From the Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118-2526
Received for publication, October 11, 2002, and in revised form, November 10, 2002
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ABSTRACT |
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Glycosylasparaginase (GA) is an amidase and
belongs to a novel family of N-terminal nucleophile hydrolases that use
a similar autoproteolytic processing mechanism to generate a
mature/active enzyme from a single chain protein precursor. From
bacteria to eukaryotes, GAs are conserved in primary sequences,
tertiary structures, and activation of amidase activity by
intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr
sequence is cleaved to generate a newly exposed N-terminal threonine,
which plays a central role in both autoproteolysis and in its amidase
activity. We have recently determined the crystal structure of the
bacterial GA precursor at 1.9-Å resolution, which reveals a highly
distorted and energetically unfavorable conformation at the scissile
peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not
understood how the polypeptide chain distortion was generated and
preserved during the folding of GA to trigger autoproteolysis. An
obstacle to our understanding of GA autoproteolysis is the uncertainty
concerning its quaternary structure in solution. Here we have revisited
this question and show that GA forms dimers in solution. Mutants with
alterations at the dimer interface cannot form dimers and are impaired
in the autoproteolytic activation. This suggests that dimerization of
GA plays an essential role in autoproteolysis to activate the amidase
activity. Comparison of the melting temperatures of GA dimers before
and after autoproteolysis suggests two states of dimerization in the
process of enzyme maturation. A two-step dimerization mechanism to
trigger autoproteolysis is proposed to accommodate the data presented
here as well as those in the literature.
Glycosylasparaginase
(GA)1 is an amidase involved
in Asn-linked glycoprotein degradation (1). A deficiency in the human GA leads to severe clinical symptoms, known as aspartylglycosaminuria (2). GA is widely distributed in vertebrate tissues (3), insect cells
(4), and in bacteria (5). GA joins the proteasome and penicillin
acylase as a novel class of enzymes, called N-terminal nucleophile
(Ntn) hydrolases that catalytically use a processed N-terminal
threonine, serine, or cysteine as both a polarizing base and a
nucleophile (6). The crystal structures of human and bacterial GA show
a similar structural frame of an Crystal structures of GA precursor have provided us a structural basis
to propose a detailed mechanism of intramolecular autoproteolysis (15).
The GA precursor undergoes a two-step mechanism to break up the
polypeptide chain, through an N-O acyl shift and an ester intermediate.
In the first step, the nucleophile Thr-152 is activated by a base
Asp-151 that is held in an unusual but precise geometry by Thr-203 to
deprotonate the nucleophile Thr-152. After the nucleophilic attack, a
transitional tetrahedral anion intermediate is stabilized by a polar
group of Thr-170 that could further be polarized to carry positive
charge by Arg-180, possibly through a bound solvent molecule. Collapse
of the tetrahedral anion intermediate shifts the linkage from an amide
bond to an ester bond (N-O acyl shift). Usually the equilibrium of N-O
acyl shift favors the amide bond, resulting in a peptide bond that does
not break often at threonine, serine, or cysteine. However, the
strained, tight turn conformation at the scissile peptide bond observed
in the GA precursor structures could drive the equilibrium toward an
ester bond formation (15). The second step of autoproteolysis is a
simple hydrolysis of the ester intermediate by a neighboring water
molecule, resulting in an active amidase with It is not understood how the polypeptide chain distortion to trigger
autoproteolysis is generated and why these local constraints are not
removed during the folding of the GA precursor. It seems plausible that
the structural constraints are generated only at a late stage of
protein folding, for example, during the formation of the native
quaternary structure. Dimerization of GA precursors has been suggested
to be a prerequisite to trigger autoproteolysis (8, 17, 18). However,
the native quaternary structure of GA was controversial. Among several
crystal forms, the same dimer structure is observed for the human and
bacterial GA, either as precursors or autoproteolyzed enzymes (7-9,
15). However, in solution, GA was reported as monomeric for mammalian
and bacterial enzymes (3, 9, 11, 19), whereas others reported dimeric GA for the human (17, 20), chicken (3), and insect enzymes (4).
This study was designed to re-examine the native quaternary structure
of GA in solution and to study the significance of the quaternary
structure in the intramolecular autoproteolysis that is essential to
activate the amidase activity for glycoprotein degradation. By using
biochemical and biophysical methods, we confirm that GA exists as
dimers in solution. Structure-based mutants designed to disrupt the
dimer interface lose their ability to form dimers and also lose their
ability for autoproteolytic activation of amidase activity.
Protein Purification--
Proteins were expressed and purified
by existing procedures (21), with some modifications. The expression of
maltose-binding protein (MBP)-GA fusion proteins was induced in the TB1
cells by adding 1 mM
isopropyl-1-thio- Gel Filtration--
A 200-µl volume, 20 µM each
of the GA or MBP fusion proteins, was applied to an Amersham
Biosciences HiPrep 16/60 Sephacryl S-200 HR gel filtration column,
equilibrated in a solution containing 10 mM
potassium-phosphate buffer, pH 7.4, and 1 mM EDTA.
According to the manufacturer, this column effectively separates
globular proteins in the molecular mass range of 5-250 kDa. The gel
filtration standards, a mixture of five molecular weight markers, were
purchased from Bio-Rad: bovine thyroglobulin (670 kDa), bovine
Cross-linking with Glutaraldehyde and DMS--
Cross-linking was
performed at 0.1-0.2 mg/ml (1.3-6.2 µM) protein and
0.0005-1% glutaraldehyde in 10 mM potassium-phosphate buffer, pH 7.4, and incubation for 6 h at room temperature.
Reactions were quenched with 0.15 volumes of 0.5 M glycine.
Cross-linked protein samples were denatured in 1% SDS, 1%
Sucrose Density Gradient Sedimentation--
Sedimentation was
performed using a modified version of the method described by Martin
and Ames (23). Linear gradients of 10-30% sucrose in 10 mM potassium-phosphate buffer, pH 7.4, were equilibrated at
4 °C for 2-4 h prior to use. Protein was diluted in the same buffer
to 30 µM, preincubated for 30 min at 4 °C, and then
layered on the gradients using a 20-gauge needle. Gradients were
centrifuged 40,000 rpm for 12 h in a Beckman SW55 Ti rotor. Gradients were withdrawn from the top into a series of 100-µl fractions. Protein peaks in the gradient were determined by Bradford assays of each fraction with reading of absorbance at 595 nm.
