Mechanism of Chaperone Function in Small Heat-shock Proteins

PHOSPHORYLATION-INDUCED ACTIVATION OF TWO-MODE BINDING IN alpha B-CRYSTALLIN*

Hanane A. Koteiche and Hassane S. MchaourabDagger

From the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, November 20, 2002, and in revised form, December 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The consequences of alpha B-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by alpha B-crystallin phosphorylation mimics containing combinations of serine to aspartate substitutions. The T4L site-directed mutants were selected to constitute an energetic ladder of progressively destabilized proteins having similar structures in the folded state. alpha B-crystallin and its variants differentially recognize the T4L mutants, binding the more destabilized ones to a larger extent. Furthermore, the aspartate substitutions result in an increase in the extent of binding to the same T4L mutant and in the appearance of biphasic binding isotherms. The latter indicates the presence of two modes of binding characterized by different affinities and different numbers of binding sites. The transition to two-mode binding can also be induced by temperature or pH activation of the second mode. The similarity between the phosphorylation, pH, and temperature effects suggests a common structural origin. The location of the phosphorylation sites in the N-terminal domain and the hypothesized burial of this domain in the core of the oligomeric structure are consistent with a critical role for the destabilization of the quaternary structure in the process of recognition and binding by small heat-shock proteins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lens transparency is the consequence of a unique molecular architecture that involves the packing of three families of proteins, the crystallins, in a glass-like state (1-3). One of these families, consisting of the two highly homologous proteins alpha A-crystallin and alpha B-crystallin, belongs to the small heat-shock protein (sHSP)1 superfamily of chaperones and shares the common structural and functional characteristics of the superfamily (4-6). sHSP form oligomeric complexes that recognize and bind non-native protein states without the hydrolysis of ATP. The binding capacity is remarkably high with reported stoichiometries that approach one substrate of equal molecular mass per sHSP subunit. Current models of lens transparency hypothesize a critical role of the chaperone function of alpha -crystallins in delaying aggregation of damaged proteins and the onset of opacity in the fiber cells that lack the machineries necessary for protein turnover (3, 7).

One of the two alpha -crystallin subunits, alpha B-crystallin, is also widely expressed in other tissues such as cardiac (8) and skeletal muscles and the brain (9, 10), where it appears to be involved in transduction pathways activated during growth and differentiation and in response to various forms of stress (11). Evidence for the participation of alpha B-crystallin and other mammalian sHSP in these pathways includes their phosphorylation by mitogen-activated protein kinase-activated protein kinases (12-15). alpha B-crystallin is phosphorylated at three serine residues during ischemia and in response to cytotoxic signals and mitogenic and inflammatory agents (16, 17). Phosphorylation is presumably used to modulate the cellular role of these proteins, the details of which remain unclear. The importance of this role has been highlighted by the identification of an inherited mutation in alpha B-crystallin that is associated with desmin-related myopathy (18). In the lens, alpha B-crystallin phosphorylation by cAMP-dependent kinase is age-related and is induced in response to stress (19, 20).

The consequences of the phosphorylation of mammalian sHSP involve both the oligomeric structure and the chaperone activity. Equilibrium sedimentation analysis and size exclusion chromatography of phosphorylated alpha B-crystallin and its serine to aspartate mimics reveal a reduction in the size of the oligomer (16). This is consistent with the effects of phosphorylation on a related mammalian sHSP, HSP27, where the oligomer dissociates to a tetramer (21, 22). In several cellular systems, the phosphorylation of both alpha B-crystallin and HSP27 is accompanied by translocation of these proteins from the cytoplasm to the nucleus (23).

On the functional level, phosphorylation of alpha B-crystallin has been reported to marginally reduce its efficiency in suppressing the aggregation of model substrates in vitro (16). In these assays, complex formation between alpha B-crystallin and the substrate is indirectly detected through the reduction in light scattering. Because of the non-equilibrium nature of the assay, it is not always possible to determine the origin of the change in efficiency, i.e. whether it reflects lower affinity or a change in the kinetics of binding and/or scattering by the resulting complex between the substrate protein and phosphorylated alpha B-crystallin. In general, little mechanistic insight can be obtained from these studies.

