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Originally published In Press as doi:10.1074/jbc.M602722200 on June 24, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25723-25733, September 1, 2006
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Size Matters for the Tripeptidylpeptidase II Complex from Drosophila

THE 6-MDa SPINDLE FORM STABILIZES THE ACTIVATED STATE*

Gönül Seyit, Beate Rockel, Wolfgang Baumeister, and Jürgen Peters1

From the Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

Received for publication, March 22, 2006 , and in revised form, June 20, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Tripeptidylpeptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases, a key component of the protein degradation cascade in many eukaryotes, which cleaves tripeptides from the N terminus of proteasome-released products. The Drosophila TPP II is a large homooligomeric complex (~6 MDa) that is organized in a unique repetitive structure with two strands each composed of ten stacked homodimers; two strands intertwine to form a spindle-shaped structure. We report a novel procedure of preparing an active, structurally homogeneous TPP II holo-complex overexpressed in Escherichia coli. Assembly studies revealed that the specific activity of TPP II increases with oligomer size, which in turn is strongly concentration-dependent. At a TPP II concentration such as prevailing in Drosophila, equilibration of size and activity proceeds on a time scale of hours and leads to spindle formation at a TPP II concentration of ≥0.03 mg/ml. Before equilibrium is reached, activation lags behind assembly, suggesting that activation occurs in a two-step process consisting of (i) assembly and (ii) a subsequent conformational change leading to a switch from basal to full activity. We propose a model consistent with the hyperbolic increase of activity with oligomer size. Spindle formation by strand pairing causes both significant thermodynamic and kinetic stabilization. The strands inherently heterogeneous in length are thus locked into a discrete oligomeric state. Our data indicate that the unique spindle form of the holo-complex represents an assembly motif stabilizing a highly active state.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Intracellular proteolysis is one of the vital functions of life. Eventually, all proteins are channeled through the proteolytic degradation cascade. One cellular strategy to cope with the complexity of a crowded cytosol is sequestration of proteolytic substrates into large protein nanocompartments. In eukaryotes, the proteolytic cascade starts with ATP-dependent cleavage of ubiquitinylated proteins by the 26 S proteasome, the paradigm of a self-compartmentalized protease (1). Downstream, tripeptidylpeptidase II (TPP II),2 a 6-MDa complex, as well as some small peptidases such as thimet oligopeptidase and prolyl oligopeptidase, and a number of aminopeptidases, act in an energy-independent manner and generate either T-cell epitopes or amino acids that can be reused (2, 3). In the recent years, TPP II has gained particular attention because of its implication in pathogenesis-related processes such as muscle sepsis (4, 5), in apoptosis (6-8), in processing of antigenic peptides for the presentation by the major histocompatibility class I complex (9-11), and in cerebral neurotransmitter degradation (12, 13) and, particularly, because of its reported capability of substituting for some metabolic functions of the proteasome (14-17).

TPP II is a serine protease belonging to the family of subtilases, and it cleaves tripeptides processively from the N terminus of larger peptides (18), but it was also reported to possess endopeptidase activity (10, 11, 15). However, monomers of TPP II and, likewise, of the archaeal subtilases pyrolysin (19) and STABLE (20) are much larger than the subtilisins, which essentially consist of the canonical catalytic domain, and homology is restricted to the N-terminal half of TPP II. Moreover, the first two residues of the catalytic triad are interrupted by an insert of ~200 amino acids that, like the C-terminal half of the polypeptide, is not homologous to any protein present in the data base. The gene for TPP II has been found exclusively in eukaryotes, and the protein has been isolated from rat, human (21), Drosophila (22), and Arabidopsis (23). The monomer molecular masses range from 138 kDa for the human, mouse and rat to 150 kDa for plant, worm, and insect homologues.

In contrast to pyrolysin and STABLE, and its eukaryotic functional relatives, TPP II is organized into a large assembly comprising 40 subunits (24), which is unique in several respects. Oligomeric protease complexes generally have a self-contained and, therefore, discrete subunit architecture, either circular, with up to four rings stacked, as in the 20S-proteasome (1), tetragonal, as in TET (25), or spherical, as in the Tricorn capsid (26). The basic architecture of the TPP II holo-complex, however, is a stack of dimers in twisted strands that appear not to be subject to any geometrical constraints limiting their subunit number (24). Thus, because of mass action equilibria, the length of single strands should be intrinsically heterogeneous, and this is indeed observed under different conditions in vitro (24, 27, 28). Nevertheless, in vivo, TPP II is composed of two stacks of 10 dimers each, forming a twisted, spindle-shaped structure as shown for Drosophila TPP II by cryo-electron microscopy (24). The cryo-electron microscopy structure also reveals a reciprocal interaction of two terminal dimeric subunits that may stabilize the spindle. The strands appear to enclose a novel variant of a proteolytic nanocompartment, a tunnel having lateral openings on one side, reminiscent of an arcade (24).

The TPP II holo-complex has been reported to decay under conditions such as dialysis (29) or freezing/thawing (30), with a concomitant decrease of activity. In the latter case, the activity dropped to about one-tenth and recovered to some extent upon partial reassembly. This hinted at a correlation between oligomeric state and specific activity and elicited speculations on a regulation of the enzyme activity via association/dissociation (2, 29, 30).