Site-directed Mutagenesis--
DNA manipulation and
site-directed mutagenesis (Kunkel methods) were carried out as
described previously (11). Wild-type Flavobacterium
glycosylasparaginase coding sequence was cloned into either the pMAL
system or the Litmus system for site-directed mutagenesis, following
the manufacturer's protocols (New England Biolabs). DNA clones with
desired mutations were confirmed by sequence analyses using an ABI
automatic DNA sequencer.
CD Spectrum Determination--
CD spectra were recorded from 185 to 250 nm using an AVIV 62DS spectrometer equipped with a
thermoelectric sample temperature controller, with a 1-mm path length
cuvette. Protein concentrations were 2-5 µM in 10 mM potassium-phosphate buffer, pH 7.4, and 1 mM
EDTA and were determined by absorbance measurements in 6 M guanidine hydrochloride (22). The spectra were collected at 25 °C as
the average of 10 scans, using a 15-s integration time at 1.0 nm
wavelength increments. Following the buffer base-line correction, the
spectra were normalized to the protein concentration and are expressed
as molar ellipticity.
Thermal Denaturation--
Thermal unfolding curves were recorded
at 222 nm upon heating from 5 to 95 °C at a rate of 60 °C/h, with
a 1 °C increment, 30-s accumulation time. The melting temperature
(Tm) was determined from the differential melting
curves d[ GA Eluted as a Single Peak from Gel Filtration
Column--
Previous reports concerning the native GA quaternary
structure drew conclusions mainly based on the apparent molecular
masses on sizing chromatography or native gel electrophoresis, often using samples from cell extracts and visualizing a trace amount of GA
by Western blot methods. Contradicting observations were reported from
different groups; some concluded GA monomers (3, 9, 11, 19),
whereas others reported dimers for GA precursor or autoproteolyzed GA
(3, 4, 17, 20). These contradictory observations have prompted us to
re-investigate the quaternary structure of GA more closely using
purified precursors and the autoproteolyzed enzyme of
Flavobacterium GA.
Wild-type GA spontaneously autoproteolyzes in cells into the mature
form amidase with two (
The purified GA proteins were first examined by chromatography on a gel
filtration column. Because the native autoproteolyzed amidase still has
the two newly generated ( MBP-GA Fusion Proteins Behave More Like Dimers on Gel Filtration
Column--
In an attempt to demonstrate the sensitivity of gel
filtration chromatography to the shape of proteins, we re-examined the behavior of GA fusion proteins on the gel filtration column. We reasoned that attaching an MBP to the N-terminal end of GA protein would change the shape of a monomer from a quite compact GA structure to an irregular shape of a fusion protein. Therefore, if existing as a
single species in solution, these fusion proteins might behave differently from GA protein alone and thus provide better resolution to
distinguish monomers from dimers. Otherwise, if GA dimers are in
equilibrium with monomers, the fusion proteins would still be eluted as
single peaks with apparent molecular masses of about 1.4-fold of a monomer.
Fig. 1B shows the gel filtration chromatograms for purified
MBP-GA fusion proteins, before and after the GA domain was
autoproteolyzed. As a control, MBP protein alone was eluted as a single
peak with an apparent molecular mass of 25 kDa, which is smaller than
that calculated for a monomer of 43 kDa. Nonetheless, it indicates that
MBP does not dimerize in solution. For a monomeric MBP-GA fusion
protein, either as a precursor or an autoproteolyzed amidase, the
calculated molecular mass is 75 kDa. The MBP-T152A (GA precursor) fusion protein was eluted as a single peak, with an apparent molecular mass of 138 kDa. Likewise, MBP fusion with T152C mature GA (MBP-T152Cm) was eluted with an apparent molecular mass of 138 kDa that is 1.8-fold
of the calculated molecular mass for a monomer. This shift of molecular
mass from 1.4- to 1.8-fold of a monomer indicates that the unusual
mobility of GA is not a result of fast equilibrium. Thus, considering
that MBP alone was eluted at 0.6-fold of its calculated molecular
weight, the MBP-GA fusion proteins eluted at 1.8-fold of the calculated
molecular masses are more likely to be dimers than monomers. These
results also suggest that the dimerization of fusion proteins is
mediated by the GA domain and not the MBP domain.
GA Dimers Are Confirmed by Cross-linking Experiments--
To
confirm GA dimers in solution, we performed cross-linking with
glutaraldehyde and DMS. Cross-linking of T152A precursor with
0.001-1% glutaraldehyde (amine-amine cross-linking) at room temperature and 0.1 mg/ml (3.1 µM) protein indicated that
the GA precursor was dimeric under these conditions (Fig.
2). Following SDS-PAGE, the T152A
precursor yielded a characteristic pattern of two bands corresponding
to cross-linked monomer and dimer (Fig. 2A), whereas an
uncross-linked control (i.e. without glutaraldehyde treatment) gave a single band corresponding to a monomer. Similar cross-linking results were obtained with autoproteolyzed enzyme of
wild-type GA (Fig. 2B), although the pattern is more
complicated because there are two subunits, Disruption of the Dimer Interface Prevents Both Autoproteolysis and
Dimer Formation--
To examine the significance of dimerization in
autoproteolysis, we designed and generated mutants that would disrupt
dimer interactions without affecting protein folding. With the aid of the crystal structures of the GA precursor protein (15) and autoproteolyzed amidase (8), we identified several targets at the
dimerization interface for mutagenesis, hoping to weaken or disrupt the
dimer formation (Fig. 3A). In
the dimers, Val-105 is sequestered out of the solvent to form
hydrophobic contacts with its equivalent from the other monomer.
