between the trans‐Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis‐matrix proteins labelling Golgi‐like structures before cis/medial enzymes. Trans‐enzyme location was preceded by trans‐matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants.     

Antagonists of Hsp16.3, a Low-Molecular-Weight Mycobacterial Chaperone and Virulence Factor, Derived from Phage-Displayed Peptide Libraries

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.     

A Mixed Model of Mutation for Electrophoretic Identity of Proteins within and between Populations

A model which is a mixture of the model of infinite alleles and the Ohta-Kimura model of stepwise mutation has been proposed for the study of eletcrophoretic variants in natural populations. Mutations which alter the mobility of a protein are divided into two classes: stepwise mutations and nonstepwise mutations. It is assumed that stepwise mutations follow the Ohta-Kimura model while nonstepwise mutations follow the infinite allele model. It is then shown that even if the proportion of nonstepwise mutations is only 5%, with the other 95% stepwise mutations, the effective number of alleles given by the present model is considerably larger than that given by the Ohta-Kimura model of stepwise mutation. The result has also been applied to study Nei's genetic distance.     

Antagonists of Hsp16.3, a low-molecular-weight mycobacterial chaperone and virulence factor, derived from phage-displayed peptide libraries

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.     

Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.     

Characterization of mammalian neurofilament subunits by circular dichroism

Critical steps in the disassembly and reassembly of neurofilaments, the intermediate filaments of neurons, have been investigated. Bovine neurofilament subunits (Mr 210 000, 160 000 and 70 000) were purified by urea-polyacrylamide gel electrophoresis and renatured by dialysis against several non-denaturing buffers. The quality of the protein renaturation was measured by circular dichroism. The spectra of renatured neurofilament subunits were interpreted in terms of secondary structure and this showed that the solubilization of proteins in guanidine-HCl buffers is more suitable than in urea buffer for a good recovery of a filamentous structure. Furthermore, it is shown that (i) the three neurofilament subunits exhibit specific CD spectra, with shapes reminiscent of those obtained for the alpha/beta class of proteins and that (ii) there is good correlation between CD spectra, the state of renaturation and the ability of the proteins to assemble into filamentous structures. We conclude that CD studies of neurofilament proteins should help in understanding the numerous variables affecting the disassembly and reassembly of neurofilaments.