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INTRODUCTION |
In response to exposure to high temperature and other forms of
stress, cells and organisms express multiple families of heat-shock proteins (HSP)1 (1). The
function of these proteins is to confer thermotolerance via a variety
of mechanisms including suppression of aggregation and refolding of
denatured proteins. The small heat-shock protein (sHSP) family consists
of proteins with molecular mass <40 kDa and possessing a stretch of
80-100 amino acids that shows sequence similarities to lens
-crystallins, the
-crystallin domain (2). The extent of sequence
similarity in this domain is variable, ranging from 20% between
distant members of the family (e.g. bacterial and mammalian
sHSP) to over 60% between mammalian sHSP (2-4). Flanking the
-crystallin domain are an N-terminal region, characterized by
extensive sequence and length variability, and a polar nonconserved C-terminal tail (2). The patterns of abundance and expression of sHSP
are also species-specific. For instance, heat-stressed plant cells
express over 25 sHSP (3), while mammalian cells express two: HSP 25/27
and
B-crystallin (5, 6). Although their cellular function is not
well understood, in vitro sHSP bind unfolding proteins in a
stable complex (7, 8). This chaperone-like function does not require
ATP, and sHSP do not have an intrinsic ability to refold their bound
substrates (9, 10).
Associated with the sequence divergence of sHSP is an oligomeric
structure characterized by different symmetries and degrees of order.
Many sHSP, particularly from archeal and bacterial species, have well
defined quaternary structures, while those of mammalian sHSP are
variable with oligomers that constantly exchange subunits (11, 12).
Recent x-ray diffraction studies of the Methanococcus jannaschii HSP 16.5 show an ordered oligomer with 24 subunits (4).
Mycobacterium tuberculosis HSP 16.3 is believed to consist of nine subunits arranged in a trimer of trimers (13). In contrast, cryo-electron microscopy studies reveal that
B-crystallin has a
variable quaternary structure indicative of a high degree of intrinsic
flexibility (11). While the role of subunit dynamics in sHSP has not
been elucidated, compelling evidence suggests that the flexibility of
the quaternary structure in
B-crystallin is essential to its
protective function (14, 15). This dynamic structure is thought to
arise from nonspecific interactions in the N-terminal domain. Initially
proposed by Augusteyn and Koretz (16), a micellar model of
-crystallin appears to be the most consistent with the known
properties of the oligomer. In this model, the hydrophobic N-terminal
domains, representing the apolar ends of the subunits, are packed in
the core of the oligomer. Wistow (17) proposed that the N-terminal
domain interactions occur between basic tetrameric units assembled by
subunit contacts in the
-crystallin domain. Wistow's model is based
on the observation of Merck et al. (18) that the recombinant
-crystallin domain of
A forms dimers and tetramers.
We have used site-directed spin labeling (19) to demonstrate the
existence of subunit interfaces in the
-crystallin domain of
A-crystallin and HSP 27 and to determine the folding pattern of a
part of this domain in
A-crystallin (20-22). In both proteins, evidence of spatial proximities between single nitroxides introduced along a highly conserved sequence led to the conclusion that this sequence may form a subunit interface (20, 21). The extent of the
interaction, particularly for HSP 27, indicates that more than 90% of
the subunits have identical local geometry, consistent with an ordered
building block involving the
-crystallin domain.
In this study, the symmetry of this subunit interface was investigated
by determining the number of strands involved and their pattern of
interaction. The data are consistent with the presence of antiparallel
-strands related by a 2-fold symmetry. The role of this interface in
the assembly of a basic multimeric unit is examined within the context
of the
-crystallin domain expressed in isolation. The effect of
sequence divergence on the local structure and subunit interactions is
evaluated in a bacterial sHSP, M. tuberculosis HSP 16.3. Site-directed spin labeling results are consistent with cryo-electron
microscopy studies indicating the presence of a 3-fold symmetry (13).
Heterologous association between trimers is mediated by subunit
interactions along the sequence. The results are compared with the
recently determined crystal structure of M. jannaschii HSP
16.5 (4).
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EXPERIMENTAL PROCEDURES |
Materials--
Resource Q media was obtained from Amersham
Pharmacia Biotech, as were the Superose 6, Superdex 75, HiTrap Q, and
HiTrap desalting columns. The POROS PEI column was obtained from
PerSeptive Biosystems. Horse liver alcohol dehydrogenase was obtained
from Sigma. Methanethiosulfonate spin label was obtained from Toronto
Research Chemicals.