Similarly, Ile-186 from each monomer interacts with each other at the
dimer interface. In addition, conserved residue Glu-220 forms charge
interactions with conserved residue Arg-238 of the adjacent monomer.
All of these residues point away from the structural core of the
monomer and thus are unlikely to disturb protein packing of each
monomer. Furthermore, because these four residues are located on the
opposite side of the scissile peptide bond (Fig. 3A), they
are not likely to be involved directly in the autoproteolysis.
Therefore, replacement of each of these residues was hypothesized to
destabilize dimer formation but not to affect the folding of the
monomer or to change active site residues.
Four dimer interface mutants were generated to study the effect of
mutation at the dimer interface, V105K, I186D, E220K, and R238D. These
mutant proteins were also expressed as MBP fusion proteins by similar
protocol published previously (11) for the wild-type and other GA
mutants. Purification of GA interface mutants alone proved to be
difficult because they were unstable and very sensitive to protease
degradation during the process of protein purification, particularly
after factor Xa digestion to separate GA mutants from the MBP domain.
However, when purified as MBP fusion proteins, these mutants were more
stable and thus allowed us to obtain sufficient amounts of proteins for
further characterization. Nonetheless, there are still a few minor
degradation bands that were eluted together with the MBP-GA full-length
protein from a maltose-affinity column (Fig. 3B, lanes
11-18). This indicates these minor degradation products may still
retain an intact MBP domain but with a degraded GA mutant domain. This
is in contrast to the wild-type GA or other active site mutants that
are very stable by the same expression and purification procedure. As
shown in Fig. 3B, wild-type GA spontaneously autoproteolyzes
into
GA retains the same autoproteolytic activity when purified as MBP
fusion proteins. MBP-T152C fusion protein partially autoproteolyzes into MBP-
Fusion proteins of all four dimer interface mutants are sensitive to
degradation during the process of protein purification, but all lose
their autoproteolytic activity. The observed degradation products are
likely to be clipped at the GA domain because they still can bind to
the maltose-affinity column. In addition to affecting the stability of
the GA domain, mutations at the dimer interface also disrupt
autoproteolytic activity of GA. Unlike the MBP-T152C active site
mutant, addition of hydroxylamine does not increase the level of
autoproteolysis for any of the dimer interface mutants. The V105K
mutant completely loses its autoproteolytic activity in the absence or
presence of hydroxylamine (Fig. 3B, lanes 11 and
12). Three other mutants were proteolyzed into two bands
that appear to be close to the autoproteolytic products (MBP- Cross-linking Detects No Dimer but Oligomers of Dimer Interface
Mutants--
Because the mutation sites described above were
specifically chosen at the dimer interface, we performed further
biochemical assays to assess if these point mutations cause changes in
quaternary structure. Cross-linking of MBP fusion proteins indicated
that the dimer interface mutations do not form dimers. As a control experiment (Fig. 4A), a fusion
protein of MBP-T152A precursor was converted from a monomer band, with
an increasing amount of glutaraldehyde, into a new band corresponding
to cross-linked dimer, whereas only an uncross-linked band of monomer
was observed without glutaraldehyde. In a second control experiment, no
cross-linked dimers were observed for the MBP protein itself (Fig.
4B), indicating that the MBP domain does not dimerize in
such conditions. Under the same conditions of cross-linking
experiments, MBP fusion protein of V105K mutant did not yield a band
corresponding to cross-linked dimers. Instead, a significant increase
of large size oligomers was retained at the top of gel with a decreased
intensity of the monomer band when the concentration of cross-linking
reagent was increased (Fig. 4C). Disappearance of the minor
degradation bands at high concentration of cross-linking reagent
suggests that these degradation products might also exist as oligomers
in solution. Similarly, cross-linking of the MBP-I186D mutant also
indicated that this mutant tends to form large size oligomers in
solutions (Fig. 4D).
Large Size Oligomers Confirmed by Gel Filtration and Sucrose
Density Gradient--
Large size oligomers of dimer interface mutants
were confirmed by gel filtration, to verify that the cross-linking
truly reflected the existence of large size oligomers in solution and
was not due to interoligomeric cross-linking. As a control, MBP-T152A precursor was eluted at a volume corresponding to 138 kDa, which approximates the predicted molecular mass of a dimer (Fig.
5A, also see Fig.
1B). However, MBP-I186D mutant was eluted at the peak of the
void volume, representing oligomers with sizes of at least several
hundred kDa. Similar results were observed for other dimer interface
mutants (data not shown).
Sucrose gradient experiments further confirmed the large size oligomers
observed in cross-linking and gel filtration (Fig. 5B). At
4 °C, MBP-T152A (75 kDa) precursor yielded a major peak between two
reference standards, bovine serum albumin (66 kDa) and Mutant Oligomers Show Secondary Structure Content Close to the
Dimers--
To investigate structural features of the large size
oligomers observed above, MBP fusion proteins of the dimer interface mutants were analyzed by far-UV CD, and the results were compared with
those of the MBP-T152A precursor and MBP-T152Cm mature enzyme (Fig.
6A). The CD
spectra showed that the dimer interface mutants retain most of the
secondary structure when compared with the dimers of MBP-T152A
precursor or MBP-T152Cm mature enzyme. The CD spectrum of the single
chain precursor MBP-T152A closely superimposed that of the
autoproteolyzed, two-subunit complex of MBP-T152Cm mature enzyme. This
is consistent with crystal structures that revealed essentially
identical secondary structure contents in both GA forms (8, 15).
Interestingly, the CD spectra also showed that the large size oligomers
of dimer-disruptive mutants retain substantial amount of intensity from
205 to 250 nm when compared with that of MBP-T152A dimer or dimer of
MBP-T152Cm mature enzymes. The largest difference of intensity is
around 222 nm where the dimer-disruptive mutants still showed 83%
intensity of the native dimers. These results suggest that these high
molecular weight oligomers are not denatured protein aggregates.
Instead, they could represent higher order oligomeric species of
properly folded GA monomers. The differences in CD signals between GA
dimers and oligomers of dimer interface mutants might be lessened if the concentrations of the latter were adjusted for the co-purified degradation products (see above and Fig. 3B). Furthermore,
the small difference in CD spectra may have also resulted from
quaternary and not tertiary structural changes in each monomer (see
below).
