A Pathway for Conformational Diversity in Proteins Mediated by
Intramolecular Chaperones*
Ujwal
Shinde,
Xuan
Fu, and
Masayori
Inouye
From the Department of Biochemistry, Robert Wood Johnson Medical
School, University of Medicine and Dentistry of New Jersey,
Piscataway, New Jersey 08854
 |
ABSTRACT |
Conformational diversity within unique amino acid
sequences is observed in diseases like scrapie and Alzheimer's
disease. The molecular basis of such diversity is unknown. Similar
phenomena occur in subtilisin, a serine protease homologous with
eukaryotic pro-hormone convertases. The subtilisin propeptide functions
as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity. Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed
"protein memory." Here, we show a mechanism that mediates conformational diversity in subtilisin. During maturation, while the
IMC is autocleaved and subsequently degraded by the active site of
subtilisin, enzymatic properties of this site differ significantly before and after cleavage. Although subtilisin folded by
Ile
48
Thr IMC (IMCI-48T) acquires
an "altered" enzymatically active conformation
(SubI-48T) significantly different from wild-type
subtilisin (SubWT), both precursors undergo autocleavage at
similar rates. IMC cleavage initiates conformational changes during
which the IMC continues its chaperoning function subsequent to its
cleavage from subtilisin. Structural imprinting resulting in
conformational diversity originates during this reorganization stage
and is a late folding event catalyzed by autocleavage of the IMC.
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INTRODUCTION |
Subtilisins constitute a family of serine proteases that have
served as model systems for understanding the structural origin of
protease function and specificity (1). Cloning of the gene for
subtilisin E from Bacillus subtilis revealed that it is
synthesized with an N-terminal precursor domain that is referred to as
the propeptide (2). Although several biochemical studies on subtilisin have indicated that the 77-mer propeptide was not part of the 275-amino
acid active protease (3, 4), Escherichia coli expression
experiments revealed that the propeptide of subtilisin was essential
for proper activation and secretion of the protease in vivo
(5-7). Soon thereafter, the propeptide of
-lytic protease (a
serine protease not evolutionarily related to subtilisin) was also
shown to be essential for proper activation of its protease domain (8).
Although these results were surprising at the time, propeptides are now
known to be required for production of a large number of proteases.
Propeptide-dependent systems have since then been
identified both in prokaryotes and eukaryotes (9). Virtually all known
extracellular bacterial proteases have propeptides (10). There are now
examples of serine (5, 11), aspartyl (12), cysteine (13), and
metalloproteases (14) synthesized as precursor pro-proteases.
Propeptides seem to perform two distinct functions: folding and
inhibition of their protease domains (15). In doing so they act as
single turnover enzymes due to their tight binding with folded
proteases and their proteolytic sensitivity. As propeptides facilitate
correct folding they were classified as molecular chaperones (16), but
due to differences that exist between molecular chaperones, propeptides
were also termed "intramolecular chaperones" (17). Although many
proteases require their propeptides to facilitate their folding, these
findings do not constitute a rule. In addition to proteases, proteins
such as growth factors, neuropeptides, hormones, and plasma proteins
also require their propeptides for correct folding (18-21). Studies on
the conformations of propeptides of subtilisin (22-24) and
carboxypeptidase Y (25) have shown them to be randomly structured. In
the vicinity of the prefolded protease domain, the propeptide of
subtilisin adopts an
-
conformation (22-24).
Anfinsen demonstrated that all the information necessary for folding of
a protein into an active conformation resides in the amino acid
sequence of that protein (26). Based on Anfinsen's observation, the
native state of a protein is generally believed to be the global free
energy minimum (27). However, there is increasing evidence that
kinetically selected states play a role in the biological function of
some proteins (28). For example, the serpin plasminogen activator
inhibitor 1 folds into an active structure and then converts slowly to
a more stable, but low activity "latent" conformation (29). Thus,
the folding of plasminogen activator inhibitor 1 is apparently under
kinetic control. When
-lytic protease (11, 30) and subtilisin (31,
32) are folded in the absence of their propeptides, they fold into
partially structured molten globule intermediates. These stable but
inactive intermediates can convert into active conformations upon
addition of their propeptides (30, 31) and suggest that propeptides promote folding of their proteases by direct stabilization of the
rate-limiting folding transition state (30, 31). Propeptides seem to be
essential only during late stages of the folding process because they
help overcome a kinetic block in the folding pathway. A similar state
is formed even in the presence of the propeptide (33), and
autoprocessing may occur in this intermediate state.