In a recent study, we reported the equilibrium binding of alpha A-crystallin to destabilized mutants of T4 lysozyme (24). The advantage of these mutants is that they do not aggregate on the time scale of binding and their equilibrium folding constants are in the 104-107 range. The differential binding of alpha A-crystallin to these mutants suggests recognition of transient excited states. Thermodynamic analysis of the binding curves reveal two different modes of binding. The T4L mutants provide an ideal model system to investigate the effects of alpha B-crystallin phosphorylation on its binding to non-native proteins and to gain insight into the mechanism of binding.

For this purpose, we have constructed five phosphorylation mimics of alpha B-crystallin consisting of different combinations of serine to aspartate substitutions. The equilibrium binding of these mutants to destabilized T4L reveals a significant increase in the extent of binding in the S/D variants relative to the WT. The increased binding results from higher affinity and the availability of a larger number of binding sites. Given the phosphorylation-induced destabilization of the alpha B-crystallin oligomeric structure, the results are consistent with the emerging hypothesis that changes in its quaternary structure are required or coupled to the recognition and binding of destabilized protein substrates (25-29).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and Site-directed Mutagenesis-- The cDNA of mouse alpha B-crystallin, obtained from American Type Culture Collection (ATCC), was cloned between the NdeI and XhoI sites of the plasmid pET-20(b+) (30, 31). The cloned DNA was verified by DNA sequencing and determined to have an identical DNA sequence to that deposited in GenBankTM under accession number M63170. Overlap-extension site-directed mutagenesis was performed to generate the following PCR fragments: S19D, S45D, S59D, S45D/S59D (alpha B-D2), and S19D/S45D/S59D (alpha B-D3). The fragments were then digested and subcloned into the pET 20(b+) vector between the NdeI and XhoI sites. All mutant constructs were sequenced to confirm the substitution and the absence of unwanted change. Single-site mutants are named by specifying the original residue, the number of the residue, and the new residue, in that order.

Protein Expression and Purification-- The T4L mutants were expressed, purified, and spin-labeled as described previously (24). alpha B-crystallin variant plasmids were used to transform competent Escherichia coli BL21(DE3). Cultures, inoculated from overnight seeds, were grown to midlog phase at 37 °C. Then the temperature was dropped to 34 °C, and the expression of alpha B variants was induced by the addition of 0.4 mM isopropyl-1-thio-beta -D-galactopyranoside. After 3 h of induction the cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris, 25 mM NaCl, 0.1 mM EDTA, 0.02% NaN3, 10 mM dithiothreitol, pH 8.0). The resuspended cultures were disrupted by sonication, and the DNA was precipitated by the addition of 0.017% polyethyleneimine. The lysates were then centrifuged at 15,000 × g. alpha B-crystallin and its single variants were purified using anion exchange chromatography followed by size exclusion chromatography as described previously (6). alpha B-D2 and alpha B-D3 were loaded on a Source Q column, washed with buffer A (20 mM Tris, 100 mM NaCl, 0.1 mM EDTA, pH 8.0), and eluted with a gradient of 0.1-1 M NaCl in buffer A. Ammonium sulfate was added to the eluted anion exchange peak to a final concentration of 1 M, and the sample then was loaded on a phenyl-Sepharose column, washed, and eluted with a 1-0 M ammonium sulfate gradient. This step was followed by size-exclusion chromatography on a Superose 6 column.

EPR Spectroscopy-- Room temperature EPR analysis of spin-labeled T4L was carried out on a Bruker E500 spectrometer equipped with a super high Q cavity. Variable temperature experiments were carried out on a Bruker EMX spectrometer using a TM110 cavity. The temperature of the cavity was maintained using a stream of nitrogen gas. Samples, consisting of 50 µM T4L and varying concentrations of alpha B-crystallin variants, were incubated at the desired temperature in a water bath for 105 min. They were then loaded in 25-µl glass capillaries and transferred to the EPR cavity 15 min before data collection.

Analysis of Binding Isotherms-- A double reciprocal representation of the binding curves was used to emphasize the difference between isotherms arising from a single set of independent binding sites versus those arising from at least two such sets. The former is expected to result in a linear curve whereas the latter leads to curved isotherms.

