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INTRODUCTION |
hsp701 is perhaps the
best studied of the major heat shock proteins. hsp70 family members
have been shown to play essential roles in a variety of cellular
activities, for example, folding of nascent polypeptides, protein
translocation across intracellular membranes, and regulation of the
activities of steroid hormone receptors and kinases (1-3). The
unifying mechanism for their action is based on their chaperoning
activity, i.e. their ability to recognize and bind to
peptide segments that are not normally exposed to the aqueous
environment because they are normally buried in the interior of the
protein or are hidden by interactions with other proteins (4-6).
Multiple members of hsp70 family occur within individual organisms, and
it is believed that these different hsp70s perform differing roles. In
Saccharomyces cerevisiae, it has been shown that different
hsp70 family members are required for specific functions,
e.g. transport across membranes or translation, and that one
member may not be interchanged for another (7-9). This division of
labor occurs despite the very high degree of amino acid sequence
identity between these hsp70 family members, usually better than
60%.
Based on strong inducibility, quantity, and presence in many cell
types, hsp110 has also been recognized for the last two decades as a
major heat shock protein, specifically in mammalian cells (10-13).
hsp110 has been recently cloned from a variety of organisms as diverse
as yeast and man (14-23). Surprisingly, the cloning of hsp110 family
members has indicated that this family does not represent a genetically
unique stress protein group, as previously seen with other heat shock
protein families such as hsp90 or 28, but that they are clearly related
to the hsp70 family (14). The hsp110s in S. cerevisiae have
been termed the Stress Seventy E (SSE) family (23). However, the hsp110
family is a distinct subset of the hsp70 family which, in addition to their significantly increased mass compared with the hsp70s, differ in
their significant sequence divergence from the archetypical hsp70s
(14). In light of the differential functions of S. cerevisiae hsp70s, the appearance of the highly diverged hsp110
family, which exists in parallel with the hsp70s in the cytoplasm and
nucleus in diverse organisms, argues strongly for related but
differential functions and properties for these two major stress
protein groups.
Initial studies have shown that hsp110 has certain functional
properties that are shared with hsp70, i.e. i) the
overexpression of hsp110 has been shown to confer cellular heat
resistance, and ii) hsp110 has been shown to have the ability to
prevent protein aggregation and keep denatured protein in a
folding-competent state; however, with apparently greater capacity
compared with hsc70 (24). To further analyze the functional properties
of hsp110 and how it may differ from the hsp70 family, a mutational investigation of hsp110 has been undertaken. By sequence alignment and
prediction of secondary structure, we have constructed several targeted
deletion mutants. We describe here the properties of these mutants and
test their ability to exhibit chaperoning functions.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
All plasmid constructs were made to
produce 6-histidine-tagged proteins. For the polymerase chain reaction
(PCR), the following primers were synthesized. Each primer sequence
contained proper restriction enzyme recognition sequences, which are
underlined and whose name is indicated in parenthesis. Primers, A, B,
C, and D are 5' (or forward primers for PCR) and primers, i, ii, iii,
iv, v, and vi are 3' (or backward primers for PCR). Primer A
(BamHI),
5'-GCTAGAGGATCCTGTGCATTGCAGTGTGCAATT-3'; primer B
(BamHI), 5'-GACGACGGATCCTCTGTCGAGGCAGACATGGA-3';
primer C (XbaI),
5'-CCGGCCTCTAGAATGTCGGTGGTGGGACTAGAC-3'; primer D
(SstI), 5'-AGCTCATGAGCTCTAACAGC-3'; primer i
(HindIII), 5'-CAGCGCAAGCTTACTAGTCCAGGTCCATATTGA-3'; primer ii
(HindIII), 5'-CCGGAGAAGCTTACAGAATTGCACACTGCAA-3'; primer iii
(HindIII),
5'-CGACAGAAGCTTAAGACCCGTCGTCTTCCTC-3'; primer iv
(BamHI), 5'-ACAGAAGGATCCGTCGTCTTCCTCTGTTGG-3';
primer v (HindIII),
5'-TCAATGAAGCTTACATGTTAAGAAGGTCTCT-3'; primer vi (HindIII),
5'-GAATTTAAGCTTACTCATATGGTCCACACAG-3'. For the