Point mutations within the propeptides can affect their folding
function (34, 35). As a result, identical protein sequences can display
altered, enzymatically active conformations with specificity toward the
propeptide that mediates folding (36). This finding helped introduce
the concept termed "protein memory" and suggests that propeptide
domains function as steric chaperones (28, 36). Altered protein folding
also occurs in prion diseases wherein the protein can exist in two
different conformations with invariant amino acid sequences, although
this folding does not require propeptides (37). Therefore, proteins can
display conformationally diverse biologically active states, and the
native state of proteins may not necessarily reside at a thermodynamic
minimum but may be ones that are most easily accessible (28, 36).
In the present manuscript we describe a mechanism that mediates
conformational diversity in subtilisin. Our results show that the
propeptide continues its chaperoning function subsequent to its
cleavage from subtilisin and that structural imprinting that results in
conformational diversity originates during this reorganization stage.
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MATERIALS AND METHODS |
Site-directed Mutagenesis--
The desired mutations
within the propeptide will be introduced through site-directed
mutagenesis using polymerase chain reaction (38). After site-directed
mutagenesis the plasmids were sequenced using an automated sequencer
(Applied Biosystems, Inc., model 310).
Protein Expression and Purification--
The plasmid
(pET11a) carrying mutant and wild-type proteins were expressed in
E. coli strain BL21(DE) that is grown on M9 medium. Propeptides were isolated from inclusion bodies by solubilizing in 15-25 ml of 6 M guanidine HCl (39). After overnight
incubation at 4 °C, the insoluble materials were removed through
centrifugation. The supernatants were then be dialyzed against 50 mM sodium phosphate buffer at pH 5.0. Purification was
carried out using a cation-ion exchange column (CM-Sephadex-50) with a
linear gradient of NaCl (0-0.4 M) followed by high
pressure liquid chromatography (C18-reverse phase) with a linear
acetonitrile gradient (0-65%). The propeptides elute in 43%
acetonitrile and are lyophilized and then resuspended into required
buffers for use in different experiments (39).
Refolding of Proteins--
Purified proteins were dissolved in 6 M guanidine HCl (0.3 mg/ml) and dialyzed step-wise against
a refolding buffer (50 mM Tris-HCl, pH 7.0, 1 mM CaCl2, 0.5 M
(NH4)2SO4) containing decreasing amounts of urea (5 to 0 M).
Estimation of Autoprocessing Activity--
The autocleavage of
the propeptide occurs under conditions in which the refolding buffer
contains 1.0 M urea. Therefore, for estimating autocleavage
efficiency as a function of pH, the precursors were dialyzed in a
step-wise manner to a final concentration of 1.5 M urea.
Proteins were dialyzed extensively, and no autocleavage occurs under
these conditions. Aliquots of the protein were further dialyzed against
refolding buffers at different pHs that contained no urea. After
24 h of dialysis, the proteins were subjected to SDS-polyacrylamide gel electrophoresis, and the extent of autocleavage was estimated densitometrically (40).
Estimation of Enzymatic Activity--
An aliquot of the sample
was incubated at 25 °C in 200 µl of 50 mM buffer at
different pH levels, containing 1 mM CaCl2 and 0.13 mM
succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (41). Enzymatic activity of subtilisin at different pH levels was estimated by monitoring the release of p-nitroaniline through changes in
absorbance at 405 nm. Reading were collected at 15 s using a
Bio-Rad UV microplate reader, and the velocity of reaction was
estimated (36).
Slow Binding Kinetics--
The propeptide of subtilisin behaves
like a slow binding inhibitor. Competitive slow binding inhibition is
usually described using two mechanisms (41).