Using the appropriate equations, both simulations and curve-fitting were performed using the program Origin (OriginLab Inc.). For non-linear least squares fits, the Levenberg-Marquart method was used. Simulations were based on the optimum overlay with the data.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To investigate the recognition and binding steps involved in sHSP chaperone function, a set of destabilized T4L mutants was constructed, the Delta Gunf of which span the 5-10 kcal/mol range (24, 32). The rationale was that progressive destabilization of a substrate protein will lead to the formation of a stable complex with the chaperone without the need for the extreme denaturing conditions used in previous assays. Because T4L binding occurs under conditions that strongly favor the native state, the threshold for binding is then a reflection of the free energy balance between association with the chaperone and refolding from the non-native state recognized. Complex formation is directly detected using fluorescent or paramagnetic probes in contrast to the indirect detection using light scattering in aggregation assays. The probes are introduced at non-destabilizing sites in T4L via cysteine mutagenesis followed by reaction with the appropriate reagent. In a previous report, we demonstrated the binding of T4L mutants to the lens chaperone alpha A-crystallin (24).

To explore the effect of phosphorylation on the chaperone activity of alpha B-crystallin, the following mutants were constructed: S19D, S45D, S59D, S45D/S59D (alpha B-D2), and S19D/S45D/S59D (alpha B-D3). All mutants form oligomeric structures as deduced from size-exclusion chromatography (data not shown). The elution volumes of the single and double aspartate mutants are indistinguishable from that of the WT. In contrast, the elution volume of alpha B-D3 is larger, presumably reflecting its smaller molecular mass as reported previously (16). Circular dichroism analysis of this variant revealed secondary and tertiary structures similar to the WT (16). Because alpha B-D3 is a combination of the single Ser to Asp substitutions, it is logical to assume that the single mutants have intact static structures at the level of a single subunit.

Phosphorylation Increases the Extent of Binding of T4L Mutants-- Table I summarizes the thermodynamic characteristics of the spin-labeled T4L mutants used in this study. All reported Delta Gunf values were obtained from non-linear least squares fit of guanidine HCl-unfolding curves as described previously (24). The T4L mutants contained a cysteine residue at the solvent-exposed site 151 in helix J, which allows the attachment of a nitroxide spin label according to Scheme 1.


                              
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Table I
Delta Gunf of the T4L mutants obtained from denaturant unfolding curves at various pH values and temperatures


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Scheme 1.  

Incubation of 50 µM T4L mutants with a 10-fold molar excess of WT alpha B-crystallin at 23 °C, pH 7.2, results in a small fraction of bound T4L such that it is not possible to construct binding isotherms. In contrast, under the same conditions alpha B-D3 binds extensively to all mutants except for the most stable, D70N. The fraction of bound T4L is deduced from the change in the EPR spectral line shape of the mutant in the presence of the chaperone (24). Fig. 1a compares the binding isotherms of L99A and L99A/A130S alpha B-D3. Both curves are linear and can be fit to the equation,
<UP>1/</UP>r<UP>=</UP>(K<SUB><UP>Da</UP></SUB>/n)×1/L<UP>+1/</UP>n (Eq. 1)
where r is the ratio of bound T4L to total alpha -crystallin, L is the fraction of free native state T4L, n is the number of binding sites, and KDa is the dissociation constant. Table II lists the number of binding sites and the corresponding dissociation constants obtained for the two mutants. Because alpha B-crystallin does not recognize the native state of T4L, the resulting KDa is an apparent dissociation constant. It differs from the intrinsic dissociation constant by a unitless equilibrium constant that characterizes the transition from the native state to the state recognized by the chaperone. Thus, the higher KDa for L99A relative to L99A/A130S, reported in Table II, presumably reflects the higher stability of the native state of the former. The number of available binding sites is similar in the range of 0.4-0.5 T4L per alpha B-D3 subunit.


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Fig. 1.   Binding isotherms to alpha B-D3 at 23°C, pH 7.2, of the moderately destabilized mutants T4L-L99A and T4L-L99A/A130S (a) and the highly destabilized mutant T4L-L99A/F153A (b). The solid line is a linear fit based on a single mode of binding. The dashed line is a numerical simulation based on two modes of binding.