constructions of BLH (375-858), LH (508-858), and BLH' (375-655),
PCR was performed with primer pairs of primer A and primer i, primer B
and primer i, and primer A and primer vi, respectively. The PCR
products were digested with appropriate restriction enzymes, and the
digested fragment was ligated to pRSETA vector (Invitrogen), which was also digested with the same restriction enzymes used for the digestion of the PCR products. For the construction of
L (
510-608), PCR was performed with the pair of primers, primer C and primer iv, and the
resulting fragment was ligated with pRSETA vector. Another PCR was
performed with primer pair primer B and primer i and cloned into the
vector in which the first PCR fragment was ligated. For AB (1-510) and
ABL (1-614), PCR using pairs of primers primer D and primer iii and
primer D and primer v was performed, and the PCR products were digested
with SstI and HindIII. pRSETC-hsp110 plasmid
that was described previously (24) was digested with SstI and HindIII, and fragments containing the
N-terminal portion of hsp110 sequences was isolated and ligated with
the PCR fragments. The results of these procedures is that constructs
BLH, LH, AB, and ABL contain N-terminal fusion of six histidines,
enterokinase recognition sequences, and additional Asp-Arg-Trp-Gly-Ser
for BLH and BLH', Asp-Arg-Trp for LH,
Asp-Arg-Trp-Ile-Arg-Pro-Arg-Asp-Leu-Gln-Pro-Ala for AB and ABL. The
construct
L contains N-terminal fusion of six histidines and
Gln-Met-Ser.
Expression and Purification--
The constructs were transformed
into Escherichia coli JM109 (DE3) cells. E. coli
transformed with the constructs BLH and LH were cultured at 37 °C
until the A600 reached 0.6, 1 mM
isopropyl-1-thio-
-D-galactopyranoside was added, and
E. coli were further cultured for 5 h. Cells containing all other constructs were cultured in a same way, except that the
expression was induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside and at 30 °C.
Cells were lysed by lysozyme treatment and sonication in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 0.5% Nonidet P-40, and protease inhibitor
mixture (Roche Molecular Biochemicals). The lysate was centrifuged at
42,000 rpm for 2 h, and soluble fraction was used for
purification. The proteins were purified using nickel nitriloacetic
acid-agarose columns (Quiagen, Inc.) following the manufacturer's
instruction. The columns were extensively washed with wash buffer
containing 20 mM Tris-HCl, pH 7.8, O.5 M NaCl,
60 mM imidazole, and 10% glycerol, and recombinant
proteins were eluted with wash buffer containing 500 mM
imidazole. The eluted proteins were dialyzed for 48 h against 20 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 10 mM
-mercaptoethanol. Proteins were concentrated using
Slide-A-Lyzer concentrating solution (Pierce). Proteins were quantified
using the Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin
as a standard. Each mutant was visibly pure as indicated by
Coomassie-stained gels, except ABL, which contained a small amount of a
truncated form of this mutant, as shown by Western blot analysis. This
mutant was not further purified.
Luciferase Aggregation and Refolding Assay--
0.15
µM luciferase (Sigma) and various test proteins were
incubated in 25 mM Hepes, pH 7.9, 5 mM
magnesium acetate, 50 mM KCl, 5 mM
-mercaptoethanol at 43 °C for 30 min, and the protein aggregation
was monitored by the increase of optical density at 320 nm. At the end
of the heating, the solution was centrifuged at 16,000 × g for 15 min, and soluble and pellet fractions were separated, run on SDS-PAGE, and subjected to Western analysis with
anti-luciferase antibody (Promega). For luciferase refolding assay,
luciferase together with various test proteins were heated in refolding
buffer (25 mM Hepes, pH 7.6, 5 mM
MgCl2, 2 mM dithiothreitol, and 2 mM ATP) at 43 °C for 30 min. The heated luciferase was
diluted 100-fold into 60% rabbit reticulocyte lysate (Promega)
containing refolding buffer and incubated at 30 °C for 2 h. For
the measurement of activity, luciferase solution was further diluted
5-fold in 25 mM Hepes, pH 7.6, containing 1 mg/ml bovine
serum albumin, and 10 µl was added to 100 µl of luciferase assay
solution (Promega); the activity was measured with Lumat LB9501 (Berthoid).