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(Eq. 1)
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|
(Eq. 2)
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Both mechanisms can be described by the same equation, where
v represent enzymatic rates at time t, and
vo and vs represent initial and steady state enzymatic rates, respectively.
|
(Eq. 3)
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The apparent rate constant k for a first order
reaction has a different significance for a one-step process and is
shown by Equation 4.
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(Eq. 4)
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However, for a two-step process the rate constant is depicted
using Equation 5.
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(Eq. 5)
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Because the initial velocity (vo)
obtained from the on-set progress curves is independent of inhibitor
concentration [I] for subtilisin E (35, 36), the results confirm that
inhibition of subtilisin by its propeptide follows Equation 1.
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(Eq. 6)
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However, if the initial velocity (vo) is
a function of the inhibitor concentration [I], then the inhibition
occurs by a two-step process (35) described by Equation 2.
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(Eq. 7)
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Integration of Equation 1 at a constant substrate concentration
[S] results in the following equation.
|
(Eq. 8)
|
where A0 and A depict the absorbance at 415 nm at
time 0 and t, respectively. Hence vs
and k can be graphically fitted using a nonlinear least
square fitting of the absorbance A versus
t.
|
(Eq. 9)
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Because the propeptide is also a substrate for the reaction,
measurement of the inhibition constants is more complex. Therefore, the
inhibition experiments were carried out under pseudo-first-order kinetics where the lowest propeptide concentrations were greater than
10-fold excess. Enzyme concentrations were set at suitably low levels
to give measurable rates of substrate hydrolysis, with observable state
of inhibitor binding over the steady state time scale. The propeptide
concentrations vary between 1 and 10 µM. Initial
substrate concentration was 3 mM, and less than 10% of the
substrate was degraded throughout the experiment. Reactions were
initiated by addition of SubWT or SubI-48T.
After thorough mixing, substrate cleavage was monitored by recording at
415 nm the release of p-nitroaniline with time (36, 42). Data from each curve were fitted to Equation 8, and the
Ki is estimated by fitting a set of inhibitor
concentrations [I] versus
[vo/vs].
Circular Dichroism Studies--
CD measurements are performed on
an automated AVIV 60DS spectrophotometer fitted with a thermostated
cell holder that is controlled by an on-line temperature control unit.
Quartz rectangular cells (Precision Cells, Hicksville, NY) with a path
length of 1 mm are used. For CD studies, modified subtilisin in 50 mM Tris-HCl, pH 7.0, containing 0.5 M
(NH2)4SO4 and 1 mM
CaCl2 will be used. Solutions are filtered through a
0.22-µm filter before measurement. Scans were carried out at
wavelengths between 260 and 190 nm in a cuvette with a 1-mm path length
maintained at 10 °C. In the thermal unfolding experiments,
temperatures were increased from 10 to 90 °C in 1 °C intervals,
with a 30-s equilibration at each temperature. Data were collected at
each temperature for 10 s. Protein solutions contain 1 mM phenylmethylsulfonyl fluoride to prevent autolysis during this procedure (36).
Fluorescence Studies--
1-Anilino-8-napthalene sulfonic acid
(ANS)1 concentration was
maintained 2.0 µM, whereas protein concentrations were
330 nM. Proteins were excited at a wavelength of 395 nm,
whereas the emission spectra was recorded between 400 and 600 nm
(33).
 |
RESULTS |
The in vitro maturation of the 352-residue
IMC-subtilisin E involves folding, autocleavage, and degradation of its
IMC domain (77-residues) to give mature subtilisin (275-residue). The
folding of pro-subtilisin occurs through a molten globule like
intermediate that becomes compact during or after the autocleavage of
the propeptide domain.
Refolding of Denatured Pro-subtilisin--
Denatured precursors
are refolded through a step-wise dialysis procedure that gradually
reduces urea concentration (see "Materials and Methods"). Fig.
1a depicts CD spectra of
denatured IMCWT-S221C-subtilisin. Extensive dialysis (48 h)
against buffer that contains 1.5 M urea, pH 7.0, induces
significant secondary structure within the uncleaved precursor (Fig.