Site-directed Mutagenesis--
The truncated
-crystallin
domain of HSP 27 was constructed using the polymerase chain reaction
(PCR). The 5' primer was designed to contain an NdeI site
flanking an 18-base sequence that starts at codon 88. The 3' primer was
the T7 terminator primer. The PCR fragment was then subcloned between
the NdeI and XhoI sites of pET-20b(+) to yield
the plasmid pET-tHSP 27. The single mutant plasmids of native HSP 27 in
the 133-142 region were digested with HincII and
XhoI and then subcloned into the pET-tHSP 27 background.
The truncated
-crystallin domain of
A-crystallin and all of the
single mutants were constructed by PCR. The cysteineless WT (WT*) was
constructed using the same strategy as pET-tHSP 27 to yield pET-t
A.
The 5' primer contained an NdeI site flanking an 18-base
sequence starting at codon 63. PCR fragments containing single-cysteine
substitutions in the 109-120 sequence were subcloned between the
NdeI and KpnI sites of pET-t
A.
HSP 16.3 was subcloned from the plasmid pMV261 (a generous gift from
Dr. Clifton Barry III) (23) into the pET-20b(+) expression vector.
Briefly, the gene was amplified, and the PCR product was digested by
the enzymes NdeI and XhoI and then subcloned.
Single-cysteine mutants of HSP 16.3 were constructed as described
previously (20). All clones were isolated and sequenced to verify the
presence of the desired mutation and the absence of unwanted changes.
Single-site mutants are named by specifying the original residue, the
number of the residue, and the new residue, in that order.
Expression, Purification, and Spin Labeling of the
Mutants--
Truncated
A-crystallin and HSP 27 mutants were
expressed and purified on an anion exchange column as described
previously (21), with the exception that protein expression was induced at 30 °C. For
A-crystallin mutants, ammonium sulfate was added to
the eluted anion exchange protein peak to a final concentration of 1 M, and this sample was loaded on a phenyl-Sepharose column, as suggested by Dr. Michael P. Bova (UCLA). The protein of interest was
eluted using a linearly decreasing gradient of ammonium sulfate. The
sample buffer was exchanged (20 mM MOPS, 50 mM
NaCl, 0.1 mM EDTA, pH 7.2) using a HiTrap desalting column.
The sample was then reacted with a 10-fold excess of the
methanethiosulfonate spin label at room temperature for 2 h and
allowed to proceed to completion overnight at 4 °C to yield the side
chain R1, as shown in Scheme 1. After
anion exchange, HSP 27 samples were further purified on a Superose 6 column and spin-labeled as described above. Protein samples were
concentrated using MICROSEP 10 filter units.
All HSP 16.3 mutants were expressed at 30 °C and purified as
described in Ref. 13, except that the first purification step was
performed on a HiTrap Q anion exchange column. Mutants S91C, E92C,
G96C, and R100C contained 1.2 M GdnHCl in the anion
exchange elution buffer. Samples were then loaded onto a PEI column and eluted with a linear gradient of sodium chloride (13). Further purification was achieved using a Superose 6 size exclusion column. Eluted samples were spin-labeled as described above.
EPR Measurements--
EPR spectroscopy was performed on a Varian
E102 spectrometer using a two-loop one-gap resonator (24). For
P1/2 measurements, samples were loaded in
gas-permeable TPX capillaries; otherwise, samples were placed in glass
capillary tubes. The EPR spectra of the mixed oligomers were recorded
under field-frequency lock. The microwave power was 2 mW incident, and
the Zeeman modulation amplitude was 1.6 G.
Power saturation studies were carried out under nitrogen, in the
presence and absence of 3 mM NiEDDA, to yield the parameter P1/2. The EPR accessibility parameter
was
calculated as described previously in Refs. 20 and 25.
Circular Dichroism--
Far-UV circular dichroism measurements
on
A-crystallin and HSP 27 truncation mutants were performed on a
Jasco 710 spectropolarimeter at a concentration of 0.15 mg/ml and 0.2 mg/ml, respectively. Protein samples were prepared in 20 mM
sodium phosphate, pH 7.1. Measurements were taken in the range of
190-260 nm at room temperature.