Differences in the Thermal Stability between Oligomers or Dimers of
Precursor and Autoproteolyzed GA--
To study thermal stability of
dimers and high molecular weight oligomers of the dimer disruptive
mutants, the heat unfolding of various GA mutants and MBP fusion
proteins was monitored by far-UV CD at 222 nm (Fig. 6, B and
C). In neutral 10 mM phosphate buffer solutions
containing 2 µM protein, the MBP-T152A precursor unfolds
from about 55-60 °C with Tm = 57.4 ± 0.5 °C, whereas the MBP-T152Cm autoproteolyzed protein unfolds from
about 58-63 °C with Tm = 60.3 ± 0.5 °C
(Fig. 6B), which is 2.9 °C (or 6 times the standard
errors) higher than Tm of the MBP-T152A precursor.
Interestingly, both MBP-V105K and MBP-I186D mutants unfold with melting
temperature ranges and Tm values almost identical to
that of the MBP-T152A precursor, even though these dimer interface
mutants are predominantly high molecular weight oligomers. Furthermore,
the widths of the single Gaussian function of the differential melting
curves d[ The data presented here clearly show that GA dimerizes in
solution. Gel filtration experiments of MBP-GA fusion proteins
suggested that GA, either as a single chain precursor or as an
autoproteolyzed amidase, forms dimers in solution. Dimerization was
further confirmed by cross-linking experiments. Site-directed mutations
at the assumed dimer interface resulted in proteins in which the
secondary structure was largely intact but in which the quaternary
structure was changed, disrupting dimer formation but forming high
molecular weight oligomers.
Previous Reports Reconciled--
The contradictory conclusions in
previous reports can be attributed to the relatively low resolution of
the methods used to separate dimer from monomer, gel filtration
chromatography or native gel electrophoresis. By using gel filtration
chromatography, GA dimers happen to be eluted in the chromatograms
right at the middle between expected fractions of dimers and monomers.
In a similar experiment (17), reported dimers of human GA had also been
eluted in fractions with much smaller apparent molecular weight (eluted
at a 60-kDa fraction whereas the human monomer is 42 kDa). In fact, a
single peak eluted at 1.5-fold of monomer size was concluded to be a
monomer in one report (11), whereas a similar abnormal elution volume
was concluded to be a dimer for human enzyme (17). These results all
seem to be due to the compact and non-elongated shape of a GA dimer
revealed in crystal structures. In addition to molecular shape and
size, the results of sizing chromatography are sensitive to other
experimental conditions such as matrix composition, pH, and ionic
strength. Interestingly, a dimer interface mutation of human GA
precursor (I240D in Ref. 17) equivalent to the bacterial I186D mutant
was also found to remain in the precursor form. In the same report
(17), a small amount of the precursor of the human dimer interface
mutant was found by immunoprecipitation to be eluted in the monomeric but not the dimeric fractions. However, that study did not look into
the fractions that could contain large size oligomers of the mutated
precursor, as did the bacterial I186D mutant in this study.
Although the dimerization appears to be important for autoproteolysis,
the autoproteolysis still could be a first-order reaction. One argument
against the significance of GA dimerization in autoproteolysis was that
the autoproteolytic reaction follows first-order kinetics (11). This
could be reconciled if the dimerization is a prerequisite, but not the
rate-limiting, step of the intramolecular autoproteolysis (see below).
Because precursors exist predominantly as dimers, dimerization does not
appear to be the rate-limiting step of autoproteolysis. Instead, the
rate-limiting step appears to be the breaking of the scissile peptide
bond (to relieve the conformational constraints). Thus, autoproteolysis
would follow a first-order reaction because the concentration of dimer
is proportional to the concentration of precursor proteins.
The crystallographic observations are also consistent with our
conclusion that GA exists predominantly as dimers in solution. The
bacterial GA precursor and autoproteolyzed amidase crystallize in
different crystal forms but share the same dimer structure (8, 15, 21).
Furthermore, the same dimer structure was also observed for the human
GA (7) crystallized in different crystal forms. All of these results
are consistent with the notion that this dimer structure is of
biological significance. Furthermore, in the crystal structures, the
surface interactions in the dimer formation are extensive and mainly
involve hydrophobic contacts and hydrogen bonds. Basically, both
monomers use the same hydrophobic surface for the dimer formation,
reminiscent of hand-shaking. Unless there is a large conformational
change as a result of dimerization, it is not likely that the extensive
hydrophobic dimer interface observed in the crystals (2485 and 1882 Å2 from each monomer of human and bacterial GA,
respectively) would be exposed to the solvent as it would in a monomer.
The compact and near globular shape of the dimer structure can also
explain why the GA behaves differently from the protein size standards that are more irregular in shape.
Prerequisite of Dimerization in Autoproteolysis--
A correlation
between disruption of dimerization and loss of autoproteolysis
indicates that GA dimerization plays an essential role in the
intramolecular autoproteolysis. Based on the crystal structures,
calculations of the solvent exposure of Val-105, Ile-186, Glu-220, and
Arg-238 side chains are 0.8, 0, 4.5, and 21% solvated, respectively,
in the dimer versus 57, 33, 44, and 48%, respectively, in
the monomer, indicating that these residues are largely buried in the
dimer but would be considerably exposed as a monomer. Thus substitutions at these positions are likely to affect the dimerization and not the secondary and presumably the tertiary structure of a
monomer. In the I186D mutant, the hydrophobic side chain of Ile-186 was
substituted with a hydrophilic side chain Asp. This mutant fails to
form a dimer but instead oligomerizes into a large size complex (likely
larger than hexamers). At the same time, the I186D mutant also loses
the ability to activate its amidase activity by autoproteolysis. In the
human GA, an amino acid substitution at the equivalent position
(Ile-240) also disrupts the dimer formation of the precursor protein
and prevents proteolytic activation of the enzyme (17, 18). Similarly,
other dimer interface mutants also fail to dimerize and lose the
ability of autoproteolytic activation to form mature amidase. It is
possible that these mutations may disturb protein folding and thus
prevent autoproteolysis. However, the CD spectra suggest that these
mutations do not cause a large change in the secondary structure
content. Therefore, the absence of autoproteolytic activation of these
mutants is likely due to a disturbance of dimerization and a change in
quaternary structure. The fact that the oligomers of dimer interface
mutants have similar CD spectra and thermal melting temperature to
T152A precursor dimer suggests that similar monomer structure and
monomer-monomer interactions are utilized in the precursor dimers and
the high molecular weight oligomers. Because these mutations were made on the opposite side of the scissile peptide bond, they are not likely
to be directly involved in the autoproteolysis. Therefore, loss of
autoproteolysis in the mutant oligomers indicates that a unique
structure required to trigger autoproteolysis, presumably a constrained
scissile peptide bond as observed in crystal structures, cannot form in
the high molecular weight oligomers. In summary, based on data reported
here and other evidence in the literature, it appears that dimerization
of GA precursor proteins is a prerequisite to trigger autoproteolytic
activation of the amidase activity.