1a). This precursor binds with ANS (Fig. 1b), a
hydrophobic dye that fluoresces upon interacting with hydrophobic
patches within proteins. Complete removal of urea through dialysis
induces the subtilisin domain to cleave its IMC. The resulting
autocleaved complex (Fig. 1c) was purified using gel
filtration and displays a secondary structure pattern similar to native
subtilisin and fluoresces approximately 8-fold less than the uncleaved
precursor (Fig. 1b). Differences in ANS fluorescence
intensities suggests that a structural reorganization that alters
solvent accessible hydrophobic surfaces within the protein coincides
with autocleavage.

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Fig. 1.
a, folding of
IMCWT-S221C-subtilisin denatured using 6 M
guanidine HCl (green line). The precursor acquires
significant secondary structure after extensive dialysis against 1.5 M urea (magenta line). Upon complete removal of
urea, the precursor begins to cleave its IMC domain. Completely
autocleaved complex (blue line) purified using gel
filtration (43) shows a well defined secondary structure similar to
mature subtilisin (black line). b, fluorescence
due to ANS binding to the protein. IMCWT-S221C-subtilisin
in 5 M urea does not bind ANS (green line). The
autocleaved complex (blue line) binds to ANS approximately
2-fold greater than mature subtilisin (black), whereas the
protein in 1.5 M urea (magenta) binds to ANS by
a factor of 8-fold greater than the autocleaved complex (blue
line). c, left-hand lane represents
IMCWT-S221C-subtilisin in 5 M urea. The protein
was dialyzed using a step-wise gradient. Extensive dialysis against 1.5 M urea (middle lane) does not result in
autocleavage. After 12 h of dialysis against refolding buffer
without urea, the precursor undergoes autocleavage to give
IMCWT-S221C-subtilisin complex (right-hand
lane). d, activity as a function of pH. Open
circles (autocleavage efficiency) and filled circles
(proteolytic activity) represent subtilisin folded using
IMCWT, whereas filled triangles (enzymatic
activity) represent subtilisin folded using IMCI-48T.
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Characterization of Autoprocessing and Degradation Activity of
Pro-Ser221Cys-subtilisin--
A Ser221
Cys
substitution at the active site of subtilisin blocks maturation after
autocleavage, resulting in a stable stoichiometric IMC-S221C-subtilisin
complex (43), whose x-ray structure was recently solved at 2 Å resolution (44, 45). Although active site residues Asp32,
His64, and Ser221 within subtilisin mediate
both autocleavage and degradation (5-8), the Ser221
Cys substitution completely abolishes degradation while allowing efficient autocleavage of its IMC domain.
Fig. 1d displays both the autocatalytic cleavage of
IMCWT-S221C-subtilisin and proteolytic activity of
wild-type subtilisin as functions of pH. The procedure used to estimate
the autoprocessing efficiencies is described under "Materials and
Methods." Maximum autocleavage occurs at pH 7.0, whereas optimum
proteolytic activity is observed at pH 8.5. Although
IMCWT-subtilisin also displays an optimum pH of 7.0 (data
not shown), IMCWT-S221C-subtilisin is used because it
allows an accurate estimation of autocleavage in the absence of any
degradation of the cleaved IMC. Substitutions at position 221 by Cys or
Ala does not alter the Km of subtilisin (46, 47).
Because S221C-subtilisin only autocleaves but does not degrade its IMC
domain, these results suggest that although mediated via the active
site of subtilisin, autocleavage and degradation activities of this
catalytic triad differ significantly before and after cleavage.
Differences in optimum pH substantiate that autocleavage and
degradation activities are different from each other.
Mapping the Effect of the Propeptide Mutation on the Maturation
Pathway--
IMCI-48V-subtilisin affects the folding
process, and although the precursor undergoes maturation, the resulting
conformation of the protease domain (SubI-48V) differs from
that SubWT. Because autocleavage and degradation represent
two activities of the same active site, effects of IMC mutations on
these activities may help elucidate the molecular basis of altered
folding. SubI-48V retains significant enzymatic activity,
and hence the mutation Ile
48
Thr was isolated in an
attempt to further reduce proteolytic activity. Crystallographic
studies of the IMC-subtilisin complex (44, 45) shows that
Ile
48 is surrounded by hydrophobic side chains (Fig.
