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INTRODUCTION |
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
A-crystallin and
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
-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
-crystallin subunits,
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
B-crystallin and other mammalian sHSP in these pathways includes
their phosphorylation by mitogen-activated protein kinase-activated
protein kinases (12-15).
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
B-crystallin that is associated with desmin-related myopathy (18).
In the lens,
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
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
B-crystallin and HSP27
is accompanied by translocation of these proteins from the cytoplasm to
the nucleus (23).
On the functional level, phosphorylation of
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
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
B-crystallin. In general, little mechanistic insight can be obtained from these studies.
In a recent study, we reported the equilibrium binding of
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
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
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
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
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).
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EXPERIMENTAL PROCEDURES |
Cloning and Site-directed Mutagenesis--
The cDNA of mouse
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
(
B-D2), and S19D/S45D/S59D (
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).
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
B
variants was induced by the addition of 0.4 mM
isopropyl-1-thio-
-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.
B-crystallin and its single variants were purified
using anion exchange chromatography followed by size exclusion
chromatography as described previously (6).
B-D2 and
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
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.
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RESULTS |
To investigate the recognition and binding steps involved in sHSP
chaperone function, a set of destabilized T4L mutants was constructed,
the
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
A-crystallin (24).
To explore the effect of phosphorylation on the chaperone activity of
B-crystallin, the following mutants were constructed: S19D, S45D,
S59D, S45D/S59D (
B-D2), and S19D/S45D/S59D (
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
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
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
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.
Incubation of 50 µM T4L mutants with a 10-fold molar
excess of WT
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
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
B-D3. Both curves are linear and
can be fit to the equation,
|
(Eq. 1)
|
where r is the ratio of bound T4L to total
-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
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
B-D3 subunit.

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Fig. 1.
Binding isotherms to
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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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
2. An alternative interpretation, based on the two-mode binding of
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
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
A-crystallin, increased pH results in significant changes
in the binding properties of
B-crystallin (24). This is illustrated
in the pH titration of the binding of T4L-D70N to
B-D3 shown in Fig.
2. Despite the lack of change in the
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 B-D3 to T4L is 8:1.
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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,
|
(Eq. 2)
|
where rt is the ratio of total T4L bound to
B-crystallin and ri is the ratio of T4L bound at
site i to
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 B-crystallin variants at pH 8.0, 23 °C
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The increased extent of binding at pH 8.0 allows a comparative analysis
of the binding properties of
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
B-D2 isotherm is shown separately in Fig.
4b along with that of
B-D3. The curves for WT
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
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 (
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 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 B-D2 and
B-D3.
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Temperature Activation of Binding--
To determine whether the
differences between
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
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
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 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 B-D3 to T4L-D70N at pH 7.2, 37 °C. At this temperature the 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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However, the increase in the extent of binding cannot be attributed
solely to the change in
Gunf of L99A. Fig.
5b shows the binding isotherm of D70N at 37 °C where it
has a
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
B-D3.
At 37 °C, the extent of binding of T4L by
B-crystallin
phosphorylation analogs is higher than the WT. Fig.
6 compares the binding isotherms of
WT-
B, S59D, and
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
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 B-crystallin
phosphorylation mimics to T4L-D70N at 37°C, pH 8. The
solid line superimposed on the B-D3 isotherm is a
numerical simulation based on the parameters of Table IV.
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 |
DISCUSSION |
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
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
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
Gunf for the
substrate may render the affinity difference between the WT and the D3
form of
B-crystallin less significant. As to the increased level of
light scattering in the presence of
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
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
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
B-crystallin chaperone
function. The Ser to Asp substitutions reveal the presence of two modes
of binding in
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
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
0.5 whereas
another fraction is bound with n
1. Similar to the
conclusion obtained from binding of T4L to
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
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
B-D3 at higher pH
detected by size-exclusion chromatography.2 Similarly,
comparative analysis of the binding characteristics of
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
B-crystallin--
Because T4L unfolding equilibrium is two-state,
the simplest interpretation of the data presented in this paper is that
B-crystallin recognizes the unfolded state, the population of which
increases as
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
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
A-crystallin to T4L.
Because the
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
B-crystallin is similar to that of
A-crystallin is
consistent with the extensive sequence and structural similarities
between the two proteins. The functional mechanism of the
-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.
A-crystallin has a higher
affinity for the T4L mutants than the WT
B-crystallin. The lower
affinity of
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
B-crystallin for these proteins might significantly
disrupt normal synthesis and folding.
Through phosphorylation,
B-crystallin can be activated to levels
that exceed the affinity and binding capacity of
A-crystallin. The
relatively small level of
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.