Two-step Dimerization Model--
With regard to the role of
dimerization and the mechanism of GA activation by intramolecular
autoproteolysis, we propose a two-step dimerization mechanism preceding
GA autoproteolysis (Fig. 7). There are
three dimer conformations involved in this model. The initial
monomer-monomer interactions mainly involve hydrophobic surfaces from
each of two precursor monomers (dimer A). This is followed by a final
mesh of the dimer interfaces with a "locking" device (dimer B),
such that stronger dimer interactions are achieved but at the expense
of creating structural constraints near the scissile peptide bond, as
observed in the crystal structures of GA precursors (15).
Autoproteolysis would then be triggered to relieve these local
constraints without changing the dimer structure, resulting in
autoproteolyzed/mature amidase (dimer C). It is intriguing that the van
der Waals surface of the dimer interface in crystal structures is
relatively flat, except that His-101 from one monomer penetrates into
the second monomer and forms hydrogen bonds with the carboxyl group of
Glu-207 and the main chain nitrogen of Met-177 (8, 15). It is thus
plausible that the intermolecular interactions between His-101 and
Glu-207/Met-177 could serve as a locking device in the second
step of dimerization for the precise orientation to create structural
constraints near the scissile peptide bond.
Wild-type GA autoproteolyzes spontaneously in cells (11). However, if
the autoproteolysis was stopped, such as in the T152A precursor or by
glycine as reversible inhibitor (15), the activated dimer B with local
structural constraints could change through an equilibrium back to the
dimer A conformation that has weaker dimer interactions and thus is
less thermostable than dimer C. Such an equilibrium between dimers A
and B is consistent with our observations that precursors stopped at
autocleavage tend to form some large size oligomers (e.g.
T152A in Fig. 5A), probably by changing back to dimer A-like
conformations. On the other hand, if the first step dimer formation was
prohibited, such as by introducing charge repulsion at the dimer
interface as in this study, the mutated monomers could still interact
with each other through hydrophobic contacts using the similar
non-polar surfaces to form oligomers. This could explain the similar CD
spectra and thermal melting temperatures between T152A precursor dimers
and oligomers of dimer-disruptive mutants. Nonetheless, such
oligomerization of dimer-disruptive mutants is non-productive in
generating the structural constraints near the scissile peptide bonds
that are required to trigger autoproteolysis.
The two-step dimerization mechanism suggests that T152A precursor will
form an equilibrium between dimers A and B, whereas the autoproteolyzed
form of T152Cm protein or wild-type GA would exist exclusively in dimer
C conformation. Thus the measurement of Tm for T152A
precursor is an average of dimers A and B weighted by their percent
population. It is not known whether dimer A or B is the predominant
form of T152A precursor in solution. Another puzzle is that the crystal
structure of precursor dimer is essentially the same as the mature
dimer C (15). One intriguing explanation is that only dimer B, not
dimer A, is stable enough to crystallize. According to the model, the
rate-limiting step of autoproteolysis could be at the autocleavage of
the constrained peptide backbone to generate a mature amidase.
Therefore, it would help explain why the autoproteolysis is a
first-order reaction even though it occurs as a dimer.
A two-step dimerization might be necessary to turn on GA
autoproteolysis at the last step to form the native quaternary
structure, by an unusual twist at the scissile peptide bond. The same
non-polar surface may interact with chaperones during an early stage of GA folding, similar to the interactions in oligomers of
dimer-disruptive mutants. Thus if local structural constraints were
generated during chaperone-GA interactions, they would either be
removed by protein refolding without a productive autoproteolysis or
would lead to premature autoproteolysis with non-native structure. The
second step dimerization with precise locking device would ensure
proper autoproteolysis with native structure. It remains to be
determined whether the two-step dimerization mechanism could be
extended to autoproteolysis mechanisms of other Ntn hydrolase family
members, most of which are found in crystals as oligomeric forms. For
example, proteasome from Saccharomyces cerevisiae is a
28-oligomer structure consisting of a four-layered barrel structure,
with seven unidentical subunits in each layer (28). Penicillin V
acylase from Bacillus sphaericus is tetrameric (29), whereas
glutamine phosphoribosylpyrophosphate amidotransferase from
Escherichia coli is a dimer (30). More work is required to
determine the significance of oligomerization in autoproteolysis of
other Ntn hydrolases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sandwich, common to all
the Ntn hydrolases (7-10). One intriguing aspect of GA is that a
single chain precursor is processed by intramolecular autoproteolysis
that generates the 17-kDa
- and 15-kDa
-subunits and exposes the
active site threonine at the newly generated N-terminal end of the
-subunit (11, 12). A similar mechanism is utilized in protein
splicing that involves two concerted autocleavages and one ligation of
the polypeptide chain (13, 14).
- and
-subunits.