2a). Theronine, a polar
residue, may disrupt the hydrophobic core and augment the phenomenon of
altered folding. Upon renaturation,
IMCI-48T-S221C-subtilisin cleaves its IMC domain (Fig.
2b) to form a complex that is further purified using gel
filtration. Similarly, IMCI-48T-subtilisin undergoes
maturation forming mature SubI-48T (Fig.
3b) with a well defined
secondary structure (Fig. 2c). The purified autocleaved
complexes IMCWT and IMCI-48T-S221C-subtilisin
interact differently with ANS, with
IMCI-48T-S221C-subtilisin exhibiting a 4-fold greater
fluorescence intensity than IMCWT-S221C-subtilisin complex.
Similarly, SubI-48T fluoresces with an magnitude
approximately 2-fold of that of SubWT (Fig. 2d).
These results suggest that subtilisin domains folded by the
IMCI-48T display exposed hydrophobic surfaces that are
absent in domains folded using IMCWT. Far ultraviolet CD
spectra indicate that SubI-48T and SubWT are
well folded and have similar but not identical conformations (Fig.
2c). The pH profile of the SubI-48T is depicted
in Fig. 1d, and it displays maximum proteolytic activity at
approximately pH 8.2, whereas autoprocessing is maximum at pH
7.0.

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Fig. 2.
a, hydrophobic core within the IMC
domain is shown in green. The Ile 48 is
displayed in yellow. b, IMCI-48T-
S221C-subtilisin and IMCI-48T-subtilisin before (5 M urea) and after refolding (buffer contains 10 mM Tris-HCl, pH 7.0, containing 0.5 M
(NH4)2SO4, 1 mM
CaCl2). c, autocleaved
IMCI-48T-S221C-subtilisin complex purified using gel
filtration (red line) compared with autocleaved
IMCWT-S221C-subtilisin purified complex (blue
line) and mature SubI-48T (black line) and
mature SubWT (cyan line). d,
fluorescence due to ANS binding. Autocleaved
IMCI-48T-S221C-subtilisin (magenta line)
displays greater fluorescence than IMCWT-S221C-subtilisin
purified complex (blue line). Similarly,
SubI-48T (black line) and mature
SubWT (cyan line) differ in florescence
intensities.
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Fig. 3.
a, thermal unfolding of precursor and
mature proteins monitored using changes in negative ellipticity at 222 nm. Unfolding spectra of SubWT (black line) and
SubI-48T (cyan line) displays higher melting
temperatures (Tm) than autocleaved precursors
IMCWT-S221C-subtilisin (blue line) and
IMCI-48T-S221Csubtilisin (red line),
respectively. b, comparison of the inhibition profiles of
SubWT and SubI-48T by IMCWT and
IMCI-48T. Concentrations of SubWT and
SubI-48T were adjusted such that they degrade the
substrates at similar velocities. Substrate concentration was
maintained at 4 mM, whereas the IMCWT and
IMCI-48T were maintained at 6.0 and 25 µM,
respectively. SubWT and SubI-48T alone are
shown in black, SubI-48T binding to
IMCI-48T is depicted in red, and
SubWT interacting with IMCI-48T is displayed in
green. Blue represents IMCWT
interacting with SubWT, and the gray line
indicates IMCWT binding to SubI-48T.
c, a comparison of autocleavage of
IMCWT-S221C-subtilisin (blue line) and
IMCI-48T-S221C-subtilisin (red line) and a
function of time. The amount of precursor autocleaved was estimated
densitometrically from SDS-polyacrylamide gel. d, a
comparison of N-succinyl-A-A-P-F-p-nitroanilide
degradation monitored by changes in absorbance at 405 nm due to release
of p-nitroaniline. Profiles due to SubWT are
shown by in blue, whereas SubI-48T is shown in
red.