                              
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Table II
Stoichiometries and dissociation constants of T4L binding to alpha B-D3 at pH 7.2, 23 °C

The isotherm of the most destabilized mutant, L99A/F153A, shown in Fig. 1b appears also to be linear. However, the intercept is significantly smaller reflecting an apparent increase in the number of binding sites. One interpretation of this isotherm is that L99A/F153A binds through a single but different mode than L99A with n congruent  2. An alternative interpretation, based on the two-mode binding of alpha A-crystallin (24), is that binding occurs through two sets of independent binding sites. The dashed curve superimposed on Fig. 1b is a numerical simulation with KDa1 = 8 µM, KDa2 = 40 µM, n1 = 0.6, and n2 = 1.2. The choice of n1 and n2 is justified by the results presented below (Fig. 5). In either case, this result implies that a second mode for binding of L99A/F153A is available presumably because of the conformation of the transient state(s) populated at this value of Delta Gunf. It should be noted that the overall fold of the native state of this mutant, determined by x-ray crystallography, is similar to that of L99A (33).

pH Activation of Two-mode Binding-- The presence of two modes of binding is revealed more directly in the binding isotherms at pH 8. Similar to alpha A-crystallin, increased pH results in significant changes in the binding properties of alpha B-crystallin (24). This is illustrated in the pH titration of the binding of T4L-D70N to alpha B-D3 shown in Fig. 2. Despite the lack of change in the Delta Gunf of D70N in the 6-8 pH range (34), the fraction of bound T4L increases significantly with increasing pH.


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Fig. 2.   Increase in the extent of binding of T4L-D70N at higher pH. The molar ratio of alpha B-D3 to T4L is 8:1.

The origin of the increase in the bound fraction can be gleaned from quantitative analysis of the binding isotherm of L99A/A130S at pH 8.0 shown in Fig. 3. The curved isotherm of this mutant, bound in a single mode at pH 7.2, reflects the presence of two sets of binding sites characterized by different affinities, referred to as binding modes. The first, a high affinity/low capacity mode, has a number of binding sites similar to that obtained at pH 7.2 but a significantly higher affinity. The second has a higher number of binding sites but a lower affinity. The superimposed solid line is a numerical simulation based on the equation describing binding to two sets of independent binding sites,
<UP>1/</UP>(r<SUB>t</SUB>)<UP>=1/</UP>(r<SUB><UP>1</UP></SUB><UP>+</UP>r<SUB><UP>2</UP></SUB>) (Eq. 2)
where rt is the ratio of total T4L bound to alpha B-crystallin and ri is the ratio of T4L bound at site i to alpha B-crystallin given by Equation 1. The parameters used for this simulation are reported in Table III.


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Fig. 3.   pH activation of two-mode binding of T4L-L99A/A130S at 23 °C, pH 8.0. The solid curve is simulated based on the parameters of Table III.


                              
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Table III
Stoichiometries and dissociation constants of T4L-L99A/130S binding to alpha B-crystallin variants at pH 8.0, 23 °C

The increased extent of binding at pH 8.0 allows a comparative analysis of the binding properties of alpha B-crystallin phosphorylation analogs. Fig. 4 compares the binding isotherms of these variants to the same T4L mutant L99A/A130S. Because of the increased binding, the alpha B-D2 isotherm is shown separately in Fig. 4b along with that of alpha B-D3. The curves for WT alpha B-crystallin and the single serine to aspartate variants are all linear suggesting predominant binding at the high affinity sites. The decrease in the slope indicates increased affinity whereas the similar intercept implies a similar number of available sites. Thus, Fig. 4 demonstrates that the S45D substitution is the most effective among the single substitution in increasing the affinity (Table III). It is noted that the determination of the number of binding sites of WT alpha B-crystallin is inherently unreliable due to the weak binding and the resulting accessible ranges of 1/L and 1/r. The combination of S45D and S59D (alpha B-D2) results in incipient curving of the binding isotherm indicating a contribution from the low affinity mode. A parallel set of experiments, carried out with L99A as the substrate, leads to similar conclusions (data not shown).


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Fig. 4.   Comparative analysis of the binding characteristics of alpha B-crystallin phosphorylation mimics. a, single mode binding of T4L-L99A/A130S by single serine to aspartate substitutions. b, activation of the second mode of binding in alpha B-D2 and alpha B-D3.