ATP Binding Assay--
ATP binding assay was essentially the
same as previously reported, with minor modification (25). ATP-agarose
(Sigma) was washed and stored in buffer B (20 mM Tris-HCl,
20 mM NaCl, 0.1 mM EDTA, 2 mM
dithiothreitol) as a 1:1 slurry. One 10-cm dish of confluent CHO cells
are lysed in 600 µl of phosphate-buffered saline, 1% Triton X-100,
and protease inhibitors, and soluble proteins are collected after
centrifugation at 16,000 × g for 5 min. 120 µl of
lysate was incubated with 200 µl of ATP-agarose beads for 16 h
at 4 °C. For ATP competition, 10 mM ATP was added to the
mixture of lysate and ATP-agarose. The ATP-agarose beads were washed 2 times with buffer B containing 500 mM NaCl and 3 mM MgCl2, 2 times with buffer B containing 5 mM GTP, and 2 times with buffer B. ATP-agarose-bound
proteins were eluted by incubation in buffer B containing 10 mM ATP and 3 mM MgCl2 for 4 h
at 4 °C. The eluted proteins were boiled in SDS-gel-loading buffer
(1% SDS, 125 mM Tris-HCl, pH 6.8, 10% glycerol, 1%
-mercaptoethanol, 1 mM EDTA, and bromphenol blue) for 10 min. The samples were run on 10% SDS-PAGE, transferred to a
nitrocellulose membrane (Bio-Rad), and probed with anti-hsp110 or
anti-hsp70 antibody (StressGen). In case of E. coli
expressing recombinant hsp110 or ATP binding domain, the cells were
cultured, and recombinant proteins are expressed in the same way as for
protein purification. The cells were lysed by sonication in buffer B
containing protease mixture and centrifuged at 42,000 rpm for 2 h,
and soluble fractions were used for ATP-agarose binding assay. 500 µg
of total proteins was used for this assay. ATP-agarose was washed 5 times with buffer B containing 500 mM NaCl and 3 mM MgCl2 and 2 times with buffer B. ATP-agarose-bound proteins were eluted and analyzed as described above.
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RESULTS |
Predicted Structure of hsp110 and the Purification of Targeted
Mutants--
In order that we might construct targeted mutations of
the hsp 110 cDNA, we built a model of hsp110 based upon its
sequence similarity with the DnaK family of stress proteins. The model (Fig. 1) is shown diagrammatically and is
compared with a similar diagram of DnaK, based upon its x-ray structure
determination (26). Residues 1-394 of hsp110 (designated as domain
A in Fig. 2) show 34%
identity in amino acid sequence to the same region of DnaK. This region
in DnaK is responsible for ATP binding. From amino acid 394 to amino
acid 509, hsp110 is predicted to exhibit seven
strands (the
sheet domain, or B in Fig. 2). This region demonstrates some
sequence similarity to, and structurally aligns with, the corresponding
region of the DnaK. It is this region of DnaK that represents its
"peptide binding domain" and consists of eight major
strands
arranged as a
sandwich. The following 98 amino acids of hsp110 are
composed of a number of negatively charged residues that computer
analysis fails to predict as having any obvious secondary structure.
