From the Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239-3098
Received for publication, November 25, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Catalytic domains of several prokaryotic and
eukaryotic protease families require dedicated N-terminal
propeptide domains or "intramolecular chaperones" to facilitate
correct folding. Amino acid sequence analysis of these families
establishes three important characteristics: (i) propeptides are almost
always less conserved than their cognate catalytic domains, (ii) they
contain a large number of charged amino acids, and (iii) propeptides
within different protease families display insignificant sequence
similarity. The implications of these findings are, however, unclear.
In this study, we have used subtilisin as our model to redesign a
peptide chaperone using information databases. Our goal was to
establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design
of tailor-made chaperones. A decision-based computer algorithm that
maintained conserved residues but varied all non-conserved residues
from a multiple protein sequence alignment was developed and utilized
to design a novel peptide sequence (ProD). Interestingly, despite a
difference of 5 pH units between their isoelectric points and despite
displaying only 16% sequence identity with the wild-type propeptide
(ProWT), ProD chaperones folding and functions as a potent subtilisin
inhibitor. The computed secondary structures and hydrophobic patterns
within these two propeptides are similar. However, unlike ProWT, ProD
adopts a well defined Subtilisin E is an alkaline serine protease isolated from
Bacillus subtilis (1). In vivo this protein is
produced as pre-pro-subtilisin (2), wherein the pre-region (signal
peptide) facilitates protein secretion and the pro-region (propeptide)
functions as an intramolecular chaperone that guides correct folding of
subtilisin (2-6). Upon completion of folding, the propeptide is
autoproteolytically removed (7). This is necessary because the
propeptide is a potent inhibitor of subtilisin activity (4).
Propeptide-mediated folding mechanisms exist in several unrelated
protease families. However, there is no sequence conservation in
propeptide domains within these families (8). This functional conservation across unrelated protease families suggests that propeptides have evolved in multiple parallel pathways and may share a
common mechanism of action (9). Subtilisin (2-6) and To establish minimum sequence requirements for a functional propeptide,
in this study we have designed and developed a peptide chaperone using
information databases on the subtilase family. This family is ideal
because enormous structural and functional information on individual
members is already available (8). The peptide sequence
(ProD)1 was designed through
a decision-based computer algorithm that maintained conserved residues
but varied all non-conserved residues from a multiple protein sequence
alignment. The DNA sequence coding for ProD was synthesized and
subsequently expressed in Escherichia coli, and the
resulting peptide was purified. Despite a difference of 5 pH units
between their isoelectric points (pI) and despite displaying only 16%
sequence identity with the wild-type propeptide (ProWT), ProD
chaperones folding and functions as a potent subtilisin inhibitor.
The computed two-dimensional structure and the distribution of
hydrophobic residues within these two propeptides are similar. However,
unlike ProWT, ProD adopts a well defined Algorithm for Designing a Peptide Chaperone--
The PSI-BLAST
(position-specific iterated basic local alignment search tool) program
(20) was used to obtain a multiple sequence alignment of proteins that
are similar to ProWT (77 residues) using a threshold E-value of 0.8. This alignment was treated as a matrix and utilized to obtain the
percentage of every individual amino acid at every position in the
77-residue polypeptide. The matrix contained 176 homologues of
subtilisin. The computed percentage was employed in a decision-based
algorithm that searched for the following criteria. If "X" is the
most abundant amino acid residue and "Y" is the second most
abundant residue at a particular position in the sequence, then residue
"X" is always chosen over Y UNLESS (frequency(X)
In the present analysis A = 2 and no two residues at any position
display identical frequencies. The aim of the algorithm was to
establish the absolute minimum sequence requirement within divergent
propeptide sequences. The computed sequence ProD, which was designed to
digress from ProWT, displays 16% sequence identity and opposite net
charge (Fig. 1A).
Gene Synthesis of ProD--
The nucleotide sequence
corresponding to ProD was obtained using the BACK-TRANSLATE module from
the Genetics Computer Group Wisconsin Package program (21) and was
based on the E. coli codon usage table. The nucleotide
sequence was synthesized using a combination of DNA synthesis and PCR.
The product was cloned into pET11a under the control of the
T7-promoter (4), and the nucleotide sequence was verified on an
ABI-310 DNA sequencer.