Size Exclusion Chromatography--
The average molecular mass
for all mutants was determined using size exclusion chromatography. HSP
16.3 and HSP 27 mutants were analyzed using a Superose 6 column, and
A-crystallin mutants were analyzed using a Superdex 75 column. All
samples were injected from a 100-µl sample volume and at a flow rate
of 0.5 ml/min. The columns were calibrated according to the
manufacturer's specifications.
Chaperone Activity Assays--
Aggregation of horse liver
alcohol dehydrogenase at 48 °C was monitored by measuring the
absorption due to scattering at 360 nm as described previously (23).
Samples were prepared in 30 mM sodium phosphate, pH 7.0, and denaturation of alcohol dehydrogenase was initiated by the addition
of 3 mM final concentration of EDTA.
Refolding of Mixed Oligomers--
A-crystallin and HSP 27 mutants were incubated at room temperature with their respective WT* in
the presence of 6 M urea. Samples were rapidly diluted to a
urea concentration of <1 M and then desalted on a HiTrap
column (26). Spin-labeled HSP 16.3 subunits were exchanged with WT at
room temperature in the presence of 0.75 M GdnHCl and then
desalted using a HiTrap column. For all exchange samples, the relative
concentrations of R1-labeled subunit and WT* were determined by
absorbance at 280 nm and confirmed by the Bradford assay.
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RESULTS |
A Conserved 2-Fold Symmetric Interface in
A-Crystallin and HSP
27--
The simplest model consistent with the observation
of spin-spin interactions at every residue along the 134-139 stretch
in HSP 27 is a 2-fold rotational symmetry that results in the hydrogen bonding of the two strands in an antiparallel fashion. One example of
such an arrangement is shown in Fig. 1.
The extent of spin-spin interactions reported by Mchaourab et
al. (21) is consistent with an interresidue separation of less
than 10 Å expected based on such geometry. Furthermore, the rules of
antiparallel packing of
-strands require a specific register that
allows hydrogen bonding of the backbone. Another constraint on the
model is the observation of disulfide bonding at Cys137,
indicating that these residues are separated by less than 8 Å (21).
This model predicts a specific pattern of proximities between
nonidentical residues and two spins along the interface of a given
oligomer. Both of these aspects can be tested.

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Fig. 1.
Schematic diagram of a packing model of the
two -strands at the isologous subunit
interface of HSP 27. Open circles indicate
residues that are more solvent-exposed, and filled
circles represent buried sites.
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The number of interacting spins can be determined experimentally by
dilution of an oligomer showing spin-spin interactions with increasing
amounts of unlabeled WT*. Because of the dramatic difference in the
normalized spectral amplitudes arising from an interface containing one
spin versus an interface containing two or more spins, the
fractional population of the former can be easily calculated from the
normalized amplitude of the central resonance line. This is illustrated
in Fig. 2a, where the addition of 0.25 molar equivalent of WT* results in a composite spectrum dominated by the sharp signal originating from interfaces containing a
single nitroxide. Fig. 2b shows the increase in the
population of monomeric R1 for different ratios of WT* to C137R1.

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Fig. 2.
a, EPR spectra of HSP 27 C137R1 refolded
in the presence of increasing amounts of WT*. The arrows
indicate spectral features arising from dipole-dipole broadening of the
spectrum. Where appropriate, a scaling factor is indicated to the
left of the spectrum. All spectra have a scan width of 200 G. b, increase in the fractional population of monomeric
spins versus the molar ratio of WT*:C137R1 ( ). The
solid line is the theoretical increase calculated
from the binomial distribution.
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The formation of an interface, consisting of N subunits, from mixtures
of WT* and C137R1 is a random process described by the binomial
distribution. Therefore, it is possible to calculate for every
stoichiometry of WT* to R1-labeled subunits the fractional population
of interfaces containing a single spin. The change in the fractional
population as a function of stoichiometry reflects the number of
interacting subunits, N, and thus the symmetry of the
interface. The calculated increase in the fractional population of
monomeric spins for a dimeric unit, i.e. consisting of two strands, is
superimposed on the experimental data points in Fig. 2b. The
close agreement demonstrates that the broadening in the EPR spectra of
C137R1 arises from the interaction of two spins. The use of spin-spin
interactions to determine oligomer symmetry was also reported recently
by Langen et al. in the context of the membrane-bound
structure of annexins (27).