This region is therefore referred to as the loop domain (residues
510-608, or domain L). Finally, distal to the loop domain,
the C-terminal residues of hsp110 are predicted to form a series of
helices (residues 608-858, or domain H). Although there is
little sequence identity in this region between hsp110 and DnaK (or
hsc70), DnaK shows three large helices, which make up a
helix-turn-helix structure. Fig. 1 correspondingly arranges the
helices of hsp110. It is in this region that the hsp110 family members
exhibit a high degree of sequence homology among themselves, which is
an identifying feature for this family (14). However, a small degree of
sequence similarity can be found between hsp110 and DnaK families in
certain of these helices, specifically
B and
C. In DnaK, the
sandwich serves as the peptide binding site, whereas the
helix-turn-helix structure, poised above the
sandwich, is thought
to regulate entry and/or exit of the peptide substrate (26).

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Fig. 1.
Proposed folding pattern for hsp 110, based
on the structure of DnaK. hsp 110 secondary structure was
predicted using the "PHD" method implemented by Rost et
al. (43, 44). A, the secondary structure and fold of
the DnaK peptide binding domains, based on the x-ray crystallographic
structure reported by Zhu et al. (26). B, the
predicted secondary structure and proposed fold for hsp 110, beginning
at residue 390, are diagrammatically represented here. The domains (A,
B, L, H), as defined in the text, are indicated on the diagram. The strands in DnaK and hsp110 domains, sharing both structural (PHD)
and significant sequence similarity by Matchbox alignment (45), are
colored yellow. The strands marked 8 and 9 in hsp110
have no structural counterpart or sequence similarity to DnaK. The
predicted helical segments in hsp110 are shown arranged in a
helix-turn-helix structure, as suggested by the helical domain in
DnaK.
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Fig. 2.
Construction and purification of deletion
mutants. A, schematic diagram of deletion mutants. The
predicted domains are denoted as the following: A, ATP
binding domain; B, -sheet domain; L, acidic
loop; H, -helices; H', first two -helices.
ND, not determined. B, SDS-PAGE of purified mutant proteins.
About 1 µg of each protein is run on SDS-PAGE and stained with
Coomassie Blue. WT, wild type.
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Based on these similarities between the predicted structural elements
of hsp110 and the actual structure of DnaK, we have constructed various
deletion mutants. These mutants were constructed to include the
specific amino acids residues defined above and to also include a small
number of additional amino acids on either side of the domain to both
increase the likelihood of correct folding of the domain and account
for small errors in the computer predictions. Fig. 2A shows
a schematic diagram of these deletion mutants. The mutants were
expressed and purified from E. coli, and their mobilities
were analyzed by SDS-PAGE (Fig. 2B). When the recombinant
full-length hsp110 is run on SDS-PAGE, its mobility (from which the
N-terminal fusion peptide is subtracted) is essentially identical to
that of native hsp110, determined by Western analysis of CHO lysate
using an anti-hsp110 antibody. However, the calculated molecular mass
of hsp110, based on the amino acid composition, is more than 10 kDa
smaller than the molecular mass determined from SDS-PAGE mobility
(Table I). This sort of discrepancy is unusual although not rare. To determine whether the mobility
retardation is caused by overall sequence or by specific region (s) of
hsp110, calculated molecular weights and mobilities of the different
deletion mutants were compared. It is apparent from Fig. 2B
that the acidic loop region, which was also noted for its unusual amino
acid composition, predicted flexibility, and lack of secondary
structure, is solely responsible for this retardation. This also
explains why mutant ABL (614 amino acids) exhibited slower mobility
than mutant
L (757 amino acids), i.e. full-length hsp110,
which lacks the loop domain.
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Table I
Size comparison of hsp110 and various mutants
N-terminal-fused amino acids are included in calculation by amino acid
composition. The DNASTAR program was used for calculation.