Expression and Purification of ProD and
S221C-subtilisin--
E. coli BL21(DE3) cells were
transformed with plasmids pET-Pro-S221C-subtilisin and pET-ProD to
obtain S221C-subtilisin and ProD, respectively. Cells were grown
in M9 medium supplemented with 50 µg/ml ampicillin (22). At an
absorbance of 0.6 A600 nm, the culture was
rapidly cooled to 12 °C. Adding isopropyl
Trans Folding of Denatured Subtilisin--
6 M
guanidine hydrochloride denatured subtilisin (0.9 µM) was
mixed with varying concentrations of ProD (0-36 µM) to a
final volume of 100 µl. ProWT was used as a control. The
mixture was dialyzed for 16 h against 50 mM MES, pH
6.5, containing 0.5 M ammonium sulfate and 1 mM
CaCl2. A 20-µl aliquot of the renaturation mixture was
treated with ~7.0 units/ml of TPCK-treated trypsin for 1 h to
degrade the excess of propeptide. This is necessary because the
propeptide can also inhibit subtilisin activity (3). Enzyme activity
was measured using N-suc-AAPF-pNA as a synthetic substrate
(23). TPCK-treated trypsin does not cleave the synthetic substrate and
hence does not interfere with the activity assay.
Inhibition of Subtilisin E Activity--
Active subtilisin (65 nM) was rapidly added to the protease assay buffer that
contained 0.5 µM synthetic substrate and 0.9 µM of ProD and ProWT. Protease activity was monitored as
described earlier (4, 22, 23).
Complex Formation between ProD and S221C-Subtilisin--
ProD
and ProWT (6.4 µM) were added to an equimolar amount of
mature S221C-subtilisin in the folding buffer (50 mM MES,
pH 6.5, 0.5 M ammonium sulfate, 2 mM
CaCl2, and 1 mM Trypsin-mediated Trans Degradation of ProD and ProWT Complexes
with S221C-subtilisin--
The trans complex was obtained as discussed
above. The ProD- and ProWT·S221C-subtilisin complexes (50 nM) were incubated with TPCK-treated trypsin (2.0 units/ml), and aliquots were removed at different time intervals. The
reaction was terminated through trichloroacetic acid precipitation as
described earlier (4), and the aliquots were separated using
SDS-PAGE. The amount of residual propeptide was estimated using
quantitative gel-scanning densitometry.
Circular Dichroism and Fluorescence Measurements--
CD
measurements were performed on an automated AVIV 215 spectrometer at
25 °C. Protein concentration was maintained between 0.05-0.2 mg/ml
(4). Spectra were taken between 190-260 nm using a 1-mm path length
cuvette (Fig. 2C) and represent averages of three
independent scans. For the fluorescence measurements, the denatured and
folded ProD (7.0 µM) were excited at 295 nm, and the
emission spectra from 310 to 410 nm were recorded as described earlier
(23).
Molecular Modeling--
Homology modeling was performed using
LOOK, Version 2.0. The sequence alignment was performed using the
BestFit module of the Genetics Computer Group software (21), and the
alignment was imported into LOOK. A structural model for
ProD·S221C-subtilisin was built, and the energy minimization was
carried out using the GROMOS 43B1 force field using the Swiss-Model
software, Version 3.7 (25). Because the ProWT and ProD contain more
than 36% charged residues, the electrostatic potentials were computed
using coulomb interactions by taking into account the charged residues
and assuming a pH of 7.0 (25). The electrostatic charges were mapped to
the molecular surface (Fig. 4B).
Criteria for Designing and Selecting ProD--
The algorithm
described under "Experimental Procedures" was used to obtain a
matrix that was analyzed using the following criteria. If "X" is
the most abundant amino acid residue and "Y" is the second most
abundant residue at a particular position in the sequence, then residue
"X" is always chosen over Y UNLESS (frequency(X) ProD Displays Low Sequence Identity with ProWT--
Fig.
1A compares the primary sequence of ProD with ProWT. N1 and
N2 represent motifs that were identified using closely related subtilases and appear to be fairly well conserved (26). The sequence
identity in Motif N1 and the C-terminal part of Motif N2 is
significant, and these motifs are located in ProD Can Inhibit Subtilisin Activity and Chaperone Folding of
Denatured Subtilisin--
It has been established that ProWT inhibits
subtilisin through slow binding inhibition kinetics (29). Because the
primary sequence and the amino acid composition between ProWT and ProD are significantly different, we examined whether ProD can inhibit the
proteolysis of the synthetic substrate and whether this inhibition follows slow binding kinetics (Fig.