Recently, both scissile peptide bonds involved in protein splicing of
PI-SceI have also been found to be in distorted
trans conformations (16). Similar to GA autoproteolysis,
relieving the distorted/strained main chain atoms is also believed to
help drive an N-S acyl shift in PI-SceI precursor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside overnight at
30 °C. Cells were pelleted by centrifugation, resuspended in 20 mM Tris buffer, pH 7.6, 50 mM NaCl, 1 mM EDTA, and lysed by sonication. Fusion proteins were
affinity-purified from the crude extracts over an amylose column
according to the protocol of the manufacturer (New England Biolabs).
All purification procedures were carried out at 4 °C. To obtain GA
proteins, the purified fusion proteins were further digested with
factor Xa at room temperature overnight, and GA was then separated from
MBP and factor Xa with a HiTrap Q-Sepharose (Amersham Biosciences)
column. Similar protocols were used to purify autoproteolysis-inactive
mutant precursors (T152A). For the precursor proteins of
autoproteolysis-active mutant precursors (T152C mutant), 10 mM glycine was included during the entire course of protein
expression and purification to inhibit autoproteolysis. Protein
concentration was determined by absorbance measurements at 280 nm in 6 M guanidine hydrochloride (22).
-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin
(17 kDa), and vitamin B-12 (1,350 Da). Thus, bovine thyroglobulin was
eluted at the point representing the void volume of the column.
-mercaptoethanol at 90 °C for 10 min, and then analyzed by
SDS-PAGE. The gels were stained with Coomassie Blue. Similar
experiments were also performed using 1-4 mg/ml dimethyl suberimidate
(DMS) as a cross-linking reagent.
220]/dT (24). ORIGIN
software (Microcal, Inc.) was used for the CD analyses and display.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
) subunits, and thus it is impossible
to isolate the precursor of wild-type GA. To study the quaternary
structure of purified GA precursor, we thus have to resort to GA
mutants. T152A precursor has the same three-dimensional structure as
other precursor proteins that are active in autoproteolysis (15). Due
to the loss of its critical nucleophile for autoproteolysis, we are
able to purify a large quantity of precursor protein of the T152A
mutant. Autoproteolysis of a second mutant, T152C, can be switched on
and off by a reversible inhibitor, glycine (15). With the exception of
a slower reaction rate, T152C resembles the wild-type GA in
autoproteolysis activity (11, 25), and the autoproteolyzed T152C enzyme
(T152Cm) has an identical crystal structure to that of the wild-type
mature enzyme (8). Throughout this study, the T152A precursor was used
to represent a native precursor structure, whereas the wild-type and/or
mature T152Cm proteins were used to represent a native structure of
mature GA amidase after autoproteolysis.
and
) subunits in tight association
(26, 27), a single chain GA precursor and a native autoproteolyzed
amidase should have similar calculated molecular masses of 32 kDa. As
shown in Fig. 1A,
autoproteolyzed amidase of wild-type GA or T152Cm protein was eluted as
a single peak with an apparent molecular mass of 43 kDa, when compared with a mixture of gel filtration size standards (Bio-Rad). Thus, the
apparent molecular mass of mature GA was about 40% larger than a GA
monomer of 32 kDa. Similarly, purified precursor T152A protein was
eluted as a single peak with an apparent molecular mass of about
1.4-fold of a monomer. The precursor was eluted at a slightly larger
volume than the mature enzyme. This is consistent with an uncleaved
loop in the precursor that becomes cleaved and thus more floppy and
bulky in the mature enzyme. Interestingly, a 43-kDa recombinant
maltose-binding protein (MBP2 from New England Biolabs) was eluted with
a substantially smaller apparent molecular mass (about 25 kDa) when
compared with the same size standards (Fig. 1B). All these
observations demonstrated the limitations of using gel filtration
chromatography to estimate molecular masses of macromolecules, because
the mobility of proteins in gel filtration columns was affected not
only by molecular weight and size but also by molecular shape. There
are various ways to interpret the single peaks of GA on chromatograms
with elution volumes at the middle between those expected for monomers
and dimers. These single peaks could represent GA monomers with a
smaller retention volume when compared with the size standards, which
may be due to an extensive hydrophobic surface observed in the crystal
structures. These single peaks could also be interpreted as GA dimers
with a larger retention volume than expected, which may be due to the compact shape of the dimer observed in the crystal structures (8). A
third explanation for the unusual mobility is that GA dimers are in
fast equilibrium with monomers during the run through the gel
filtration column. This uncertainty appears to be the source of
confusion that results in contradictory reports in the literature.
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Fig. 1.
Gel filtration chromatograms of GA and MBP-GA
fusion proteins. A, purified GA proteins. Dotted
line, autoproteolyzed wild-type GA; thin line,
autoproteolyzed T152Cm protein; thick line, T152A precursor;
and background line, a mixture of molecular mass standards
from left to right: bovine thyroglobulin (670 kDa), bovine -globulin (158 kDa), chicken ovalbumin (44 kDa), and
equine myoglobin (17 kDa). B, purified MBP-GA fusion
proteins. Dotted line, MBP protein; thin line,
autoproteolyzed MBP-T152Cm protein; thick line, MBP-T152A
precursor; and background line, the same molecular mass
standards as in a. Proteins were gel-filtrated in a solution
of 10 mM potassium-phosphate buffer, pH 7.4, and 1 mM EDTA, with a flow rate of 0.5 ml/min.
and
, in each native
mature/autoproteolyzed enzyme. Without cross-linking treatment, the
wild-type mature enzyme gave two bands for the
- and
-subunits. A
low dose of cross-linking reagent (0.001-0.01%) gave a new cluster of
bands of 30-34 kDa corresponding to two cross-linked subunits either from the same GA molecule (
-
and
'-
' of 32 kDa) or through the dimer interface (
-
',
-
',
'-
, and
-
' ranging
from 30 to 34 kDa). At higher concentrations of cross-linking treatment (0.1-1%), an additional cluster of bands was present corresponding to
completely cross-linked dimer of autoproteolyzed enzyme (~64 kDa of
-
-
'-
'). No band corresponding to three cross-linked subunits (e.g.