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Identification of Domains Required for the Chaperoning Activity of
hsp110--
Two assays were employed to identify the functional
domains of hsp110 required for chaperoning activity. These are 1) the ability of the mutants to prevent protein aggregation induced by heat
treatment, which is assessed by the suppression of the increase in
light scattering obtained upon heat treatment as well as the solubility
of the reporter protein, firefly luciferase, and 2) the ability to
sustain denatured luciferase in a folding competent state, which is
assessed by measuring the ability of the denatured luciferase to refold
(i.e. recover activity) upon the addition of folding
chaperones present in rabbit reticulocyte lysate.
First, as seen in Fig. 3, the ability of
the different mutants to prevent heat-induced aggregation is presented.
As shown in Fig. 3, A and B, mutant BLH
(i.e. containing only the
sheet peptide binding domain
(B), loop (L), and
helical domain (H)) is able to prevent the
aggregation of heat-denatured luciferase as equally well as does wild
type hsp110. However, mutant LH does not prevent luciferase
aggregation. This indicates that domain B containing amino acids from
375 to 507 (
-sheet domain) contains the substrate binding domain, in
agreement with the predictions of the sequence alignment and secondary
structure. To address whether
-sheet domain alone is sufficient for
this chaperoning activity, mutant AB (ATP binding and
sheet-peptide
binding domains) was examined. In the presence of mutant AB, heat
denaturation of luciferase resulted in a change in optical density that
was 3-fold greater than that observed with luciferase alone
(i.e. 300%, not plotted in Fig. 3A), in which
case luciferase is found almost entirely in the pellet fraction (Fig.
3B). This indicates that mutant AB is not capable of
preventing aggregation and, further, that it itself aggregates.
Although it was determined above that BLH is totally effective in
"holding" denatured luciferase and that B is required, the
additional involvement of L and/or H was still a possibility. We
therefore examined mutants ABL and BLH' (H' containing only the first
major
helix of the complete H domain). Mutant ABL is inactive (even
at a 2× molar concentration), but mutant BLH' is active, even though
its efficiency is 50% that of wild type hsp110 or mutant BLH. Thus,
the C-terminal boundary of an "effective" peptide binding domain
resides between amino acids 614 and 655, which defines the first part
of the "cap" domain. This data further indicates that the complete
holding ability of the protein requires additional C-terminal sequence
components in the cap. Finally, to examine the role of the loop domain
in this holding activity, mutant
L was next examined. It is observed that
L (containing full-length hsp110, excluding L) was also capable
of inhibiting the aggregation of heat-denatured luciferase. In addition
to luciferase, we have also tested the ability of BLH, LH, and BLH' to
inhibit the heat-induced aggregation of citrate synthase using
Coomassie Blue staining to measure the amount of citrate synthase in
the supernatant and pellet. These results were essentially identical to
those observed using luciferase. Taken together, the predicted
-sheet domain and
-helical cap domain are necessary and
sufficient for the total ability of hsp110 to prevent aggregation and,
further, that the significant component of this holding activity
requires only the first major
helix of cap. The loop domain and ATP
binding domain are dispensable for this function.

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Fig. 3.
Identification of domains for the in
vitro chaperoning activity of hsp110. A,
examination of the ability to prevent protein aggregation by light
scattering. 0.15 µM of luciferase was incubated with 0.15 µM or 0.3 µM mutant hsp110, depending on
the mutant, at 43 °C for 30 min. Aggregation was monitored by
measuring the increase of optical density at 320 nm. The molar ratio of
test protein:luciferase is indicated in each case. The optical density
of the luciferase heated alone was set to 100% OVA,
ovalbumin. B, Western blot analysis of luciferase in the
supernatant (Spt) and pellet of control (25 °C) and
heat-shocked (43 °C) solutions in the presence of wild type (ABLH)
or the various mutants designated in Fig. 2. C, folding
competency of luciferase bound to various deletion mutants. The
heat-denatured luciferase was refolded by rabbit reticulocyte lysate.
1.5 µM wild type (WT) or mutant hsp110 was
used. In case of L and BLH', two concentration, 1.5 µM
and 3.0 µM, were used. The activity of luciferase
obtained with 1.5 µM wild type hsp110 was set to
100%.