2A). From Fig. 2A,
it is evident that in 10 min the activity of subtilisin (65 nM) drops ~12- and 135-fold in the presence of 0.9 µM of ProWT and ProD, respectively. Hence under these
conditions, ProD is an ~10-fold better inhibitor than ProWT and does
not demonstrate slow binding inhibition. At lower concentrations,
however, ProD demonstrates slow binding inhibition (Fig. 2A,
inset), whereas the same concentration of ProWT is not
inhibitory (data not shown). We next examined whether ProD can
chaperone folding of subtilisin in a trans folding reaction. Denatured
subtilisin was folded using ProD and ProS as a chaperone, and the
subtilisin activity that was recovered was measured (see "Experimental Procedures"). Fig. 2B demonstrates that
ProWT and ProD can both chaperone folding of subtilisin in
trans, and the activity recovered increases with the chaperone
concentration. When a 40-fold excess of the chaperone was used, ProWT
and ProD recovered ~40 and 10 units of activity, respectively. Hence,
under these conditions ProD is ~25% as efficient as ProWT in folding subtilisin (Table I).
We next examined the kinetics of trans degradation of ProD and ProWT as
complexes with Ser221C-subtilisin by TPCK-treated trypsin. Fig.
2C depicts the kinetics measured using quantitative gel-scanning densitometry as described under "Experimental
Procedures." The S221C-subtilisin is proteolytically inactive and is
unable to degrade the inhibitory propeptides. This enables one to
monitor the kinetics of trans degradation of ProD and ProWT, in the
absence of autodegradation. The data were fitted to a single
exponential rate equation, and rate constants for degradation of ProD
and ProWT were estimated as 0.09 and 0.31 min ProD Is Structured as an Isolated Peptide and Forms a Complex with
Subtilisin--
Because ProD can function both as a chaperone and an
inhibitor of subtilisin, we next attempted to analyze the secondary
structure of ProD in its isolated form and as a complex with
S221C-subtilisin using CD spectroscopy. Equimolar concentrations of
ProD and ProWT were added to S221C-subtilisin, and the samples were
incubated overnight. The S221C substitution at the active site lowers
the proteolytic activity by 10,000-fold and is insufficient to degrade the inhibitory propeptide (28). Far-UV CD spectroscopy establishes that
unlike ProWT, which is completely unstructured (3), ProD adopts a well
defined
However, the CD spectra of the complexes of ProD and ProWT with
S221C-subtilisin appear to be similar. Because the x-ray structure of
the ProWT·S221C-subtilisin complex establishes that the conformation of S221C-subtilisin does not change through its interactions with ProWT
(28), Fig. 3A suggests that the structure of ProD as a complex with S221C-subtilisin is similar to that of ProWT.
ProD contains one tryptophan, four tyrosine, and five phenylalanine
residues. Because phenylalanine, tyrosine, and tryptophan contribute to
the fluorescence of a protein and are useful probes for monitoring the
solvent accessibility of the side chains, we next examined the
intrinsic fluorescence to detect conformational differences between the
folded and denatured states of ProD. The protein was excited at 295, and the emission spectra were recorded (Fig. 3B). ProD shows
an emission maximum at 352 and 349 nm for denatured and folded ProD,
respectively. The shift in maxima to a lower wavelength (blue shift)
suggests that the environment around the tryptophan side chain becomes
non-polar upon folding and is consistent with a conformational change
within the protein. The 10% decrease in fluorescence intensity of
folded ProD may occur because of quenching of the neighboring aromatic
residues (Fig. 4, A-C).
We next measured the tertiary structure of ProD under denaturing and
non-denaturing conditions using near-UV CD spectroscopy. The near-UV CD
profiles of denatured and non-denatured ProD differ only marginally and
suggest that ProD may not possess a well defined tertiary. However, the
lack of a tertiary structure in small peptides in not unusual (30).
Molecular Model of ProD--
From Fig. 2, A-D and Fig.
3, A-C, it is evident that (i) ProD functions as an
inhibitor and a chaperone of subtilisin similar to ProWT, and (ii) ProD
and ProS adopt similar conformations as complexes with
S221C-subtilisin. Hence we attempted to build a homology model of
ProD·S221C-subtilisin (Fig. 4A) using the x-ray structure
of the ProWT·subtilisin complex (28) as described under
"Experimental Procedures." The goal of this model is to obtain the
spatial orientation of residues within ProD and ProWT, because such
models can guide mutagenesis experiments and hypotheses about
structure-function relationships (24). From this model it is evident
that the distribution of the electrostatic potential around the
complexes formed by ProD and ProWT is remarkably different (Fig.
