From the Department of Molecular Cell Biology, Center
for Molecular Medicine, SBRI, Sungkyunkwan University School of
Medicine, Suwon 440-746, Korea, the § Bioneer Corporation,
49-3, Daejeon 306-220, Korea, and ¶ PENGEN Biotech, R&D Center,
KSBC, Suwon 442-270, Korea
Received for publication, August 9, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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HtrA (high temperature requirement A), a
periplasmic heat-shock protein, functions as a molecular chaperone at
low temperatures, and its proteolytic activity is turned on at elevated
temperatures. To investigate the mechanism of functional switch to
protease, we determined the crystal structure of the
NH2-terminal protease domain (PD) of HtrA from
Thermotoga maritima, which was shown to retain both
proteolytic and chaperone-like activities. Three subunits of HtrA PD
compose a trimer, and multimerization architecture is similar to that
found in the crystal structures of intact HtrA hexamer from
Escherichia coli and human HtrA2 trimer. HtrA PD shares the
same fold with chymotrypsin-like serine proteases, but it contains an
additional lid that blocks access the of substrates to the active site.
A corresponding lid found in E. coli HtrA is a long loop
that also blocks the active site of another subunit. These results
suggest that the activation of the proteolytic function of HtrA at
elevated temperatures might occur by a conformational change, which
includes the opening of the helical lid to expose the active site and
subsequent rearrangement of a catalytic triad and an oxyanion hole.
Protein quality control, which is essential for cell viability, is
tightly controlled by proteases and molecular chaperones (1).
Especially under stress conditions such as high temperature, heat-shock
proteins, which are mostly proteases or molecular chaperones, are
induced to protect cells from toxic denatured proteins (2). Molecular
chaperones bind to the hydrophobic patches on denatured proteins to
prevent further aggregation and help them to fold back into their
native states (3). The regulatory subunits of heat-shock proteases (4)
recognize the hydrophobic surface on unfolded proteins and eliminate
them mostly in an ATP-dependent manner (5).
High temperature requirement A
(HtrA,1 also called DegP or
protease Do) is a heat-shock protease localized in the periplasmic space of bacteria (6). It shows an ATP-independent proteolytic activity
and plays an important role in the degradation of misfolded proteins
accumulated by heat shock or other stresses (7). Therefore, its
activity seems to be essential for bacterial thermotolerance and for
cell survival at high temperatures (7). HtrA is also involved in
pathogenesis of Gram-negative and Gram-positive bacteria by degrading
damaged proteins that are produced by reactive oxygen species released
from the host defense system (8). Therefore, HtrA is considered as a
target for development of broad-spectrum antibiotics (8).
In addition to proteolytic activity, HtrA is known to have a molecular
chaperone activity (9, 10). The chaperone function is dominant at low
temperatures, whereas the proteolytic activity is turned on at elevated
temperatures (9). This temperature-dependent functional
switch is necessary for controlling protein stability as well as
eliminating denatured proteins to maintain cellular viability (9). HtrA
is a highly conserved protein found in species ranging from bacteria to
humans. Two known human homologues of bacterial HtrA (HtrA1 and HtrA2)
are also expected to be involved in mammalian stress response pathways
(11, 12). However, because HtrA2 showed proteolytic activity even at
room temperature, temperature-dependent activation of
proteolytic activity seems to be absent from mammalian HtrAs (13).
HtrA is a serine protease with a catalytic triad in its active site.
Recent crystal structure analyses revealed that Escherichia coli HtrA forms a hexameric complex composed of two trimers (14) and human HtrA2 forms a homotrimer (15). Each subunit is composed of
one protease domain at the amino terminus and one or two PDZ (named
after three proteins, PSD-95, Discs-large, and ZO-1) domains at the
carboxyl terminus. The protease domain of E. coli HtrA fully
retains the molecular chaperone activity, although the proteolytic activity is absent (9). The PDZ domains, also found in the Clp/Hsp100
family of heat-shock proteins, are known to play a role in substrate
recognition (16). In the crystal structures of E. coli HtrA
and human HtrA2, PDZ domains are proposed to mediate the initial
binding of substrates (14) or to be involved in modulation of protease
activity (15). However, PDZ domains do not participate in
multimerization in both E. coli and human HtrAs (14, 15).
Unlike other proteases of the Clp/Hsp100 family, HtrA does not have a
regulatory component or an ATP binding domain because it is an
ATP-independent heat-shock protease.