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Second, we examined the ability of the various mutants to provide a
folding competent substrate. To examine this question, luciferase was
incubated with each mutant at 43 °C for 30 min, following which
rabbit reticulocyte lysate was added. The solution was further
incubated at 30 °C for 2 h, a sufficient time to enable maximal
refolding with wild type hsp110 (approximately 70% of the activity of
unheated luciferase, designated 100% in Fig. 3C for the
comparison of mutants). As shown in Fig. 3C, it is seen that
when the luciferase is sustained by the mutant in a soluble form in
aggregation assays in Figs. 3, A and B, it was
found to be refoldable (i.e. recover activity). It is also
seen in this figure that the mutants
L and BLH', although holding
luciferase in a folding-competent state, do so less efficiently than
does wild type hsp110 (about one-half as efficient at equal
concentration). Doubling the concentration of
L and BLH' improved
the recovery of luciferase activity. In case of BLH', efficiency
was increased to a level equivalent to that of the wild type
protein. However, in case of
L, recovery of activity was still
significantly less than wild type hsp110 or BLH'. Last, it is also seen
that the ATP binding domain (A) is neither required for folding or
holding of luciferase in these studies, as discussed above. Thus, the
-sheet domain, loop domain, and parts of the
-helical domain (specifically
B) are the minimal components required for hsp110 to
function as a molecular chaperone that can hold substrate in a
folding-competent state.
hsp110 Has a Functional ATP Binding Domain--
Sequence analysis
has indicated that hsp110 exhibits an ATP binding domain, and moreover,
the domain shares significant sequence homology to hsp70. Because hsp70
binds ATP and ATP is required for its chaperoning function, the ATP
binding ability of hsp110 was examined using ATP-agarose. CHO cell
lysates were incubated with ATP-agarose beads, and bound proteins
eluted with free ATP. The eluted proteins were then run on SDS-PAGE and
subjected to Western analysis with anti-hsp110 or anti-hsp70 antibody
(Fig. 4A). It is seen that
hsp70 is a very good ATP-agarose binder (as has been long recognized),
but that hsp110 is not. Free ATP competition during the ATP-agarose
incubation shows the specificity of this assay. Because hsp110 has all
the known consensus sequences for ATP binding, the possibility of the
structural blocking of the ATP binding site in hsp110 was examined by
using the mutant containing only the ATP binding domain (A). The
E. coli lysates expressing recombinant full-length hsp110 or
the ATP binding domain alone were subjected to the same assay as the
CHO cell lysate just described. The proteins bound to ATP-agarose were
then analyzed with anti-oligohistidine antibody on a Western blot. The
recombinant, full-length hsp110 still did not bind ATP-agarose.
However, it is seen that the ATP binding domain (A) mutant was able to
bind ATP-agarose. This indicates that the ATP binding domain of hsp110
is functional, but that its ability to bind ATP is masked by some part
of its more C-terminal regions.

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Fig. 4.
ATP binding domain of hsp110 has intrinsic
ATP binding ability. A, the ability of native hsp110 in
CHO lysate to bind ATP-agarose. hsp110 and hsc/p70 were detected by
Western analysis with anti-hsp110 or anti-hsc/p70 antibodies.
B, the ability of recombinant hsp110 or ATP binding domain
fragment to bind ATP-agarose. The recombinant proteins were detected by
Western analysis with anti-His antibody. The smaller bands detected
with anti-His antibody represent the truncated or degraded recombinant
proteins. Lysate, CHO or E. coli lysate before
the incubation with ATP-agarose; Unbound, unbound proteins;
Wash 1 and 2, proteins washed off from
ATP-agarose; ATP-eluted, proteins eluted with ATP;
Beads, proteins remaining bound to agarose after ATP
elution.