4B). Whereas ProD appears to be completely negative
(acidic), ProWT displays a strongly positive electrostatic potential.
This is consistent with the finding that ProD contains a large number of acidic amino acids and ProWT displays a bias for basic residues (Table I; Fig. 1A). However, it is interesting to note that
the hydrophobic core within ProD and ProWT appears to be maintained (Figs. 1A and 4C). Hydrophobic interactions can
make significant contributions to folding and stability of proteins.
The average hydrophobicity of ProD is greater than ProWT.
Furthermore, the computed free energy for the transfer of the
10-residue hydrophobic core (Fig. 4C) from an
aqueous to a hydrophobic environment stabilizes ProD by ~2.5
kcal (Table I).
We have used the subtilase family as a model to analyze the
relation between sequence, charge, structure, and function within propeptides. An indigenously developed algorithm provided a novel peptide, ProD, that displays only 16% sequence identity and has a pI 5 pH units lower than ProWT (Table I). However, the overall hydrophobic
patterns, the computed secondary structures, and the potential folding
nucleation motifs (N1 and N2) appear to be conserved in ProD (Fig. 1,
A-C). These properties seem to be important for function
because, despite low sequence identity, ProD and ProWT can function as
peptide chaperones and potent protease inhibitors (Fig. 2, A
and B). However, ProD is a less efficient chaperone and a
more potent inhibitor than ProWT. This confirms that the chaperone and
inhibitory functions are not obligatorily related (22). The differences
in the amino acid composition are also reflected in the secondary
structures of the isolated ProWT and ProD measured using CD
spectroscopy (Fig. 3A). Unlike ProWT, ProD adopts a well
defined It is important to note that although the sequence similarity between
ProWT and ProD is low, the sequence similarity of the residues that
form interface with subtilisin (as judged by three-dimensional structure 1SCJ) is at least 70%. As a result, one would expect the net
binding energy of ProWT and ProD to subtilisin to be very similar.
However, in the case of ProWT its binding to subtilisin is coupled to
its folding. Hence a part of the interaction energy will be spent on
ProWT folding, thereby lowering the ProWT-subtilisin affinity (or
inhibition constant). The ProD is already folded; it does not need to
expend as much energy on its folding when compared with ProWT. This in
turn may result in a higher affinity and hence a 10-fold tighter
inhibition constant.
Because propeptide domains appear to have emerged through convergent
evolution (9), this raises another important question. Are there common
structural features that define propeptide function in different
protein families? To examine this possibility we compared the
structures of the propeptides from subtilisin (PDB identifier, 1SCJ),
conformation as an isolated peptide and
forms a stoichiometric complex with mature subtilisin. The CD spectra
of this complex is similar to ProWT·subtilisin. Our results
establish that despite low sequence identity and dramatically different
charge distribution, both propeptides adopt similar structural
scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not
absolute charge, dictate propeptide function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lytic
protease (10-13) constitute the best-studied examples of propeptide-mediated protein folding. Bacterial subtilisins are models
for the subtilase family that spans prokaryotes and eukaryotes. Amino
acid sequence analysis of this family establishes that although the
subtilisin domain is conserved, the cognate propeptides can vary
significantly (8). Because propeptides perform functions different from
the catalytic domains, they may be subjected to different mutational
frequencies because of different functional constraints (14-16).
However, given that they impart structural information to their
catalytic domains (17-19), propeptides within one family could adopt
similar structural scaffolds despite digressions in their polypeptide
sequences. This makes propeptides attractive models for protein
redesign and for understanding the relation between sequence and structure.
-
conformation as an
isolated peptide. The CD structure of ProD·subtilisin was found to be
similar to ProWT·subtilisin. Hence, despite low sequence identity and
opposite net charge, ProD and ProWT adopt similar structural scaffolds.
Our results suggest conserved structural scaffolds and
hydrophobicity, but not absolute charge, dictates propeptide function.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A*
frequency(Y)) AND residue X is present in ProWT, where "A"
represents a "cutoff factor."