An antiparallel arrangement also results in a specific pattern of
proximities between R1 introduced at nonidentical residues. Although
this depends to some extent on the exact relative alignment of the
strands and their right-handed twist, short range spin-spin interactions are expected in many of the possible combinations. Therefore, the pattern of proximities between the two strands was
examined by forming mixed oligomers from two subunits where R1 is
introduced at different residues. For each pair, equimolar amounts of
each mutant were mixed in the presence of 6 M urea. The
co-oligomers were refolded following the protocol of Ref. 26. On a
statistical basis, 50% of the oligomers consist of mixed subunits of
the two R1-labeled mutants. Thus, spectral subtraction was used to
separate the 25% contribution of oligomers consisting of each mutant.
Fig. 3 shows that in HSP 27 the pattern
of pairwise spin-spin interactions among residues S135R1, C137R1, and
T139R1 is consistent with the antiparallel arrangement of Fig. 1. The separation of R1 in the 135/139 mixed oligomer is less than in the 139 homo-oligomers, as deduced from the increased broadening in the
spectrum of the former. The pairs S135R1/C137R1 and C137R1/T139R1 show
extensive spin-spin interactions, which indicates separations of less
than 10 Å.

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Fig. 3.
EPR spectra of HSP 27 mixed oligomers
refolded in a 1:1 molar ratio. All spectra have a scan width of
200 G.
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A 2-fold symmetry also appears to be involved in the assembly of the
A-crystallin oligomer. A similar pattern of change in the monomeric
spin population was observed when the E113R1 oligomer was titrated with
WT*
A-crystallin. As shown in Fig.
4b, except for the zero point,
the data follow a binomial distribution expected for a two-spin basic
unit. The origin of the deviation in the zero point appears to be the
incomplete refolding of a small population of this particular mutant.
That
A-crystallin can form co-oligomers with HSP 27 has been
established by Merck et al. (28) using immunoprecipitation
analysis. Fig. 5b demonstrates
that these co-oligomers also have the expected 2-fold symmetry. When
the C137R1 mutant of HSP 27 is refolded in the presence of increasing amounts of WT*
A, the increase in the monomeric population follows the trend expected from a dimeric basic unit.

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Fig. 4.
a, EPR spectra of A-crystallin E113R1
refolded in the presence of increasing amounts of WT*. Where
appropriate, a scaling factor is indicated to the left of
the spectrum. All spectra have a scan width of 200 G. b,
increase in the fractional population of monomeric spins
versus the molar ratio of WT*:E113R1 ( ). The
solid line is the theoretical increase calculated
from the binomial distribution.
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Fig. 5.
a, EPR spectra of HSP 27 C137R1 refolded
in the presence of increasing amounts of A-crystallin WT*. Where
appropriate, a scaling factor is indicated to the left of
the spectrum. All spectra have a scan width of 200 G. b,
increase in the fractional population of monomeric spins
versus the molar ratio of A-crystallin WT*:HSP 27 C137R1
( ). The solid line is the theoretical increase
calculated from the binomial distribution.
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The 2-Fold Symmetric Interface Is Present in the Recombinant
-Crystallin Domain--
To examine whether this conserved interface
mediates the formation of a building block of the quaternary structure,
its presence was investigated in the
-crystallin domain of
A-crystallin and HSP 27 expressed in isolation. For this purpose, a
truncated form of
A-crystallin consisting of residues 63-173 and a
truncated form of HSP 27 consisting of residues 88-205 were
constructed. Previously, Merck et al. demonstrated that
these domains are folded and form multimeric structures (18). Twelve
sequential cysteine mutants of each truncated domain were constructed.
All mutants were overexpressed and remained in the soluble fraction,
and the molecular mass of a subunit, determined by SDS-polyacrylamide gel electrophoresis, showed no evidence of proteolysis (data not shown). Using far-UV circular dichroism, it was verified that all
mutants except
A-crystallin L120R1 have a predominantly
-sheet structure, and the spectra in the 210-220 nm region were
superimposable on that of the truncated WT*. L120R1 appears to have
increased random coil content. In the native oligomer as well as in the truncated domain, this residue is in a buried environment. Thus, it is
possible that the introduction of R1 at this site results in the local
disruption of secondary structure (data not shown).