-
-
', etc.) appeared. This might be
due to a preferential cross-linking of contacts either through dimer
interface or within a monomer. Thus, by the time two subunits were
cross-linked through the minor cross-linking surface, they were also
cross-linked through the major cross-linking surface, resulting in the
64-kDa species. The cross-linking pattern of mature T152Cm protein
(Fig. 2C) is the same as the wild-type enzyme and confirms a
dimeric structure of mature enzyme. Similar results were also obtained using DMS as the cross-linking reagent (data not shown). All together, results here confirm that both GA precursor and mature enzyme form
dimers in solution.
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Fig. 2.
SDS-PAGE of cross-linked GA precursor and
autoproteolyzed amidase. Cross-linking was performed at 0.1 mg/ml
(3.1 µM) of T152A precursor (A),
autoproteolyzed wild-type GA (B), and autoproteolyzed T152Cm
protein (c), with various amounts of glutaraldehyde at room
temperature for 6 h. Two different percentages of acrylamide gels
were used in b and c to separate a wider size
range of protein fragments and cross-linked products. Lane M
is a mixture of molecular weight markers, and lane 0 is a
sample treated under the same condition except no cross-linking reagent
was added. The rest of the lanes are samples treated with, from
left to right, an increasing concentration of
glutaraldehyde: 0.1-1% in a, and 0.001-1% in
b and c.
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Fig. 3.
Structure-based design of mutations to
disrupt the dimer interface. A, stereo view of the
dimer interface with the substituted amino acids. One GA precursor
monomer is drawn as a molecular surface, colored according to
electrostatic potential (blue, positively charged;
red, negatively charged), whereas the other monomer is drawn
as a cyan polypeptide backbone. In the monomer closer to
viewer, the side chains of substituted amino acids (shown in
green with the amino acid numbers adjacent to the residues)
are on the dimer interface, away from the viewer, whereas the
autoproteolytic site (the scissile peptide bond marked in
magenta) is on the opposite side of the monomer, toward
viewer. The image was made with the program MOLMOL (31) and rendered
with Photoshop. B, SDS-PAGE analysis of autoproteolysis of
wild-type GA, active site mutants (T152C and T152A), and dimer
interface mutants (V105K, I186D, E220K, and R238D). The dimer interface
mutants are very sensitive to protease degradation and thus could only
be partially purified in the context of MBP fusion proteins. Purified
protein samples without ( ) or with (+) further incubation at 30 °C
for 4 h in 10 mM potassium-phosphate buffer, pH 7.4, and 0.5 M hydroxylamine (NH2OH) were separated
on a 15% SDS-acrylamide gel. Lane M is a mixture of
molecular weight markers.
- and
-subunits during protein purification (lane
2). The T152C mutant also partially autoproteolyzes (lane
3) during the protein purification, resulting in the same
- and
-subunits as the wild-type GA. Furthermore, it has been shown that
autoproteolysis of T152C mutant can be accelerated by incubation with
hydroxylamine (lane 4; see Ref. 11), indicating a thioester
intermediate involved in the autoproteolysis of the T152C mutant.
Conversely, T152A mutant loses its critical nucleophile for
autoproteolysis (lane 5), and hydroxylamine does not have
any effect on autoproteolysis of the T152A mutant (lane
6).
and -
subunits (Fig. 3B, lane 7),
and hydroxylamine can also stimulate its autoproteolytic activity
(lane 8). MBP-T152A fusion does not autoproteolyze either
without or with hydroxylamine (lanes 9 and 10).
Thus, expression of GA as MBP fusion proteins does not alter their
autoproteolytic activity but can stabilize the dimer interface mutants
that are extremely sensitive to protease degradation if isolated as
individual GA mutants. We therefore characterized all the dimer
interface mutants as MBP fusion proteins and compared them to the
controls of MBP-T152A precursor and autoproteolyzed MBP-T152C
(MBP-T152Cm) as precursor and mature enzyme, respectively.
and
-
fragments) (Fig. 3B, lanes 13-18). However,
these partially proteolyzed enzymes do not have any amidase
activity,2 suggesting that
the clip site could be different from the autoproteolytic site that
generates an N-terminal threonine critical for amidase activity. In
other words, the processed products might be an act of proteases and
not autoproteolysis. It remained a possibility, however, that
autoproteolysis did occur in these mutants but with no amidase activity
due to some structural changes at the active site, although CD spectra
show that these mutants retain the wild-type fold (see below). Similar
proteolytic trimming near the autoproteolytic site is also observed for
the non-autoproteolytic mutants of human GA precursor (18).
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Fig. 4.
SDS-PAGE of cross-linked MBP-T152A precursor
and MBP fusion proteins of dimer interface mutants. Cross-linking
was performed at 0.1-0.2 mg/ml (1.3-2.6 µM) of
MBP-T152A precursor (A), MBP protein (B),
MBP-V105K mutant (C), and MBP-I186D mutant (D),
with increasing amounts of glutaraldehyde at room temperature for
6 h. A slightly higher concentration (0.2 mg/ml) of protein
samples was used for dimer interface mutants (C and
D) to compensate for the co-purified degradation products
(see text). Lane M is a mixture of molecular weight markers,
and lane 0 is a sample treated under the same condition
except no cross-linking reagent was added. Subsequent lanes are samples
treated with, from left to right, an increasing
concentration of glutaraldehyde from 0.0005-0.05%.
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Fig. 5.
Characterization of MBP fusion proteins of
dimer interface mutants. A, gel filtration
chromatograms of MBP-GA fusion proteins. Thick line,
MBP-T152A precursor; thin line, MBP-I186D mutant; and
background line, a mixture of molecular mass standards from
left to right: bovine thyroglobulin (670 kDa),
bovine (BSA) -globulin (158 kDa), chicken ovalbumin (44 kDa), and equine myoglobin (17 kDa). Proteins were gel-filtrated in a
solution of 10 mM potassium-phosphate buffer, pH 7.4, and 1 mM EDTA, with a flow rate of 0.5 ml/min. B,
sucrose density gradient profiles of MBP-GA dimers (T152A and T152Cm)
and dimer interface mutants (V105K and I186D) at 4 °C for MBP-T152A
precursor (
), MBP-T152Cm mature enzyme (
), MBP-V105K mutant
(
), and MBP-I186D mutant (
). Sedimentation distances of two
reference proteins,
-globulin (158 kDa) and bovine serum albumin (66 kDa), are indicated by arrows. Aliquots (100 µl) of
protein (30 µM) were layered on 10-30% sucrose
gradients and centrifuged at 40,000 rpm for 12 h in a Beckman SW55
Ti rotor. Protein was detected by Bradford assays of a series of
100-µl fractions taken from the top of the gradients. Sedimentation
is depicted from left to right.