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DISCUSSION |
It has been long recognized that the major heat shock proteins
(hsps) of mammalian cells are observed at 28, 70, 90, and 110 kDa (10,
12, 27-29), and other hsp families, e.g. hsp60 (GroEL) and
hsp40 (DnaJ), were later identified. All of these hsps except hsp110
have been intensively studied, and their interactions in numerous
cellular processes are today broadly recognized. The situation with
hsp110 is somewhat curious. Studies from the beginning of the 1980s
from this laboratory as well as virtually every other laboratory that
examined the induction of heat shock proteins described a major hsp of
100 to 112 kDa size ( Refs. 10, 12, and 27-29, among many
references). These studies demonstrated that hsp110 was inducible
by a broad variety of stresses that induced other hsps and that it was
a major hsp whose expression strongly correlated with the expression of
thermotolerance (10, 12, 28).
The cloning of hsp110 from hamster, mouse, yeast, arabidopsis, sea
urchin, and a variety of other species has been described in the last
few years (15-23, 30). Surprisingly, although hsp110 appears as a
conserved and distinctive protein species in countless heat shock
studies, its sequence shows that it is a significantly enlarged and
diverged relative of the hsp70 family of proteins. Therefore, questions
arise as to the differences and similarities between the hsp70 and
hsp110 families and purpose for having these two major subgroups
expressed in parallel in organisms as diverse as yeast and man. The
present study continues our investigation of this question and
identifies the functional domains of hsp110 in comparison with the
available knowledge of hsp70 organization and function. It is shown in
the present study that 1) the ATP binding domain of hsp110 is
functional, but appears to be normally masked, 2) the ATP binding
domain is not required for the activity of hsp110 as a holding
chaperone, 3) the
-sheet domain and C-terminal
-helical cap
appear to compose the substrate binding pocket.
hsp110 has been previously shown to be an efficient holding chaperone,
a characteristic that includes most identified molecular chaperones.
Moreover, we have shown previously that this holding function is
independent of ATP (24). Thus, it is not surprising to find here that
the ATP binding domain is not required for the holding activity of
hsp110. A somewhat similar situation is seen with other holding
chaperones such as hsp90 and hsp25 (31-33). This may also be true for
hsp70 because recombinant hsc70, without its ATP binding domain, is
capable of binding reduced carboxymethylated lactalbumin (34).
Moreover, it has been shown that an hsp70 mutant, which lacks its ATP
binding domain, was able to confer thermoresistance in tissue culture
cells as well as does wild type hsp70 (35). Taken together with the
in vitro substrate binding studies, this suggests that the
ATP domain of hsp70 has a dispensable role in holding substrates in a
folding-competent state and that the chaperoning function of the
constructs without this domain is sufficient to protect cells from
stress. This is analogous to the present results obtained with hsp110.
Therefore, hsp70 and hsp110 may be similar with respect to the lack of
involvement of their ATP binding domains in their basic
chaperoning/holding functions. However, wild type hsp70 is also capable
of folding substrate in the presence of DnaJ co-chaperones (albeit, to
a limited degree only (24)). However, this does require ATP and, logically, the hsp70 ATP binding domain. Although a folding function for hsp110 has not been identified to date, there is still the possibility of hsp110 being a folding chaperone in conjunction with
other co-chaperone(s) that have yet to be identified. It would then be
conceivable that its ATP binding domain could play an essential role.