The molecular mass of the truncated multimers was determined by size
exclusion chromatography. As previously reported, the elution peaks of
the truncated
-crystallin domains are asymmetric, reflecting the
heterogeneity of these oligomers (18). Despite the sequence similarity
along the
-crystallin domain of
A and HSP 27, the average
molecular mass indicates that the truncated multimers of HSP 27 have
further subunit contacts, allowing the formation of a higher order
structure than
A (Tables I and
II). Truncated
A-crystallin elutes in
a range of molecular masses consistent with the formation of dimers and
tetramers (18).
All R1-labeled mutants have average molecular masses in the range of
that of the WT*. In both truncated
A-crystallin and HSP 27, R1
substitution along the subunit interface results in an apparent
increase in the molecular mass, as was observed in the native oligomer
(20, 21). It is likely that this effect is due to changes in the
hydrodynamic radius that result from local readjustments to accommodate
the increased molar volume of R1.
Regardless of the difference in molecular mass, both
-crystallin
domains have subunit interactions along the target sequence. For HSP
27, the EPR spectra of R1 at residues 134-138, shown in Fig.
6a, indicate spin-spin
interactions of similar magnitude to those observed in the native
oligomer. The absence of a dominant sharp component indicates that
90-95% of the multimers have this subunit interface. Dilution of
these oligomers with excess WT* eliminates the spectral feature arising
from spin-spin interactions (Fig. 6a, thin
traces). The magnitude of spin-spin interactions can be
qualitatively deduced from the decrease in spectral amplitude observed
in the fully labeled oligomer relative to the spin-diluted oligomer.
This is reflected in the scaling factor in Fig. 6. Similarly, the EPR
spectra of residues 110-113 of
A (Fig. 6b) are
consistent with the oligomeric assembly resulting in close proximity
between R1 on different subunits. Thus, all dimers and tetramers of
truncated
A-crystallin have a similar packing interface. Broadening
arising from spin-spin interactions was the dominant feature in the
spectra of 112R1 and 113R1 at concentrations as low as 0.2 mg/ml, the smallest detectable concentration in our EPR spectrometer using a flat
cell (not shown).

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Fig. 6.
EPR spectra of R1-labeled C-terminal
truncation mutants of HSP 27 (a) and
A-crystallin (b). Spectra
exhibiting dipole-dipole interactions are shown in thick
lines, while their respective spin-diluted spectra are
represented by thin lines. Where appropriate, a
scaling factor is displayed to the left of the spectrum and
is relative to the thin lines. Except where noted, all
spectra have a scan width of 100 G.
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As in the native oligomer, subunit interactions in the truncated
domains are mediated by the antiparallel packing of
-strands. Fig.
7 shows the sequence-specific
accessibility,
, of R1 to NiEDDA along both sequences. NiEDDA is a
highly polar compound exclusively soluble in the aqueous phase. In both
nitroxide scans, a periodicity of 2 is observed, which is consistent
with a
-strand configuration. Accessibility to NiEDDA at residues
110-113 in
A-crystallin and 134-138 in HSP 27 was measured after
refolding these oligomers in the presence of a 3-fold molar excess of
their WT*. It was not possible to obtain a spin-diluted oligomer of the
A-crystallin I110R1, because the sample precipitated at ambient temperature and did not interact with the WT*. For
A F114R1, the
presence of the sharp component interfered with the measurement of both
P1/2 and
(
H0)
1. However, at both sites
the dominant component of the line shape is consistent with an
immobilization of R1 as expected at buried sites.

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Fig. 7.
Variation in versus
residue number of R1-labeled C-terminal truncations of HSP 27 ( ) (a) and A-crystallin
( ) (b). The dotted lines
indicate mutants for which power saturation data are not
available.
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The subunit contacts along this strand mediate the formation of a
2-fold symmetric unit. The titration of HSP 27 C137R1 with WT*, shown
in Fig. 8a, reveals that the
basic interaction unit consists of two spins. A similar conclusion is
reached from the titration of the truncated
A E113R1, as shown in
Fig. 8b.

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Fig. 8.
The increase in the fractional population of
monomeric spins versus the molar ratio of WT*:C137R1
(a) and WT*:E113R1 (b). The
solid line is the theoretical increase calculated
from the binomial distribution assuming a dimeric interface
(n = 2).