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Thermostability of Autoprocessed Complexes--
Thermostabilities
of mature protease domains SubWT and SubI-48T
and the corresponding autocleaved complexes were determined by
monitoring changes in negative ellipticity at 222 nm (Fig.
3a). The autoprocessed complexes used in these experiments
were separated from the unautoprocessed precursors using gel filtration
chromatography as described earlier (41). SubI-48T
(Tm 49.5 °C) was found to be substantially
more unstable than SubWT (Tm
58.5 °C), whereas the IMCI-48T-S221C-subtilisin
autocleaved complex (Tm 46.8 °C) was also
more unstable than the IMCWT-S221C-subtilisin complex
(Tm 53.2 °C). Both protease domains complexed
with the IMC domains melt at lower temperatures than their
corresponding protease domains. This suggests that the presence of IMC
domains destabilize the corresponding complexes and conversely that the
degradation of the IMC domains enhances stability of cognate protease
domains. It is important to note that phenylmethylsulfonyl fluoride
covalently interacts with the active site Ser221 residue.
Crystallographic results have shown that such covalent linkage may
cause local perturbations but does not seem to affect the rest of the
backbone. The introduction of an bulky residue into the active site may
therefore slightly destabilize the mature protein, causing it to lower
the Tm of the mature protease. Therefore the
difference between the Tm of subtilisin and the
complex with its propeptide may actually be greater.
Characterization of the Binding of Propeptide with Mature
Subtilisin--
Fig. 3b describes the IMCI-48T
and IMCWT binding with SubWT and
SubI-48T. From these curves it is evident that
IMCI-48T binds to SubI-48T more strongly (and
is degraded more quickly) than with SubWT, suggesting more
specific recognition of SubI-48T than SubWT by
IMCI-48T. Rates of autocleavage of the IMCI-48T
and IMCWT-S221C-subtilisin precursors are estimated as
described under "Materials and Methods." Relative rates of
autocleavage of the two precursor proteins appear similar (Fig.
3c), whereas proteolytic activities of SubWT and
SubI-48T toward a peptide substrate
N-succinyl-A-A-P-F-p-nitroanilide differ
significantly (Fig. 3d). Enzymatic activities of
SubWT and SubI-48T were estimated as described
earlier (12, 26), and SubI-48T displays a
Km of 0.24 × 10
3 M,
whereas SubWT displays a Km of 2.0 × 10
3 M. Because autocleavage occurs at
similar rates whereas the proteolytic specific activity of
SubI-48T is only 9.0% of that of SubWT, the
results indicate that Ile
48
Thr substitution seems to
selectively affect proteolytic activity (final conformation) but not
autocleavage of the maturation intermediate.
 |
DISCUSSION |
Based on the above results a mechanism that leads to altered
folding is proposed (Fig. 4). It is
unclear how the IMC initiates this folding process, but it has been
speculated that two helices between residues 100 and 144 within the
protease domain may be involved (44, 45). The IMC domain facilitates
the precursors adopt an intermediate state that is capable of
autocleavage, a process mediated by the active site of subtilisin
(5-8). The properties of this active site differ from that of the
mature protease because (i) it displays an optimum pH that is 1.5 units less than the mature protein; (ii) IMC-S221C-subtilisin can only autocleave its IMC but cannot degrade it to release mature
S221C-subtilisin; and (iii) both IMCI-48T and
IMCWT uncleaved precursors bind strongly with ANS, whereas
the autocleaved complexes do not. This is consistent with structural
reorganization that occurs after proteolytic cleavage (33).
IMCI-48T and IMCWT S221C-subtilisin both cleave
their IMC domains at similar rates, indicating that the
Ile
48
Thr substitution has little effect on
autocleavage. Because SubWT and SubI-48T differ
in their enzymatic activity after cleavage and subsequent degradation,
we conclude that altered folding and protein memory that result from
IMC-mutations occurs after autocleavage, after which the IMC continues
to functions as a chaperone. Therefore, autocleavage does not imply
that the folding process has been completed and may represent the
transition state of the folding reaction. Autocleavage and degradation
are closely coupled in wild-type subtilisin, and occurrence of these
two processes renders the folding process irreversible.