So far two crystal structures of HtrAs have been reported (14, 15), and
they seem to differ in structural architecture, multimerization, and
activation mechanism. For a better understanding of the dual role of
HtrA and the activation mechanism of the proteolytic function, we have
solved the crystal structure of the protease domain (PD, residues
24-262, Fig. 1) of Thermotoga
maritima HtrA (Tm HtrA), which displays both molecular chaperone
and proteolytic activities. The crystal structure indicates that the
rearrangement of the active site of bacterial HtrA is necessary for the
proteolytic activity and that oligomerization architecture of HtrA
might vary depending on the presence of the lid covering the active
site.
Protein Preparation and Crystallization--
The protease domain
of HtrA from T. maritima (PD, residues 24-262, Fig. 1) was
cloned, purified, and crystallized as described elsewhere (18). The
putative signal sequence (residues 1-23) was deleted in the construct.
For the translational start, a methionine residue was added in front of
Asp24. Intact HtrA was also prepared in the same way as
HtrA PD (18). HtrA PD was crystallized in the cubic space group
P213, with the unit cell parameters a = b = c = 120.55 Å by the hanging
drop vapor diffusion method at 22 °C from a reservoir solution
containing 100 mM phosphate-citrate (pH 4.4), 110 mM Li2SO4, and 5% (v/v) PEG 1000 (18). There are two molecules in an asymmetric unit.
Light-scattering Measurement--
The chaperone-like activities
of HtrA and HtrA PD were measured as described previously (19) with
some modifications using pig heart citrate synthase (CS) as the
substrate (Sigma). CS (final 65 µM monomer) was denatured
in a solution containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 6 M guanidinium hydrochloride, and 40 mM dithiothreitol for at least 2 h at room
temperature. Chemically denatured CS was rapidly diluted 250-fold into
a refolding buffer containing 10 mM Tris-HCl (pH 8.0) and
HtrA or HtrA PD to reach the indicated molar ratios (Fig. 2). Light
scatterings from aggregated proteins were monitored by measuring the
absorbance at 320 nm with Spectra MAX Plus (Molecular Device) at
25 °C. Initial velocities of aggregate formation were calculated by
measuring the absorbance change for the initial 15 s where the
rate of aggregate formation is constant.
Proteolytic Activity Measurement--
Thirty micrograms of the
reduced form of Structure Determination and Refinement--
Multiwavelength
anomalous diffraction data were collected from a frozen crystal of HtrA
PD at the Pohang Accelerator Laboratory beamline 6B with a MacScience
2030b area detector. Data collected at three wavelengths (edge, peak,
and remote) were processed and integrated by DENZO and scaled by
SCALEPACK using the HKL program suite (20). Native data of HtrA PD were
also collected at the Pohang Accelerator Laboratory and processed using
the HKL program suite (Table I). Two selenium sites (one methionine in
one subunit) of PD were found and used for phase calculation in the
program SOLVE (21). A relatively low figure of merit (0.25) is
explained by the presence of only one selenium atom per 238 residues in the subunit.
Solvent flattening and 2-fold noncrystallographic symmetry (NCS)
averaged by RESOLVE (22) resulted in a high quality electron density
map sufficient for model building. Amino acids were assigned using the
program O (23). Several cycles of rigid body refinement, positional
refinement, and simulated annealing were performed at 3.0-Å resolution
with CNS (24). The refinements were continued at 2.8-Å resolution
using the data collected from the native HtrA PD. Successive refinement
with temperature factors and addition of solvents resulted in an
R-value of 22.2% and an Rfree value of 28.4%, with a bulk solvent correction and overall anisotropic thermal factor refinement. Rfree was calculated
with 10% of the reflections. NCS restraints were enforced during the
refinement except flexible regions (LA and L2),
in which two subunits in an asymmetric unit showed different conformations.
The final model includes residues 24-48 and 51-251 and 58 water
molecules (Table I). Structural evaluation of the refined model using
PROCHECK (25) reveals that the structure has good geometric parameters
(Table I), and no residue falls in the disallowed region of the
Ramachandran plot. Statistical analysis of B-factor distribution was
performed by t test and the Wilcoxon rank sum test.
p < 0.001 was considered to be significant. The
figures in the article were drawn using the programs MOLSCRIPT (26) and
GRASP (27). The final coordinates and structure factors have been
deposited in the Protein Data Bank (PDB; accession number 1L1J).