-D-thiogalactopyranoside to a final concentration of 1 mM induced protein expression. After 16 h at 12 °C,
the cells were harvested as described earlier (4). Under these
conditions Pro·S221C-subtilisin and ProD remain in the soluble
fraction. The samples were then centrifuged at 20,000 × g to remove the insoluble cell debris. The supernatant that contained Pro·S221C-subtilisin was then incubated at 4 °C to allow the degradation of the inhibitory ProWT through cellular proteases. The
cell lysates were dialyzed overnight (using a 3-kDa cutoff) against 100 vol of 50 mM Tris-HCl, pH 8.6, and 1 mM CaCl2. The proteins were loaded onto a HighQ
column (BioRad) equilibrated with 50 mM Tris-HCl, pH 8.6, and then eluted using a 0-1 M NaCl gradient. Fractions
containing the desired proteins were pooled together and dialyzed
against 50 mM Tris-HCl, pH 7.0, containing 0.5 M ammonium sulfate, 2 mM CaCl2 and
concentrated by ultra-filtration using a 3-kDa cutoff membrane.
Concentrated ProD was further purified using a Superdex-75
gel-filtration column (Amersham Biosciences). Both ProD and
S221C-subtilisin were purified using the above approach. The only
difference was that a Superdex-200 gel-filtration column was used to
obtain mature S221C-subtilisin.
-mercaptoethanol). The
mixtures were incubated overnight at 4 °C to allow complex formation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A*frequency(Y)) AND residue X is present in ProWT, where "A"
represents a "cutoff factor." Fig.
1D depicts the relation between the cutoff factor "A" and the sequence identity of the computed sequences to ProWT. It is evident from Fig. 1D that
as the cutoff factor increases the percent identity decreases according to two different slopes. In the present analysis the value of the
cutoff factor was selected as 2 because the transition in slopes occurs
when A = 2. If for example, frequency(X) = 70%, then
frequency(Y)
30%; then according to our criterion X was selected because (frequency(X)
2* frequency(Y)). If, however, frequency(X) = 60% and frequency(Y) = 40% AND residue X was
present in ProWT, then residue Y was selected over X because
(frequency(X)
2 * frequency(Y)). However, if frequency(X) = 60% and frequency(Y) = 40% AND residue X was NOT present in
ProWT, then residue X was selected in the computer-generated sequence.
Although it is possible that the frequencies of X or Y at any position
may be identical, during the analysis of 176 subtilisin homologues this
scenario did not arise when A = 2. Because no two residues at any
position displayed identical X or Y frequencies, this algorithm
provided us with a unique sequence for ProD (Fig. 1A).
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Fig. 1.
Design criteria and computational
characterization of proD. A, primary sequence alignment
between the subtilisin propeptide (ProWT) and the redesigned
propeptide (ProD). Identical residues (black
background), highly conserved substitutions (gray
background), and less conserved substitutions
(boldface) were identified using the program BestFit (21).
The secondary structure of ProWT (obtained from the x-ray structure)
when it forms a complex with subtilisin is depicted above
the sequence alignment; the secondary structure computed using
PREDICT-PROTEIN (34) is depicted below the sequence
alignment. C, E, and H denote coils,
-sheets, and
-helices, respectively. The predicted structure in
boldface represents predictions that do not match with the secondary
structure obtained from the crystallized complex of ProWT·subtilisin.
Motifs N1 and N2 represent the conserved domains within the subtilase
family (18). Asterisks denote residues that constitute the
hydrophobic core within the propeptide. B, the difference in
the amino acid composition between ProWT and ProD. Positive
values represent those residues that are in excess in ProWT;
negative values indicate residues that are in excess in
ProD. C, hydropathy plot comparison between ProD
(black line) and ProWT (gray line). D,
change in sequence identity of the computed peptide with ProWT as a
function of Cutoff Factor (A).
1 and
2
4, as
seen from the x-ray crystallographic structure of ProWT (28). NMR
structure analysis suggests that
1 and
2-
4 display
conformational rigidity and may represent potential folding
nucleation sites in ProWT (27). Hence, motifs that nucleate the folding
process appear to be conserved in ProD. When the difference in the
total number of individual amino acids between the two propeptides is examined (Fig. 1B), it is clear that ProWT (pI = 9.76)
has an excess of basic residues, whereas ProD (pI = 4.52) is rich
in acidic amino acids. However, the hydrophobic patterns within these two peptides are similar (Fig. 1C), and the residues that
constitute the buried hydrophobic core in ProS and ProD are always
non-polar (Fig. 1A). Properties of ProD and ProWT are
summarized in Table I.