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Fig. 4.
Schematic representation of folding mediated
by the IMC domain. Red depicts the IMC domain, whereas
orange represents the protease domain. Green
represents mutation within the IMC domain.
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|
Although proteolytic activity of subtilisin at pH 8.5 is approximately
2-fold greater than pH 7.0 (Fig. 1d), incubation of the
IMCWT-S221C-subtilisin complex at pH 8.5 does not
facilitate degradation of the IMC domain (data not shown). Changes in
the optimum pH alone cannot explain the absence of proteolytic activity
toward the noncovalently bound IMC domain. The protease domain in the autocleaved complex is almost identical to the wild-type protease domain with the exception of the active site Ser221
Cys
substitution that causes a 10,000-fold drop in the proteolytic activity. Because uncleaved precursors display exposed hydrophobic surfaces relative to cleaved complexes, it is reasonable to speculate that distribution of charge on cleaved and uncleaved protein surfaces are different. It is known that altering surface charges through mutagenesis produces subtilisin with altered specificities and pH
activity profiles with enhanced catalytic activities (48, 49).
Therefore, the active site within uncleaved precursor may also display
enhanced autocleavage activity. After cleavage, the resulting
conformational changes alter the catalytic properties of this active
site. Because Ile
48
Thr substitution within the IMC
does not affect cleavage but alters enzymatic activity of the protease
domain, our results indicate that conformational diversity that leads
to protein memory occurs late during the folding pathway and the IMC
functions even after its autocleavage.
A sequence alignment of propeptides from the subtilisin family shows an
interesting observation. Although the overall sequence identity is low,
two regions dispersed over the N and C termini display significant
sequence conservation (Fig. 5). These
regions designated as motifs N1 and N2 contain the hydrophobic core
residues (Val12, Phe14, Ile30,
Val37, Leu51, Val56,
Leu59, Val65, and Val68) within the
subtilisin propeptide, apart from Ile30. We speculate that
motifs N1 and N2 may be critical for nucleation of the folding process,
whereas the nonconserved segments between N1 and N2 may be crucial for
specific interactions with their cognate protease domains, during the
propagation of folding and subsequent inhibition of activity. This
speculation is supported by four of our findings: (i) A substitution at
position Ile30 by a Val allows the propeptide to exert its
chaperoning function but alters the final conformation of the protein
(36). This Ile30 residue is located in the nonconserved
region flanked by motifs N1 and N2 (note that Ile30 was
previously termed Ile
48). (ii) By using an
Ile30
Thr substitution, we have shown that the
structural imprinting, which results in protein memory, occurs late in
the folding pathway, after the propeptide has been cleaved from the
protease domain through autocatalysis. Therefore, a substitution at
position 30 within the propeptide does not affect folding nucleation
but dramatically alters the final conformation of the protein. (iii)
Results of a random mutagenesis procedure that helped identify
mutational hot spots within the propeptide show that substitutions that
blocked folding of subtilisin are localized within motifs N1 and N2
(34). (iv) NMR spectroscopy on the propeptide of subtilisin E shows that residues located within motifs N1 and N2 display residue-specific conformational rigidity,2 a
criteria that classifies a polypeptide segment as a potential nucleation site in a protein folding reaction.

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Fig. 5.
Sequence alignments of various IMC domains
showing conserved motifs N1 and N2 flanking a variable region.
Bold letters represent conserved residues.
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ACKNOWLEDGEMENTS |
We thank Drs. S. Phadatare and K. Madura for
comments and suggestions, J. Liu for technical help, and Yuyun Li for
cloning IMCI-48T-S221C-subtilisin in pET11a.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Robert Wood Johnson Medical School-UMDNJ, 675 Hoes Ln., Piscataway, NJ
08854. Tel.: 732-235-4115; Fax: 732-235-4559; E-mail: inouye{at}rwja.umdnj.edu.
2
U. Shinde, A. Buevich, X. Wang, M. Inouye, and
J. Baum, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
ANS, 1-anilino-8-napthalene sulfonic acid;
IMC, intramolecular
chaperone.
 |
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