Biochemical Activities of Tm HtrA and Tm HtrA PD--
Tm HtrA and
Tm HtrA PD were overexpressed in E. coli and purified. The
chaperone-like activities of both Tm HtrA and Tm HtrA PD were measured
by their abilities to suppress the aggregation of CS, which has been
widely used for molecular chaperone assays (19). The aggregation of CS
was monitored by light scattering at 320 nm after chemically denatured
CS was diluted in the refolding buffer. Tm HtrA or Tm HtrA PD
suppressed CS aggregation, decreasing the initial velocity of aggregate
formation (Fig. 2, A and
B). By addition of 2- and 4-fold molar excesses of Tm HtrA
to CS, the initial velocities of aggregation were decreased to 40.6 and 13.7%, respectively, compared with the initial velocity in the absence
of HtrA or HtrA PD. It was decreased to 68.3% when a 4-fold molar
excess of Tm HtrA PD was added, whereas 2-fold addition of Tm HtrA PD
essentially did not change it (data not shown). An 8-fold excess of
either Tm HtrA or Tm HtrA PD was enough to decrease the aggregation
rate to about zero. Under the same assay conditions, the initial
velocity of the reaction is not decreased when bovine serum albumin was
used for the control protein (Fig. 2, A and B).
These results indicate that Tm HtrA PD as well as the intact Tm HtrA
have the chaperone-like activity to inhibit the aggregation of CS.
However, because the enzyme activity of CS was not recovered (data not
shown), it seems that Tm HtrA does not assist the refolding of CS,
although the aggregation of denaturated CS was completely suppressed by
incubation with Tm HtrA. It can be inferred that Tm HtrA exhibited only
chaperone-like activity against CS, as is observed for other heat-shock
proteins such as
The molar ratios of chaperones to substrates required for suppression
of CS aggregation are different between intact HtrA and HtrA PD (Fig.
2), which is also observed for E. coli HtrA and E. coli HtrA PD. In the case of E. coli HtrA, a higher
concentration of the protease domain was required for refolding of MalS
protein than intact HtrA (9). Such a requirement for higher molar
ratios of HtrA PD might be explained by the absence of PDZ domains,
which are known to be involved in substrate recognition (14-16).
Compared with Tm HtrA, Tm HtrA PD showed a relatively weak proteolytic
activity, although its activity also increased with temperature. The
degradation products of Tm HtrA PD were long enough to be visible on
17% SDS-PAGE, whereas intact Tm HtrA completely degraded the substrate
into short peptides that are not shown in the gel (Fig. 2C).
Such a weak proteolytic activity of Tm HtrA can also be explained by
the absence of a PDZ domain. In both E. coli HtrA and human
HtrA2, PDZ domains play key roles in substrate binding and formation of
the chamber near the active site (14, 15). Tm HtrA PD generates longer
degradation products, because substrates are freely released from Tm
HtrA PD after cleavage. In contrast, within the chamber of intact Tm
HtrA, substrates are cleaved into small peptides simultaneously at
adjacent active sites.
Overall Structure of Tm HtrA PD--
The crystal structure of Tm
HtrA PD (residues 24-262) has been solved by multiwavelength anomalous
diffraction at 3.0 Å resolution and refined to 2.8-Å resolution
(Table I). The experimental electron density map calculated with multiwavelength anomalous diffraction phases and improved by solvent flattening and NCS averaging was of
sufficient quality to locate most main chains and some side chains. Tm
HtrA PD is composed of two
It is notable, however, that several structural differences exist
between Tm HtrA PD and Tm HtrA Trimer and Structural Comparison with Other
HtrAs--
Overall structure of Tm HtrA PD turns out to be similar
with the protease domains of E. coli HtrA and human HtrA2
with an r.m.s.d. of 2.2 Å for 179 C
Trimeric interactions found in E. coli HtrA hexamer or human
HtrA2 trimer seem to be conserved in Tm HtrA PD (Fig.
7), in which three subunits of Tm HtrA PD
related by 3-fold crystallographic rotation symmetry are tightly packed
by hydrophobic interactions. A hydrophobic patch composed of the
residues near
The main differences among HtrAs might be the size and conformation of
LA (Figs. 1, 4, and 5). E. coli HtrA has a long
loop reaching the active site of the opposite subunit (Figs.