-globulin
(158 kDa), indicating an unusual sedimentation velocity midway
between those expected for monomer and dimer, similar to its unusual
mobility on a gel filtration column (Fig. 1). Autoproteolyzed fusion
protein (MBP-T152Cm) also yielded a major peak near that of the
MBP-T152A precursor. In contrast, the majority of both MBP-V105K and
MBP-I186D mutant proteins sedimented at the bottom of the gradient,
representing species of large size oligomers. No distinct peaks
representing dimers were observed for these dimer interface mutants.
Continuous trace amount of proteins were detected throughout the
sucrose gradient, presumably representing various size oligomers of
these dimer interface mutants.
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Fig. 6.
Far-UV CD spectra of precursor dimer,
autoproteolyzed amidase dimer, and dimer-interface mutants.
A, the spectra were recorded from 2 µM protein
solutions in 10 mM potassium-phosphate buffer, pH 7.4, and 1 mM EDTA at 25 °C:
MBP-T152A ( ), MBP-T152Cm (
), MBP-V105K (
), and MBP-I186D
(
). Following the buffer base-line correction, the spectra were
normalized to the protein concentration and are expressed as molar
ellipticity. B, thermal unfolding of MBP-GA fusion proteins
monitored at 222 nm upon heating the sample solutions from 5 to
95 °C. Line codings are as in a. Inset, first
derivative d[
220]/dT to
determine the melting temperature Tm at the centers
of the derivative peaks. C, thermal unfolding of GA
wild-type amidase and mutants, monitored under the same condition as in
B: autoproteolyzed wild-type GA (
), T152A precursor
(
), and autoproteolyzed T152Cm protein (
).
220]/dT
(inset in Fig. 6B) are nearly identical (about
6 °C), suggesting that unfolding cooperativity is identical among
the oligomer of dimer-disruptive mutants and dimer of MBP-T152A
precursor. Because the difference in the melting temperatures between
the precursor and the autoproteolyzed protein was detected for MBP
fusion proteins, we performed additional thermal unfolding studies of
isolated GA proteins to assess if the difference in thermal stability
resides in the GA domains (Fig. 6C). Similar to the MBP
fusion proteins, autoproteolyzed dimers of T152Cm unfolded 3.9 °C
higher than the precursor dimer of T152A. The wild-type mature protein
unfolded at an even higher temperature (more than 6 °C higher than
the Tm of the T152A precursor dimer). The difference
in melting temperature between the mature T152Cm protein and the mature
wild-type GA might be due to some residual amount of T152C protein
remaining in the precursor form (see Fig. 3B, lane
4). Thus the apparent melting temperatures of the predominantly
mature enzymes of T152C mutant were lowered by this residual precursor.
We were unable to separate this residual precursor from the T152C
mature form, and we could not make the autoproteolysis of the T152C
mutant to 100% completion. Nonetheless, these results indicate that
the autoproteolyzed GA dimers (both wild-type and T152Cm mature form) are thermally more stable than the dimer of the T152A precursor. On the
other hand, the dimeric T152A precursor has a similar thermal stability
to the high molecular weight oligomers that are unable to form native
dimers and remain in the precursor form. CD studies have shown that the
GA precursor has similar secondary structure content to the
autoproteolyzed GA. Crystal structure studies have also shown that GA
precursor has essentially the same dimer structure as the
autoproteolyzed amidase, with an exception of a non-structured link in
the precursor between
- and
-subunits (15). Because this
precursor link is on the opposite side of the dimer interface, it
cannot explain why the precursor dimer has a lower melting temperature
than the autoproteolyzed dimer. One intriguing explanation for the
difference in thermal stability is that it is due to stronger monomer-monomer interactions in the autoproteolyzed dimers than in the
precursor oligomers (either dimeric or oligomeric) (see below).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
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Fig. 7.
Two-step dimerization is essential for
autoproteolytic activation of GA amidase. The - and
-subunits of GA are initially joined by a loop that is
autoproteolyzed to activate the amidase active site. In the first step
of dimerization, two molecules of wild-type GA form a dimer
(dimer A) through an extensive hydrophobic surface from each
monomer. This is followed by an equilibrium with dimer B,
which has stronger dimer interactions but at the expense of creating
structural constraints near the scissile peptide bond, as observed in
the crystal structures of GA precursors (15). As a result,
autoproteolysis is triggered to remove such structural constraints and
generate the N-terminal threonines essential for the amidase activity
(dimer C). Single amino acid substitutions at the dimer
interface (indicated by an asterisk) prevent dimer formation
but result in oligomerization through similar monomer-monomer
interactions as in dimer A. However, such oligomerizations are
non-productive in generating the structural constraints required to
trigger autoproteolysis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Bullitt and Gursky for critical reading and comments on the manuscript, members of the lab for helpful suggestions, and C. Guan for assistance in protein purification.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant DK53893 from the NIDDK, National Institutes of Health.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 Physiology
and Biophysics, Boston University School of Medicine, 715 Albany St.,
Boston, MA 02118-2526. Tel.: 617-638-4023; Fax: 617-638-4041; E-mail:
hcguo@bu.edu.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M210431200
2 C. Guan, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GA, glycosylasparaginase; AGU, aspartylglycosaminuria; Ntn, N-terminal nucleophile; T152Cm, autoproteolyzed/mature form of the T152C mutant protein; MBP, maltose-binding protein; DMS, dimethyl suberimidate; PI, protein intervening sequence.
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