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Effects of Sequence Divergence: Nitroxide Scanning of the
Equivalent Sequence in HSP 16.3--
The extent to which this
interface and the associated 2-fold symmetry are conserved in distant
sHSP was examined in M. tuberculosis HSP 16.3. This protein
forms a highly ordered oligomer characterized by the presence of two
3-fold symmetry axes (13). Nitroxide scanning between residues 91 and
105 was carried out to determine the local structure and possible
quaternary contacts. The sequence alignment shown in Fig.
9 reveals significant divergence,
although residues 98, 100, and 102 are nonetheless conserved.
Structural and Functional Consequences of the Mutations--
The
single cysteine mutants of HSP 16.3 were overexpressed and remained
water-soluble. Except for S91C, E92C, F93C, Y95C, G96C, and R100C, all
mutants formed oligomers of molecular mass similar to that of WT (Table
III). The apparent molecular mass of the
WT, estimated from gel filtration analysis, is 221 kDa, consistent with
the value reported by Chang et al. (13). It was noted,
however, by those authors that sedimentation analysis and dynamic light
scattering reveal a smaller oligomer consisting of nine subunits. Fig.
10 shows the gel filtration profiles of F93C and Y95C. SDS-polyacrylamide gel electrophoresis analysis demonstrated that both peaks 1 and 2 are composed primarily of HSP
16.3. For F93C and Y95C, the molecular masses were estimated to be 162 and 177 kDa, respectively, for the first peak and 66 kDa for both
second peaks. When these mutants were spin-labeled, the equilibrium
shifted toward peak 2. The molecular mass of peak 1 strongly indicates
that it arises from the native oligomer, while peak 2 consists of a
dissociation product at about one-third of the molecular mass. If
indeed HSP 16.3 consists of a trimer of trimers (13), the cysteine
substitutions must be disrupting contacts at the interface between
trimers. While gel filtration analysis of S91C and E92C also revealed
dissociation to a trimeric species, the yield of these mutants was not
enough to allow further analysis. The elution profile of G96C and R100C
did not show distinct multiple peaks. Nevertheless, the width of the
peak suggests a broad distribution of molecular masses. Therefore, the
apparent molecular masses for these mutants reported in Table III might not represent a unique molecular species.

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Fig. 10.
Size exclusion chromatographs of HSP 16.3 mutants. Traces are for WT, F93R1, F93C, Y95R1, and Y95C.
Calibrated molecular mass markers (MDa) are provided at the
top.
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All cysteine mutants in the 93-105 stretch suppressed the aggregation
of alcohol dehydrogenase at 48 °C (Table
IV). Except for residues 93, 95, and 100, the mutants had chaperone efficiencies similar to the WT. Whether the
lower efficiency observed at 93 and 95 reflects intrinsic changes in
chaperone function or is due to the lower thermal stability of the
trimer cannot be determined using this type of assay.
Secondary Structure and Subunit Interactions along the
Sequence--
Analysis of the EPR spectral line shape of R1 along the
sequence 93-105 (Fig. 11) indicates
the absence of strong spin-spin interactions near the N terminus of the
sequence. Instead, a broadened spectrum was observed at residue S103R1.
That this broadening was due to interaction between R1 side chains from
different subunits was verified by refolding S103R1 in the presence of
a 5-fold molar excess of WT as shown in Fig. 11 (thin
trace).

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Fig. 11.
EPR spectra of R1-labeled HSP 16.3 mutants. Except where indicated, all spectra have a scan width of
100 G. The spectrum of S103R1, exhibiting spectral broadening due to
dipole-dipole interactions, is shown in thick
lines and is superimposed on the spin-diluted spectrum
indicated by a thin line. The thick line
spectrum is scaled by the factor indicated to the left
of the spectrum.
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To determine the local secondary structure, the accessibility of R1 to
NiEDDA was measured at every residue along the sequence and is reported
in Fig. 12. For R100R1, the presence of
a sharp spectral component interferes with the determination of
P1/2. Given the change in the gel filtration profile
of this mutant, the origin of the sharp component might well be an
unfolded population. The results in Fig. 12 show a pattern with a
period of 2, consistent with the presence of a
-strand. One face of
the strand consisting of the even residues is buried as in
A-crystallin and HSP 27. The amplitude of the oscillatory function
decreases dramatically near the 99-105 stretch, indicating a decrease
in the solvent exposure of the odd sites.

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Fig. 12.