4C, 6C, and 7B). In addition,
Two molecules of HtrA PD in an asymmetric unit related by 2-fold NCS
are associated by the minimal hydrophobic interactions among a few
residues (Tyr25, Pro28, Val32, and
Ala35; figure not shown) in the NH2-terminal
helix ( Hydrophobic Patches on the Surface of HtrA PD--
Most molecular
chaperones have hydrophobic substrate binding sites on their surfaces
to recognize and bind to the exposed hydrophobic patches of substrates
(3). However, in Tm HtrA PD trimer, most of the hydrophobic surface
near the active site is buried and no noticeable hydrophobic region is
exposed (Fig. 7D). Therefore, certain conformational changes
might occur to expose the hydrophobic substrate binding site when Tm
HtrA or Tm HtrA PD shows the chaperone-like activity.
Proteolytic Active Site of HtrA PD--
Most hydrophobic residues
in the helical lid of LA form wide contacts by hydrophobic
interactions with Leu80 in
In addition to the fact that the active site is blocked by the helical
lid, several residues that are essential for proteolytic activity are
positioned differently from those in the
Another crucial factor for the proteolysis by chymotrypsin-like
proteases is the stabilization of a negative charge of carbonyl oxygen
on the reaction intermediate (oxyanion hole) by hydrogen bonds from the
amide nitrogen atoms of two peptide bonds in the backbone. In Tm HtrA
PD, nitrogen atoms of Ser206 and Gly204 are
assumed to be the hydrogen donors to the putative oxyanion hole.
However, the NH group of Gly204 in Tm HtrA PD and its
counterpart in the Considering the helical lid, catalytic triad, and oxyanion hole in
the crystal structure of Tm HtrA PD, it can be referred that the
current crystal structure determined at room temperature represents an
inactive conformation of Tm HtrA PD (Figs. 4A and 6A). This is also true in E. coli HtrA, in which
the distortion of the active site loops and the intervening
LA* are assumed to prevent the proper position of the
catalytic triad and the formation of an oxyanion hole, resulting in an
inactive conformation of the protease domain (Figs. 4C and
6C). However, because Tm HtrA PD shows protease activity at
high temperatures (Fig. 2C), a structural change should
occur to activate its proteolytic activity. LA appears to be
flexible because of the high temperature factors in the crystal
structure and different conformations in two molecules in the
asymmetric unit; therefore, it is tempting to propose that LA becomes more flexible and flips up at elevated
temperatures. By these plausible conformational changes, Tm HtrA
becomes ready for proteolysis by exposing the substrate binding site
and rearranging the residues in the active site. A similar
temperature-dependent activation mechanism involving
conformational changes was suggested based on the biochemical
properties of E. coli HtrA (9). In addition, a
conformational change of the loop covering the catalytic site of HtrA
is also expected from the crystal structure of E. coli HtrA
(14).
In support of our assumptions, an infrared spectroscopic study (31) and
1-anilino-8-naphthalenesulfonate binding experiment (30) of E. coli HtrA suggest that a conformational change and exposure of the
hydrophobic region could occur when HtrA is activated. In human HtrA,
which does not have the helical lid (Figs. 1 and 6D),
proteolytic activity is evident even at room temperatures because of
the exposed active site (13, 15). However, human HtrA might also
experience some conformational changes near the active site by binding
to the substrate, because its active site also seems to be imperfectly
formed in the crystal structure (Fig. 6D) (15). The proposed
activation mechanism of HtrA is reminiscent of that found in lipase,
whose hydrolytic action is achieved by activation at an oil-water
interface (37). Although triggered by different factors, both lipase
and HtrA may undergo quite similar conformational changes: the opening
of the lid and rearrangement of the active site.
In the crystal structures of E. coli and human HtrAs, the
protease domains exclusively are involved in oligomerization (14, 15).
Therefore, we expect that the oligomerization interaction only occurs
in the protease domain of Tm HtrA (Fig. 7A). Structural differences among the three HtrAs mainly originate from the size and
conformation of LA (or LA* in E. coli
HtrA). This loop participates in the dimerization of HtrA trimers in
E. coli (Fig. 7B). But, LA forms a
helix and covers the active site of the same subunit in Tm HtrA PD
(Fig. 7A) and is very short in human HtrA2 (Fig. 7C). Consequently, E. coli HtrA forms a hexamer,
whereas Tm HtrA PD and human HtrA2 are found as trimers (14, 15).