Temperature Activation of Binding-- To determine whether the differences between alpha B and its phosphorylation analogs persist under physiological temperatures, binding isotherms were obtained at 37 °C. Fig. 5a shows the binding isotherm of L99A to alpha B-D3 at this temperature. The data do not conform to the linear behavior expected for independent equivalent binding sites (Equation 1). Therefore, a non-linear least squares fit to Equation 2 was performed resulting in the superimposed solid line of Fig. 5. The corresponding parameters obtained from this fit are reported in Table IV. Because of the decrease in the stability of L99A at 37 °C relative to 23 °C, binding by the high affinity mode is expected to increase. Furthermore, the curvature suggests that the reduction in Delta Gunf is sufficient to activate the low affinity mode similar to L99A/F153A at 23 °C (Fig. 1b).


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Fig. 5.   a, two-mode binding of alpha B-D3 to T4L-L99A at pH 7.2, 37 °C. The solid line is a non-linear least squares fit based on the parameters of Table IV. b, two-mode binding of alpha B-D3 to T4L-D70N at pH 7.2, 37 °C. At this temperature the Delta Gunf of this mutant is similar to that of T4L-L99A/A130S at 23 °C (Fig. 1a). The solid line is a numerical simulation based on the parameters of Table IV.


                              
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Table IV
Stoichiometries and dissociation constants of T4L-D70N binding to alpha B-crystallin variants at 37 °C

However, the increase in the extent of binding cannot be attributed solely to the change in Delta Gunf of L99A. Fig. 5b shows the binding isotherm of D70N at 37 °C where it has a Delta Gunf similar to L99A/A130S at 23 °C. Comparison of the 1/r values at similar 1/L indicates a higher extent of binding for D70N. The incipient curving of the isotherm of this mutant reflects an activation of the second mode of binding at 37 °C and not 23 °C. Furthermore, the affinity of the low capacity mode increases by almost an order of magnitude (Tables II and IV). Taken together, these results strongly suggest that activation of binding at high temperature is partly associated with changes in the binding characteristics of alpha B-D3.

At 37 °C, the extent of binding of T4L by alpha B-crystallin phosphorylation analogs is higher than the WT. Fig. 6 compares the binding isotherms of WT-alpha B, S59D, and alpha B-D3 to D70N at pH 8. The decrease in the slope of the S59D isotherm relative to the WT indicates increased affinity, assuming that a similar number of sites are available. The three Ser to Asp substitutions result in a significant increase in the amount of T4L bound per alpha B-crystallin subunit as a consequence of the activation of the second mode of binding (Table IV). It is noted that comparison of the isotherms of D70N in Figs. 5 and 6 indicates that binding is also pH-activated at 37 °C.


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Fig. 6.   Comparative analysis of the binding characteristics of alpha B-crystallin phosphorylation mimics to T4L-D70N at 37°C, pH 8. The solid line superimposed on the alpha B-D3 isotherm is a numerical simulation based on the parameters of Table IV.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To develop a mechanistic understanding of the chaperone activity of sHSP, it is central to determine whether conformational changes occur during or as a control of recognition and binding. Because the phosphorylation of mammalian sHSP has been associated with changes in their oligomeric assembly (16, 21), the functional consequences are important not only in the context of their cellular role but also as a test of the hypothesized coupling between transient dissociation of the oligomeric structure and the binding of non-native proteins (25-27).

Phosphorylation as a Mechanism for Activation of Mammalian sHSP-- The main conclusion of this study is that phosphorylation of alpha B-crystallin results in a significant enhancement of its binding to non-native states of T4L. The activation occurs in the single phosphorylation analogs that tend to be the predominant form physiologically, in particular at residue Ser-45 (16). This conclusion does not necessarily contradict the results of Ito et al. (16) that alpha B-D3 is marginally less efficient in suppressing the aggregation of alcohol dehydrogenase at 50 °C. At this temperature, alcohol dehydrogenase is predominantly in the unfolded states whereas in our binding assay T4L is predominantly in the folded state. The results of Fig. 1b show that substantially destabilized substrates (L99A/F153A) can activate binding at both modes. Thus, it is conceivable that a negative Delta Gunf for the substrate may render the affinity difference between the WT and the D3 form of alpha B-crystallin less significant. As to the increased level of light scattering in the presence of alpha B-D3, it may be a consequence of the lower thermal stability of this mutant (16) resulting in co-precipitation with the substrate at 50 °C (35).