Variation in versus residue number of R1-labeled HSP 16.3 mutants. The dotted line indicates the
mutant for which power saturation data are not available.
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To determine whether the spin-spin interactions observed at site 103 arise from the assembly of the overall oligomer or from the constituent
trimer, S103R1 was titrated with increasing amounts of GdnHCl. HSP 16.3 has an equilibrium folding intermediate consisting of a
trimer (13). For the WT, this state is populated in the presence of
~1 M GdnHCl. As shown in Fig.
13a, spin-spin interactions are eliminated in the presence of 0.7 M GdnHCl, indicating
that the observed spin-spin interactions at residue S103R1 are due to
the assembly of trimers. Furthermore, in the presence of 2 M GdnHCl, the sharp spectral line shape is consistent with
a predominantly unfolded environment. The complete unfolding curve is
shown in Fig. 13b. Two cooperative transitions are reported
by R1: the first from an oligomer to a trimer and the second from a
trimer to an unfolded monomer.

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Fig. 13.
a, EPR spectra of HSP 16.3 S103R1 in
the presence of increasing amounts of GdnHCl. All spectra were recorded
with a 160 G scan width except the spectrum in the presence of 2 M GdnHCl, which has a 100-G scan width. b,
GdnHCl denaturation curve obtained from measurement of the change in
the high field line amplitude of S103R1 plotted versus the
molar concentration of GdnHCl. c, EPR spectra of HSP 16.3 S103R1 refolded in the presence of increasing amounts of WT. Where
appropriate, a scaling factor is displayed to the left of
the spectrum. All spectra have a scan width of 160 G. d,
increase in the fractional population of monomeric spins
versus the molar ratio of WT:S103R1 ( ). The
solid line is the theoretical increase calculated
from the binomial distribution assuming a trimeric interface
(n = 3).
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That the interacting unit involves three spin labels was confirmed by
titration of the S103R1 oligomer with increasing amounts of WT. The
mixed oligomer was refolded from a 0.7 M GdnHCl solution. This has the effect of exchanging labeled with unlabeled trimer without
dissociating the trimer into its constituent monomers. Fig.
13d shows that the increase in the monomer population
follows that expected from a basic unit consisting of three interacting spins (Fig. 13d).
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DISCUSSION |
There is increasing evidence that the evolution of sHSP has
resulted in different size, symmetry, and flexibility of their oligomeric assemblies. It is logical to assume that such variations are
the result of a tuning mechanism at the level of the primary sequence
that optimizes the function of these proteins in their respective
cellular environment. While sHSP share an in vitro chaperone-like function, their role in the response to stress appears
to be organism-specific. For instance,
B-crystallin and HSP 27 are
involved in transduction pathways activated in response to a variety of
stressful and cytotoxic stimuli (14, 15). The putative protective and
regulatory functions of these proteins are associated with
phosphorylation and thermally induced changes in their oligomerization
and cellular localization. Thus, the dynamic and heterogeneous nature
of the oligomers regulates the response of these proteins to cellular
stimuli. On the other hand, plant sHSP do not appear to be
phosphorylated (3), while some bacterial sHSP appear to lack the
dynamic oligomeric structure (4, 13). Identifying the sequence
determinants of the structural polymorphism in sHSP is an important
step in understanding the mechanistic aspect of their diverse cellular function.
Short of obtaining and comparing atomic resolution structures, which
for mammalian sHSP has proved to be difficult, one approach toward
achieving this goal is to explore the effects of sequence divergence on
the structure and subunit interactions in the conserved
-crystallin
domain. Of particular importance are sequences that participate in
subunit contacts. One such sequence has been identified (20, 21). The
type of symmetry and the detailed packing across this sequence are
investigated in this study in three members of the sHSP family.
Among the many models of the oligomeric structure of sHSP, the rhombic
dodecahedron model proposes that subunit interactions in the
-crystallin domain mediate the formation of a fundamental basic
tetrameric unit (17). Our results clearly demonstrate that in
A-crystallin and HSP 27 the basic units result from subunit interactions along a highly conserved
-strand. The interface involves a 2-fold symmetry that extends the
-sheet of the
interacting monomers. The extensive sequence similarity among
A-crystallin,
B-crystallin, and HSP 27 suggests that a similar
interface exist in
B-crystallin as well as in the native lens
-crystallin oligomer. However, it appears that both
B-crystallin
and HSP 27 have further subunit contacts in this domain, since both
truncations form higher order structures (18).