Therefore, trimer formation of HtrA is mediated by hydrophobic residues
in its protease domain and further multimerization occurs by way of
LA and neighboring Current crystal structure cannot provide the structural basis for the
chaperone activity of Tm HtrA PD, because no remarkable substrate
binding sites are found on the surface of Tm HtrA PD (Fig.
7D). To display the chaperone-like activity seen under the experimental conditions (Fig. 2A), we propose that a
hydrophobic groove necessary for chaperone function is exposed by
certain conformational changes. The most important finding in the
crystal structure of the protease domain of Tm HtrA is the presence of the helical lid covering the active site; this lid is expected to open
at high temperatures for proteolytic action. However, we cannot rule
out the possibility that LA may not have the same conformation in intact HtrA as the current crystal structure of protease domain, in which LA extends to the opposite subunit
instead of covering its own active site and contributes to the
formation of hexamer in solution. Similarly, in E. coli
HtrA, LA* blocks the active site of the subunit in the
opposite trimer (Figs. 6C and 7B) (14).
Considering the common hydrophobic patches composed of the loops near
the active site in bacterial HtrAs, however, the active site of Tm HtrA
could be blocked by either LA in the same subunit or
LA* in the other subunit, and the active site is moved away
for proteolytic activity. Therefore, a temperature-dependent activation mechanism might be a general feature of bacterial HtrAs. To
explore this possible conformational change of LA and to
confirm the proposed activation, the crystal structure of intact Tm
HtrA and its biochemical characterization will be required.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Multiple sequence alignment of
HtrAs. The sequences of the protease domains of HtrAs from
Thermotoga maritima (Tm), E. coli
(Ec), Haemophilus influenza (Hi), and
HtrA2 from human mitochondria (Hs) are used for alignment
using the program CLUSTALW (17). The secondary structures of Tm HtrA PD
are indicated by a cylinder for -helix and by an
arrow for
-strand. The amino acids in the catalytic triad
are marked with blue triangles. The helical lid covering the
active site is colored magenta. In the alignment, identical
residues are boxed in red and homologous residues
are boxed in yellow.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactalbumin (Sigma) and 28 µg of HtrA PD or 48 µg of HtrA (at 1:2 molar ratio of protease to substrate) were
incubated in 60 µl of reaction buffer containing 5 mM
Tris (pH 7.5) and 1.5 mM dithiothreitol at 25, 45, 65, and
85 °C. The reduced form of
-lactalbumin was prepared by
incubating 3 mg/ml protein in 5 mM Tris (pH 7.5) containing 10 mM dithiothreitol at 4 °C for 2 days. HtrA and HtrA
PD were preincubated at each indicated temperature prior to the
addition of the substrate protein. The reaction was performed in
100-µl tubes to minimize the evaporation effect. After incubation for 30 min at each temperature, 17 µl of 5× SDS-PAGE sample buffer was
added to stop the reaction. Then, the samples were analyzed by 17%
SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-crystallin (28).
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Fig. 2.
Biochemical activities of Tm HtrA and Tm HtrA
PD. Suppression of the aggregation of CS by Tm HtrA PD
(A) and intact Tm HtrA (B). Pig heart CS
denatured in 6 M guanidinium hydrochloride was diluted in
10 mM Tris buffer and incubated at 25 °C with various
concentrations of Tm HtrA PD (CS only ( ), HtrA PD:CS = 4:1 (
), 8:1 (
) and bovine serum albumin:CS = 5:1 (
)), or
intact Tm HtrA (CS only (
), HtrA:CS = 2:1 (
), 4:1 (
), 8:1
(
), and bovine serum albumin:CS = 5:1 (
)). The aggregation
of CS was monitored by measuring the apparent light scattering
(absorbance at 320 nm). Bovine serum albumin was used as a negative
control. C, proteolytic activities of Tm HtrA PD and intact
Tm HtrA. Proteolytic activity was monitored at each temperature by
incubating the reduced form of
-lactalbumin (LA) with Tm
HtrA PD or intact Tm HtrA. The molar ratio of Tm HtrA (or Tm HtrA PD)
to
-lactalbumin was adjusted to 1:2. The first and
sixth lanes contain molecular weight markers in
kilodaltons.