If the primordial role of sHSP is to respond to an increase in temperature, it is logical to have a stress-activated switch built into the oligomeric structure that serves to regulate binding affinity and stoichiometry. However, the narrow ranges of physiological pH and temperature in mammals requires the use of an alternative mechanism, i.e. phosphorylation, to activate the switch. It is noted in this context that phosphorylation sites in both HSP27 and alpha B-crystallin are located in the N-terminal domain. Current structural models suggest the burial of this domain in the core of the oligomer (27, 29). Thus, the introduction of negative charges is likely to destabilize the oligomer. In support of this hypothesis, a variant of HSP27 containing three serine to aspartate substitutions dissociates into a tetramer (21, 36). Furthermore, equilibrium sedimentation analysis demonstrates that the alpha B-D3 oligomer is smaller than the native oligomer (16).

Previous studies have indirectly implicated a transient dissociation of the oligomeric structure in the mechanism of binding of non-native proteins by sHSP (25, 26, 28, 37-40). The underlying dynamic processes presumably expose binding regions otherwise inaccessible to the substrate. Therefore, the dual effects of the triple Ser to Asp substitutions on the oligomeric structure and on the binding to T4L can be interpreted in terms of activation of these dynamic processes rather than a direct participation of the phosphorylated sites in the interaction with the substrate. In support of this interpretation, it is noted that the effects of the Ser to Asp are not only to enhance the affinity but also to increase the available number of binding sites similar to what is observed with increased temperature and pH. This suggests changes in the oligomeric structure, albeit it of dynamic nature, that expose these additional sites. The lack of detectable change in the elution volumes of the single Ser to Asp variants in size-exclusion chromatography suggests the need for different experimental approaches to fully assess the consequences of phosphorylation not only on the static oligomer but also on its dynamics.

Two-mode Binding of the Phosphorylated Analogs-- The results also provide a mechanistic perspective on alpha B-crystallin chaperone function. The Ser to Asp substitutions reveal the presence of two modes of binding in alpha B-crystallin. This is inferred from the curved isotherms observed for a number of mutants. Besides the two types of binding isotherms emphasized in this paper, the two-mode model predicts a range of changes in the shape of the isotherms as the binding transitions from one mode to two modes. In particular, numerical calculations reveal that in a range of KDa1 and KDa2, the curvature can either be weak or can occur in a range that is not accessible experimentally. An example of the former is the binding isotherm of L99A/A130S to alpha B-D2 (Fig. 4b). In the case of linear isotherms, the intercept may vary in the range between 1 and 2. We experimentally observed this type of isotherm for other T4L mutants (data not shown), and their interpretation is consistent with the two-mode model.

The term "mode" is used to emphasize that the data presented in this paper do not address whether the two sets of binding sites are physically separate, overlapping, or identical. In the latter case, the appearance of a second mode of binding would result from the increased availability of the binding sites at higher temperature, higher pH, or as a consequence of the substrate conformation presented. Therefore, the analysis of the data in this paper can be considered phenomenological. Regardless of the origin of the two modes, for the same T4L mutant, a fraction is bound with n congruent  0.5 whereas another fraction is bound with n congruent  1. Similar to the conclusion obtained from binding of T4L to alpha A-crystallin, the more destabilized mutants seem to activate binding at the high capacity site. This is revealed by the isotherm of L99A/F153A at 23 °C, pH 7.2. In contrast, at the same temperature and pH, L99A and L99A/A130S bind exclusively at the high affinity site.

Both modes of binding have higher affinity at higher pH and temperature. For the case of D70N, a mutant with a pH-independent Delta Gunf, the pH effect reflects either an electrostatic contribution to the interaction or chaperone-specific effects that further enhance binding. The latter is supported by the reduction in the apparent molecular mass of alpha B-D3 at higher pH detected by size-exclusion chromatography.2 Similarly, comparative analysis of the binding characteristics of alpha B-D3 to mutants with similar stability at different temperatures supports a temperature activation of binding, in particular an increase in the affinity of the high capacity mode. This is likely to be a consequence of changes in the oligomeric structure of similar origin to those induced by phosphorylation.