Properties of the isolated peptide chaperones
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Fig. 2.
Biochemical characterization of ProD.
A, inhibition of subtilisin activity as a function of time.
Subtilisin (65 nM) rapidly degrades
N-suc-AAPF-pNA to produce p-NA, which
can be monitored spectroscopically (black line without
symbols). The ProWT (open circles and
triangles represent 0.8 and 1.2 µM of ProWT,
respectively) can inhibit proteolysis of the substrate, and this
inhibition follows slow binding kinetics. However, 0.8 (filled
circles) and 1.2 µM (filled triangles) of
ProD almost completely blocks subtilisin activity. Inset
depicts slow binding inhibition of subtilisin by low concentrations of
ProD (inverted triangles, 0.45 µM;
filled diamonds, 0.30 µM). B, units
of subtilisin activity recovered by refolding denatured subtilisin E
using ProD (filled circles) and ProWT (open
circles) as peptide chaperones. Refolding was carried out as
described under "Experimental Procedures," and the amount of
denatured subtilisin was maintained at 0.9 µM, whereas
the concentrations of ProD and ProWT were varied from 0 to 36 µM. C, kinetics of trypsin mediated
degradation of ProD (filled circles) and ProWT (open
circles) from their cognate inhibition complexes with
S221C-subtilisin. Amount of residual ProD and ProWT were estimated from
SDS-PAGE using quantitative gel-scanning densitometry as described
under "Experimental Procedures." D, SDS-PAGE analysis of
a trans refolding reaction. Mature subtilisin (band A) was
refolded using ProWT (band C) and ProD (band D)
as described under "Experimental Procedures." The
subtilisin-propeptides (1:20 ratio) (lanes 2 and
5) were dialyzed against refolding buffer. After 16 h,
subtilisin folded by ProWT and ProD (lanes 3 and
6) were treated with trypsin (band B) as
described under "Experimental Procedures." Trypsin degrades the
excess of ProWT and ProD within 1 h (lanes 4 and
7) to give active subtilisin.
1,
respectively. This suggests that in a complex with S221C-subtilisin, ProD is degraded ~3-fold slower than ProWT, which may be
attributed to increased structure in the isolated ProD, higher affinity
for subtilisin, and fewer cleavage sites for trypsin. To examine
whether lower activity recovered by folding mediated by ProD occurs
because of incomplete degradation of ProD, an SDS-PAGE analysis of the refolding reaction before and after digestion by TPCK-treated trypsin
was performed. Fig. 2D establishes that both ProD and ProWT
(at 20-fold molar excess) are completely degraded by 1 h of
trypsin digestion. Moreover, the amount of active subtilisin obtained
after refolding and trypsin treatment of the ProD folding reaction
(Fig. 2D, lane 7) is lower when compared with
that of the ProWT reaction (Fig. 2D, lane 4). It
is also important to note that active subtilisin produced during
folding can also degrade ProD and ProWT. However, allowing the folded
subtilisin to degrade the excess of ProD and ProWT does not
significantly improve the units of activity recovered (data not shown).
Hence, the data presented in Fig. 2D suggest that ProD folds
subtilisin less efficiently than ProWT.
-
conformation (Fig.
3A). Deconvolution of this
spectrum suggests that ProD contains 20%
-helices and 26%
-sheets. Hence, as shown in Equation 1, the equilibrium
(Keq) between the folded (Profolded)
and unfolded states (Prounfolded), which normally favors
the unfolded state in ProWT (5), appears to have shifted toward
the folded state in the case of ProD,
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Fig. 3.
Biophysical characterization of ProD.
A, CD spectroscopy of ProD (thick black) and
ProWT (gray) alone (dotted) and as stoichiometric
complexes (solid) with mature S221C-subtilisin (thin
black line). The difference spectra between
ProD·S221C-subtilisin and mature subtilisin alone give the spectra of
ProD (black dashes) in a complex with subtilisin. The gray
dashed line represents the structure of ProWT when complexed with
S221C-subtilisin obtained using the difference spectra. B,
tryptophan fluorescence spectra of denatured (dotted line)
and folded ProD (dashed line) excited at 295 nm.