-Lactalbumin, which is commonly used for the assays of heat-shock
proteases such as 20 S proteosome (29) and E. coli HtrA (30), was employed as a substrate to investigate the proteolytic activity of Tm HtrA. Clearly, Tm HtrA displayed the proteolytic activity at elevated temperatures, and maximal activity was observed at
85 °C (Fig. 2C). As reported for E. coli HtrA
(9, 31), the proteolytic activity of Tm HtrA increased with
temperature, and Tm HtrA is autodegraded at high temperatures (Fig.
2C).
-barrel domains connected by a long loop
between
6 and
7 (Figs. 3 and
4A). Residues in the catalytic
triad, Asp127-His97-Ser206, are
located in the cleft of two
-barrels. The structural comparison by
DALI server (32) reveals that its topology is similar to proteases in
the chymotrypsin family (33). Among the members of the chymotrypsin
family,
-lytic protease (PDB accession number 1qq4; Ref. 34) can be
superimposed on Tm HtrA PD with the lowest r.m.s.d. of 2.5 Å for 161 C
atoms of 198 C
atoms of
-lytic protease (Figs. 4B
and 5). However, its fold is different from those of proteolytic cores
of ATP-dependent proteases such as ClpP (35) and HslV
(36).
Data collection, phasing, and refinement statistics
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Fig. 3.
Structure of Tm HtrA PD. A stereo C
trace of Tm HtrA PD. Every 20th residue and NH2- and
COOH-terminal residues are indicated as black balls and
labeled. Overall structure of Tm HtrA PD is similar to the structure of
proteases in the chymotrypsin family.
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Fig. 4.
Ribbon diagrams of Tm HtrA PD
(A), -lytic protease
(B), E. coli HtrA PD
(C), and human HtrA2 PD (D).
-Lytic protease, E. coli HtrA PD, and human HtrA2 PD were
positioned in the same orientation with Tm HtrA PD. Two
-barrel
domains are green and blue. The regions
(LA) connecting
1 and
2 in Tm HtrA PD, E. coli HtrA PD, human HtrA2 PD, and
-lytic protease are
magenta and labeled. In E. coli HtrA PD the loop
LA* containing residues 38-79 is pink.
LA* is the loop belonging to the subunit in the opposite
trimer; E. coli HtrA PD forms a dimer by an interaction at
the interface between the loop LA and LA*.
Residues 48 and 51 are connected by a dashed line because
residues 49 and 50 are not modeled in Tm HtrA PD. The residues in the
catalytic triads (Asp, His, and Ser) are drawn as orange stick
models. These residues in Tm HtrA PD and E. coli HtrA
are blocked by LA or LA*, whereas they are open
to the solvent in
-lytic proteases and human HtrA2. Disulfide bonds
of
-lytic protease are drawn as yellow lines. These bonds
are not found in HtrAs, however. Each secondary structure and loop of
Tm HtrA PD is labeled.
-lytic protease. The most significant of them
is the length of LA (loop A connecting
1 and
2,
according to the nomenclature in Ref. 33) in two proteins (Figs. 4 and 5). LA of Tm HtrA (residues
47-76), including an amphipathic helical lid (
2, residues 55-66)
and
3, is located on top of the catalytic residue Ser206
(Figs. 4A and 6A). Interestingly, the residues
corresponding to the helical lid of Tm HtrA PD are found only in
bacterial HtrAs, not in human homologues (Fig. 1), and this lid is
expected to have the important functional or structural roles in
bacterial HtrAs. There are differences in several other loops
connecting
-strands (Fig. 5). Among them, structural changes near
the loop containing the putative oxyanion hole and the catalytic
residue Ser206 (residues 202-206) seem to be significant
for explaining the functional differences of Tm HtrA and
-lytic
protease (Figs. 5 and 6). Three conserved
disulfide bonds found in almost all members of the chymotrypsin family
of serine proteases are absent from all known HtrAs (Fig. 4).
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Fig. 5.
Comparison of three HtrA PDs
and -lytic protease. A stereo C
trace
of Tm HtrA PD (green), E. coli HtrA PD
(blue), human HtrA2 PD (red), and
-lytic
protease (magenta). Three residues of the catalytic triad of
Tm HtrA PD are drawn as black stick models. Most structures
in the core region containing the central
-barrel and
-helices
are well conserved in the four proteins, whereas loop regions display
differences. The loop LA of each HtrA, where a large
conformational movement is observed, is labeled and emphasized by a
thicker line.