Nature of the T4L States Recognized by alpha B-crystallin-- Because T4L unfolding equilibrium is two-state, the simplest interpretation of the data presented in this paper is that alpha B-crystallin recognizes the unfolded state, the population of which increases as Delta Gunf decreases. This interpretation predicts that the change in apparent dissociation constant between two mutants should reflect the change in their folding equilibrium constant. Comparison of the change in KD for L99A and L99A/A130S reveals a 2-fold decrease. Based on the change in the Delta Gunf the change should be closer to 5.

Similarly, the changes in KDa for the more destabilized mutants such as L99A/F153A at 23 °C or L99A at 37 °C do not support this simple interpretation. A second mode of binding is employed rather than an increase in the affinity of the low capacity mode. A similar conclusion was obtained from the analysis of binding of alpha A-crystallin to T4L.

Because the alpha B-crystallin variants do not bind the folded state of T4L, one interpretation of the results of this paper is that they recognize high energy states of proteins that are transiently populated under native conditions, also known as excited states (41, 42). The existence of these states in T4L has been demonstrated in WT T4L (43) and in the L99A mutant (44). In the context of this interpretation, the activation of the second mode of binding by extremely destabilized mutants, such as L99A/F153A at 23 °C, might reflect a conformational preference in the binding at each mode. Thus, the low capacity mode (n = 0.5) is used to bind native-like excited states, i.e. characterized by limited unfolding, which are populated in relatively stable proteins such as L99A. As the stability of the native state is further reduced, the dynamic population of globally unfolded states becomes more favorable. The high capacity mode (n = 1) is selective to such states. Because of the intrinsic lower affinity of this mode, the apparent KD is in the same range observed for the more stable mutants bound by the high affinity mode.

Concluding Remarks-- That the general outline of the mechanism of binding in alpha B-crystallin is similar to that of alpha A-crystallin is consistent with the extensive sequence and structural similarities between the two proteins. The functional mechanism of the alpha -crystallins, whereby the affinity toward non-native states is bracketed whereas the binding capacity is increased in response to the appearance of extensively unfolded states, has been interpreted previously by us as a reflection of the need of sHSP to avoid the role of unfoldases under physiological conditions (24). Such activity would occur if the affinity for globally unfolded states is set too high.

The differences in the details are suggestive of the different roles that the two proteins have acquired. alpha A-crystallin has a higher affinity for the T4L mutants than the WT alpha B-crystallin. The lower affinity of alpha B-crystallin may reflect the more stringent requirement on its activation in non-lenticular tissues. Unlike the lens fiber cells, in which relatively little protein synthesis occurs, in typical cells unfolded proteins continuously emerge from the ribosomes. A high affinity of alpha B-crystallin for these proteins might significantly disrupt normal synthesis and folding.

Through phosphorylation, alpha B-crystallin can be activated to levels that exceed the affinity and binding capacity of alpha A-crystallin. The relatively small level of alpha B-crystallin phosphorylated at the three serine residues is a reflection of the remarkable affinity of this form to bind non-native states. The absence of a tight control of the level of this protein might lead to an undesirable unfoldase activity in the cell.

The concomitant destabilization of the oligomeric structure and increase in the extent of binding of T4L with increased phosphorylation is consistent with the requirement of conformational changes during function. Thus far the manifestations of the dynamic oligomeric structure of mammalian sHSP include subunit exchange (45), heterogeneous oligomeric structure (46), and modulation of substrate binding. Understanding the molecular mechanism mediating these processes and the extent of their coupling is an important step toward solving the structure-function puzzle of sHSP.

    ACKNOWLEDGEMENTS

We thank Drs. Hasige Sathish and Chuck Cobb for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by Grant EYR0112683 from the NEI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University, 741 Light Hall, Nashville, TN 37232. Tel.: 615-322-3307; Fax: 615-322-7236; E-mail: hassane.mchaourab@vanderbilt.edu.

Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M211851200

2 H. A. Koteiche and H. S. Mchaourab, unpublished results.

    ABBREVIATIONS

The abbreviations used are: sHSP, small heat-shock protein(s); T4L, T4 lysozyme; WT, wild type; alpha B-D2, S45D/S59D; alpha B-D3, S19D/ S45D/S59D.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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