Analysis of crystal structure of DnaK, the E. coli homologue
of hsp70, revealed the substrate binding pocket of DnaK, and sequence
alignment of DnaK, bovine hsp70, and BiP predicts that these hsp70
family members have a common substrate binding pocket (26). This is
supported by several deletion mutation studies of hsp70. Boice and
Hightower (36) reported a mutational analysis of hsc70 guided by
secondary structure predictions showing that hsp70 and DnaK have very
similar peptide binding domains. Although this domain represents the
peptide binding cleft, the precise boundaries within hsp70 required for
peptide binding remain uncertain. Earlier studies demonstrated that an
18-kDa fragment of hsp 70 immediately after the ATP binding domain has
essentially the same affinity for synthetic peptide as does wild type
hsc70 (37). This result is analogous to the results presented here for
hsp110. It has also been shown that a 60-kDa fragment of bovine hsc70, which excludes the extreme C-terminal 10-kDa domain, can bind the
clathrin cage but cannot act in its disassembly (38). This latter
result suggests that hsp70 substrate binding activity does not involve
the C-terminal 10-kDa region, but its regulation does. Yet, a third
study has shown that the highly conserved C-terminal EEVD motif at the
extreme C-terminal of hsp70 is required for binding to reduced
carboxymethylated lactalbumin (34). Thus the actual roles of the
C-terminal regions of hsp70 in regulation of its chaperoning functions
is complex. Indeed, different substrates, e.g. reduced
carboxymethylated lactalbumin, synthetic peptide, and clathrin, may
contribute to the differing conclusions regarding the varying
requirements for parts of this region of hsp70 in its function.
The results obtained in the present study on hsp110 indicate that it
has a similar substrate binding pocket to DnaK. The loop domain and the
extension of the expanded C-terminal
-helical cap domain are not
absolutely required for the chaperoning activity of hsp110 as defined
here. However, they are both required for hsp110 to be fully efficient
in its activity. There are at least two possibilities for the role of
these two domains. 1) These two domains may be required for maintaining
structural stability of hsp110, and/or 2) they may be required for
hsp110 to bind and release substrates efficiently. Curiously, the loop
domain in hsp110/SSE family members is essentially absent in hsp70
family members. We have shown previously that hsp110 is more efficient in solubilizing luciferase than is hsc70 at an equal molar
concentration. The deletion of the loop would make hsp110 more similar
in its holding capacity to hsc70. The loop and the expansion of the
C-terminal
-helical domain could alter the peptide binding
characteristics of hsp110 relative to hsp70 in ways that have yet to be
clearly defined.
We have shown here that hsp110 does not bind ATP in vitro,
despite the conservation of its ATP binding domain, whereas its ATP
binding domain mutant (A) does. The inability to detect ATP binding
activity experimentally seems to be due to the structural blocking of
the ATP binding site by more C-terminal domains. Nonetheless, the ATP
binding ability of the ATP binding domain mutant and the high degree of
conservation of ATP binding motif in hsp110 argues strongly that hsp110
does in fact exhibit ATP binding properties in vivo. If so,
then the binding and putative hydrolysis of ATP could be expected to
have regulatory implications in hsp110 holding and (possibly) folding
functions and/or interactions with co-chaperones. Moreover, the
apparent constriction on ATP binding in hsp110 would indicate an
additional regulatory feature of this chaperone that would appear to
differ from the present models for hsp70.
As with the hsp70 family, multiple members of hsp110 family have been
identified in individual organisms with three members having been
identified in mice (15-17, 21, 22). Although hsp110 appears to be the
principal family member expressed in most murine tissues, apg-1
(another family member) is also abundantly expressed in testes and
heart. That such a multiplicity of genes exist for each family, hsp70
and hsp110, further argues for important differences in the general
functions of each. Indeed, a different requirement for hsp110 and hsc70
in cells is suggested by the fact that they are differentially
expressed in different tissues and different regions of the same tissue
(e.g. brain)2 (14,
15). Last, in addition to hsp110 and its family members, there is a
second large relative of the hsp70 family that resides in the
endoplasmic reticulum, known as grp170 (39-41). Although grp170 does
contain an hsp70/hsp110-like ATPase domain and a large hsp110-like loop
domain, it and its family are as diverged from the hsp110 family as it
is from the hsp70 family (i.e. hsp70, hsp110 and grp170
segregate into three major subfamilies on a phylogram) (41, 42).
Further studies of hsp110 and grp170 will provide additional insight
into the purpose for these unusual and unexpected relatives of the
hsp70 family and how they interact and cooperate with each other
and hsp70 in cellular function.