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Fig. 6.
Active sites of Tm HtrA PD
(A), -lytic protease
(B), E. coli HtrA
(C), and human HtrA2 (D). The
amino acid residues in the catalytic triads and three residues
preceding the active site Ser are drawn in ball and
stick models and labeled. The putative oxyanion holes of Tm
HtrA PD and
-lytic protease are indicated by red balls.
Dashed lines represent possible hydrogen bonds in the active
site. Hydrogen bonds in the catalytic triad and the oxyanion hole of
-lytic protease, which are necessary for the protease activity, are
formed. However, those in the three HtrAs are not fully formed,
representing the inactive conformations. LAs of Tm HtrA and
E. coli HtrA are magenta, and LA*, a
loop reaching into the active site of E. coli HtrA PD from
the opposite subunit, is pink. LA and
LA* cover the active sites of Tm HtrA PD and E. coli HtrA, respectively. In Tm HtrA PD, hydrophobic residues
involved in lid interaction are also drawn and labeled. Hydrophobic
residues in LA are light yellow to distinguish
them from the residues on the other side.
atoms of 215 C
atoms of
E. coli HtrA and 1.9 Å for 181 C
atoms of 196 C
atoms
of human HtrA2, respectively (Figs. 4 and 5). The large r.m.s.d. among
HtrAs are mostly caused by the structural difference near the active
site and loop LA (Fig. 5). Interestingly, Tm HtrA shows more
structural resemblance with human HtrA than E. coli
HtrA.
1,
7,
8, and
11 is involved in the
hydrophobic packing in a trimer (Figs. 4A and
7A). Those hydrophobic residues are quite well conserved in
most other HtrAs (Fig. 1), implying that hydrophobic packing in a
trimer is a general feature of HtrAs. By trimerization, the 6700 Å2 surface area of Tm HtrA PD is buried, which is
comparable with the 6044 Å2 in human HtrA2 (15). Taken
together, it appears that the Tm HtrA forms a trimer by hydrophobic
interaction mediated by the protease domain, as observed in E. coli HtrA and human HtrA2 (14, 15). Analytical ultracentrifugation
and gel filtration experiments also support the existence of Tm HtrA PD
as a trimer in solution (data not shown).
View larger version (54K):
[in a new window]
Fig. 7.
Trimeric packing of Tm HtrA PD
and comparison with other HtrA PDs. Bottom and
side views of Tm HtrA PD (A), E. coli
HtrA PD (B), and human HtrA2 PD (C) trimers were
drawn in ribbon diagrams with the same color schemes used in
Fig. 4. LA, LA*, 1,
2, and NH2-
and COOH-terminal residues in one subunit are labeled. In E. coli HtrA PD trimer, three LA loops (LA*)
protruding into the active sites from the opposite trimer are drawn.
The NH2-terminal helix and COOH-terminal barrel domain are
involved in trimer formation by hydrophobic packing. LA and
neighboring strands,
1 and
2, show the most different
conformations in three HtrAs. D, surface charge distribution
of Tm HtrA PD trimer. The red and blue areas
represent negatively and positively charged surfaces, respectively.
White patch, representing the hydrophobic surface, is not
found, implying the substrate binding region necessary for chaperone
activity is not observed in the current structure. Ser206
residues are not visible in this figure because they are buried by
LA. However, their positions are indicated by black
arrowheads.
1 and
2 in E. coli HtrA is long enough to make a
-sheet with
two other
-strands from the trimer in the other side, leading to a
hexamer structure (Fig. 7B) (14). In contrast, LA
in Tm HtrA PD is mainly composed of a helix (
2) covering the active
site of the same subunit in the current structure (Figs. 4A,
6A, and 7A) and is shorter than its counterpart
in E. coli HtrA (Figs. 1 and 7). Interestingly, human HtrA2
has a very short LA, which is not involved in the
dimerization of trimers or the covering of the active site (Figs.
4D, 6D, and 7C).
1). Therefore, the presence of two molecules in the
asymmetric unit seems to have no biological relevance. The two
molecules in the asymmetric unit show identical conformations except
the regions near LA and L2 (loop 2 connecting
11 and
12), suggesting those regions are relatively flexible.