Arrows indicate the fluorescence maxima. C,
near-UV CD spectra for denatured (dotted line) and folded
ProD (dashed line). Denatured (thick solid line)
and folded (thin solid line) subtilisin is used as a control
for folded and unfolded protein.
where Keq = kf/ku.
(Eq. 1)
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Fig. 4.
A, ribbon diagram depicting x-ray
structure of the ProWT· subtilisin complex (left) and
the homology model for ProD·subtilisin (right).
B, the electrostatic potential mapped to the molecular
surfaces of the two complexes. Red depicts negative
charges. Blue indicates positive charges. C, the
side chains that contribute to the hydrophobic core in ProWT and ProD.
The extended C terminus interacts with the substrate binding loops to
inhibit subtilisin activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
conformation as an isolated peptide and can form a
stoichiometric complex with mature subtilisin. The CD spectra of these
two complexes are similar and suggest that ProD and ProWT adopt similar
structural scaffolds. Based on our results, we propose that the
chaperoning activity of the propeptide of the subtilisin family appears
to be encoded in the ability to adopt a specific conformation.
-lytic protease (1P02), and carboxypeptidase B (1KWM). Fig.
5A suggests that all three
proteins display similar structural scaffolds despite there being no
sequence and structural similarities between their cognate protease
domains (31). Interestingly, potent subtilisin inhibitors (chymotrypsin inhibitor II (2SNI), streptomyces subtilisin inhibitor (3SSI), and
eglin C (1CSE)) that have no known chaperone function also display
similar structural scaffolds without any sequence similarity (Fig.
5B). Does this structural similarity imply a common
evolutionary domain? In other words, have propeptides emerged from a
primordial protease inhibitor? The conservation of the structural
scaffold in ProD, a chaperone designed to digress from the ProWT
sequence while maintaining function, suggests that the ability to adopt a specific
-
conformation along with certain specific
interactions appears to be crucial for propeptide function. These
functions are to chaperone and to inhibit the protease domain. We
suggest that the structural scaffold may contribute to the inhibitory function and certain residue specific interactions may be important for
its chaperone function. Such specific interactions may be provided by
motifs N1 and N2 in the subtilase family and may vary between families.
Although the structural similarities are striking in Fig. 5,
A and B, it is also possible that they are purely
co-incidental.
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Fig. 5.
The ribbon diagrams for (A)
propeptides of subtilisin, -lytic protease, and carboxypeptidase B
and (B) ProWT, chymotrypsin inhibitor II,
streptomyces subtilisin inhibitor, and eglin C.
The functional conservation in ProD, although having a difference of 5 pH units, suggests that charge per se may not be important for propeptide function. So why is there a large bias for charge in
propeptide domains? Also, if the propeptides within a family adopt
similar structures and perform similar functions, why do the primary
sequences of propeptide diverge faster than their cognate catalytic
domains? One possibility is that the charged N terminus increases the
solubility of the precursor protein. Another possibility is that the
environment in which these proteins are required to become active
influences sequence digressions. For example, subtilisin is secreted
out by B. subtilis for the purpose of scavenging food,
whereas mammalian cells secrete the eukaryotic subtilisin homologue
furin into the trans Golgi network for processing precursor proteins
(32). Although the catalytic domains of bacterial subtilisin and their
eukaryotic counterparts are similar and robustly stable (8), the
propeptides in the above example are removed in dramatically different
environments (4). We propose that the amino acid differences between
their propeptides allow these proteins to respond to different
environments to modulate activation precision of proteases. Another
noteworthy point is that our work raises questions regarding the lower
limit for sequence identity that is acceptable for two proteins to
perform similar functions.
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ACKNOWLEDGEMENT |
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We thank the Medical Research Foundation of Oregon for support.
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FOOTNOTES |
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* 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.
Present address: Laboratoire des Aminoacides Peptides et Proteins,
Faculté de pharmacie, 15 Av Charles Flahault, BP 14491, 34093 Montpellier, France.
§ To whom correspondence should be addressed. Tel.: 503-494-8683; Fax: 503-494-8393; E-mail: shindeu@ohsu.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M212003200
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ABBREVIATIONS |
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The abbreviations used are: ProD, propeptide designed using databases; ProWT, propeptide of subtilisin E; MES, 4-morpholineethane-sulfonic acid; pI, isoelectric point; N-suc-AAPF-pNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; TPCK, n-tosyl-L-phenylalanine chloromethyl ketone.
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