Subunit interactions along this particular interface are observed when
the
-crystallin domains of
A and HSP 27 are expressed in
isolation. Because truncated-
A forms dimers and tetramers, this
result strongly suggests that this subunit interface mediates the
assembly of an ordered basic dimeric unit, the oligomerization of which
leads to the overall quaternary structure.
There is evidence from difference adiabatic scanning microcalorimetry
to support a dimeric structure as the minimal cooperative unit in
mammalian sHSP (29). The crystal structure of HSP 16.5 from M. jannaschii reveals the presence of a 2-fold symmetric interface
where the interactions between dimers occur on the edge of the
-sheet (4). However, one of the strands involved in the dimeric
interface is deleted in mammalian sHSP, suggesting a different mode of
dimerization. Based on the x-ray structure of HSP 16.5, weak spin-spin
interactions are expected at sites 110-113 and 134-139 in
A and
HSP 27, respectively, and would arise from subunit contacts around a
3-fold symmetry axis (as opposed to the 2-fold symmetry observed).
Consequently, these interactions are not expected to persist in the
dimeric unit. Furthermore, the structure of HSP 16.5 predicts a 25-Å
separation between the
-carbon of residues 137 across the 3-fold
symmetric interface, clearly above the cut-off limit for the
experimentally observed disulfide bond formation (21). Thus, the
oligomeric structure of mammalian sHSP is significantly different from
that of HSP 16.5.
In HSP 16.3, sequence divergence leads to a change in the oligomer
symmetry, although the data suggest that the region between residues 91 and 105 is involved in subunit contacts. At sites 91, 92, 93, and 95, cysteine substitutions result in the dissociation of the nonamer. The
observation of spin-spin interactions at residue 103 is consistent with
the expected heterologous association that results in a 3-fold
symmetry. In this type of association, the actual subunit interfaces
are not identical or overlapping; however, the symmetry operation
results in residues distant from the interaction surface being in close
proximity. Remarkably, residue Lys110 from M. jannaschii HSP 16.5, the equivalent residue to Ser103
in the sequence alignment, is in close proximity in the 4-fold symmetric unit (4). Furthermore, the decrease in solvent accessibility observed near the C terminus of the strand in HSP 16.3, but not observed in
A and HSP 27, is consistent with the structure of HSP
16.5, showing an increase in quaternary interactions at the odd
residues. Thus, despite the different symmetries of the HSP 16.5 and
HSP 16.3 oligomers, they appear to be more similar to each other than
to the mammalian sHSP.
It is instructive to compare the tolerance of
A-crystallin, HSP 27, and HSP 16.3 to mutations. It has been established that flexibility is
a requirement for proteins to be able to accommodate changes in their
amino acid sequence (30). To date, more than 150 cysteine mutants of
A-crystallin and 20 mutants of HSP 27 have been constructed in our
laboratory. None of these mutations result in the dissociation of the
oligomeric structure. Mutations in
A and HSP 27 cause shifts in the
average molecular mass and in some cases affect the molecular mass
distribution. On the other hand, six of 15 mutations in HSP 16.3 appeared to have considerable effects on the oligomeric structure.
Residue Arg100 is buried in the protein interior, and
therefore the substitution might have resulted in the countercharge
being buried in a low dielectric medium destabilizing the nonamer,
trimer, and monomer. Substitutions at the subunit interface at residues
91, 92, 93, and 95 in HSP 16.3 resulted in the dissociation of the
oligomer to the constituent trimers. Overall, these observations are
consistent with the reportedly flexible quaternary structure of
mammalian sHSP versus the rigid and ordered structure of
bacterial and archeal sHSP.
In conclusion, the data presented in this paper support the general
notion that the
-crystallin domain forms a common structural framework in sHSP. In all three sHSP investigated in this study, the
sequence has a
-strand configuration similar to that observed in the
crystal structure of M. jannaschii HSP 16.5. However, the role of the
-crystallin domain in subunit interactions appears to be
different for distant members of the sHSP family. Sequence divergence
along this domain results in different oligomer symmetry. This is not
unexpected, considering that the quaternary structure of mammalian sHSP
has evolved a dynamic dimension that seems to mediate their response to
changes in the cellular environment.