2, Pro163 and
Leu164 in LD (loop D connecting
7 and
8),
Pro203 and Gly204 in L1 (loop 1 connecting
9 and
10), and Ala223 and
Ile224 in L2 (Fig. 6A). Because
LA* of E. coli HtrA makes intimate contact with
L1 and L2 (the asterisk denotes the
loops in the neighboring subunit, see Figs. 4C,
6C, and 7B; Ref. 14), the hydrophobic interactions between the loops near the active site and lid seem to be
common in bacterial HtrAs. However, in other HtrAs residues interacting
with LA appear to vary depending on the size of the lid
(Fig. 1). Possible substrate binding sites of Tm HtrA (S3, S2, S1, S1',
S2', and S3' defined in Ref. 33) are completely blocked by the lid and
inaccessible to the solvent (Fig. 6A). Because the average
temperature factor of the residues in LA is relatively
higher (64.9 Å2) than that for the whole protein (54.8 Å2), it appears that the lid is rather flexible and its
interaction with the loops are not tight. B-factor difference between
LA and other regions is significant (p < 0.001). Flexibility of the lid is also inferred from different
conformations of the lids of two molecules in an asymmetric unit. When
the molecules in the asymmetric unit are superposed, 28 C
atoms in
LA (residues 47, 48, and 51-76) give a r.m.s.d. of 1.52 Å,
whereas other C
atoms (except another flexible region at residues
225-232) give 0.45 Å (Fig. 5). We also suspect that residues 49 and
50 do not show electron density because of the flexibility of
LA. Structural flexibility of LA found both in Tm
and E. coli HtrAs (14) suggests that this loop could undergo
a conformational change in bacterial HtrAs.
-lytic protease (Fig. 6,
A and B). For hydrolytic cleavage of a peptide bond, the residues in the catalytic triad need to be aligned close enough for electron transfer from Asp to Ser through His. However, in
the current crystal structure of Tm HtrA PD, distances between the
N
2 atom of His97 and the O
atom of Ser206 of each molecule in the asymmetric unit are
3.69 and 3.40 Å (Fig. 6A, Table
II), whereas the N
2 atom
of His36 and the O
atom of
Ser143 in the
-lytic protease are hydrogen-bonded at a
distance of 2.96 Å (Fig. 6B, Table II). Consequently,
His97 is not expected to function as a general base to
remove the proton from Ser206. In the crystal structures of
E. coli and human HtrAs, the distance between the
N
2 atom of His and the O
atom of Ser in
the catalytic triad cannot be measured because Ser mutated to Ala (Fig.
6, C and D) (14, 15). However, the distance
between the N
2 atom of His and the C
atom
of the mutated Ala in catalytic triads of E. coli HtrAs is
too long compared with the corresponding distance in the
-lytic protease (Fig. 6, Table II), although a little conformational change in
the Ala mutant is expected. Therefore, it is obvious that both Tm and
E. coli HtrAs are inactive because of the distortion of the
loops near the active site, whereas
-lytic protease is in an active
state.
Distances between the atoms in the catalytic triads of HtrAs and
-lytic protease (Å)
-lytic protease (the NH group of
Gly141) point to opposite directions (Fig. 6, A
and B). In this conformation it is impossible for Tm HtrA PD
to form an oxyanion hole. This unique conformation of the loop near the
oxyanion hole is also found in two other HtrAs (Fig. 6, C
and D). Similarly, triacylglycerol lipase and
Staphylococcus aureus epidermolytic toxin A do not have
pre-formed oxyanion holes (37, 38), leading to inactive states, as seen
in HtrA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strands, depending on their size and
structure. Furthermore, the proteolytic activity of HtrA may be
modulated by the structure of LA and the following
multimerization state.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. H. S. Lee and scientists at beamline 6B of Pohang Accelerator Laboratory, Korea, for assistance during data collection.
![]() |
FOOTNOTES |
---|
* This work was supported by Korea Research Foundation Grant KRF-2000-015-FS0003.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1L1J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.:
82-31-299-6136; Fax: 82-31-299-6159; E-mail:
kkim@med.skku.ac.kr.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M208148200
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ABBREVIATIONS |
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The abbreviations used are: HtrA, high temperature requirement A; CS, citrate synthase; NCS, noncrystallographic symmetry; PD, protease domain; r.m.s.d., root mean square deviations; Tm, Thermotoga maritima..
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