To obtain clues as to the biological meaning of the enormous size of the TPP II holo-complex as well as its supposedly size-dependent activity, we have studied the stability of the spindle-shaped complex as well as the time course of assembly and activation using TPP II overexpressed recombinantly in Escherichia coli in its native form. The relationship between oligomer size and specific activity established here led us to propose a model of assembly and activation of the TPP II holo-complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Strains and Cloning—E. coli strain BL21(DE3) was used for expression of TPP II. From a Drosophila expressed sequence tag clone (Umea Drosophila Stock Center, University of Umea, Sweden) the structural gene for TPP II was cloned between the NdeI and the NotI sites of the vector pET-30b (Novagen). Cells were grown on LB agar plates containing kanamycin (30 µg/ml).

Expression in E. coli—Single colonies were inoculated into Terrific broth (TB)-rich medium supplemented with 30 mg/liter kanamycin, and precultures were grown at 37 °C for 12 h. 5 ml of preculture was transferred into 500 ml of fresh medium (Terrific broth) and grown with vigorous shaking at 37 °C to an A600 of 2.5. After cooling down to 18 °C, the cultures were induced with 0.1 mM IPTG and grown at 18 °C for 24 h.

Activity Measurements—Exopeptidase activity measurements of TPP II were carried out at 30 °C. The fluorogenic substrate Ala-Ala-Phe-7-amino-4-methylcoumarin (AAF-AMC, Bachem) was used at a concentration of either 0.2 mM or 2.0 mM in 100 mM potassium phosphate, pH 7.5, 1 mM dithiothreitol (DTT), 5% glycerol, 5% Me2SO, 0.1 mM bestatin. 50-µl reactions were stopped after 30 s to 10 min with 450 µl of 100 mM Tris/Cl, pH 9.5, 1% SDS. The release of AMC was measured in an SFM25 fluorometer (Kontron Instruments) at {lambda}ex360 nm and {lambda}em460 nm calibrated using free AMC.

SDS-PAGE and Densitometric Evaluation—For SDS-PAGE, the buffer system of Schägger and Jagow (31) was used. The acrylamide concentration was 9%. Samples were mixed with 0.2 volume of 5% SDS, 100 mM Tris/Cl, pH 8.5, 50 mM DTT, 50% glycerol and solubilized at 70 °C for 5 min. Cross-linked samples were centrifuged at 19,000 x g prior to loading. Dilute samples were subjected to precipitation with trichloroacetic acid (10% final concentration) on ice.

For native PAGE, samples were mixed with gel loading buffer to give final concentrations of 40 mM Tris/Cl, pH 6.8, 5% (v/v) glycerol, 2.5% (v/v) beta-mercaptoethanol, and 10% (w/v), bromphenol blue, and subjected to electrophoresis on Novex® Pre-Cast 4-12% Tris/glycine gels (Invitrogen) using 1x Tris/glycine native running buffer (25 mM Tris/Cl, pH 8.5, 190 mM glycine). Electrophoresis was performed overnight at 4 °C. Staining was performed with colloidal Coomassie (Coomassie BioSafe, Bio-Rad). Bands were scanned on an HP 1680 Pro Scanner and evaluated using the software Image (National Institutes of Health). For calibration of the TPP II concentration, 0.5-3 µg of purified TPP II were loaded on the same gel. Protein concentration was determined using the Bradford protein assay (Bio-Rad).

Kinetic Studies—Induced E. coli cultures were harvested at different times and suspended in 1-3 volumes of 80 mM potassium phosphate, pH 7.5, 5% glycerol, 10 mM DTT. These suspensions were frozen dropwise in liquid nitrogen, and the resulting beads were stored at -80 °C. For cell disruption, thawed out samples were sonicated in an ice/water bath in 15-ml Falcon tubes using a sonicator (Bransonic, cylindrical tip, 1-cm diameter, 3 x 5 pulses, setting "8" at 50% pulse duration), centrifuged at 19,000 x g for 2 min, and the supernatant was subjected to chromatography on a Superose 6 HR 10/30 column at room temperature using 80 mM potassium phosphate, pH 7.5, 2 mM DTT, 5% glycerol as running buffer at a flow rate of 0.4 ml/min. Fractions eluted were immediately subjected to activity assays.

For assembly studies, crude extract from cells harvested after 2 h of induction was thawed on ice and incubated at 30 °C for different periods of time, and 200 µl of sample was then subjected to size-exclusion chromatography (SEC) on Superose 6 (Amersham Biosciences). Activity was assayed immediately after elution. From the specific activity of fully assembled TPP II, the concentration of TPP II in the cell extract was calculated and adjusted to 0.1 mg/ml before incubation using the phosphate buffer.

Purification of TPP II—TPP II was initially purified from E. coli using the method described for native Drosophila TPP II (28), and an additional run on Superose 6 was performed to separate dissociated complexes from intact material. Later, the following novel method was used: E. coli crude extract was prepared by passage of the cells suspended in 1 volume of 50 mM potassium phosphate, pH 7.5, 10 mM DTT, through a Cell Disrupter (EmulsiFlex-C5 Homogenizer, Avestin) twice. Then the crude lysate was centrifuged at 30,000 x g for 15 min, and the sediment was resuspended in 3 volumes of 100 mM potassium phosphate, 5 mM DTT. After centrifugation as before, 1 M triethanolamine, pH 7.7, was added to the supernatant to 100 mM final concentration, then polyethyleneimine (PEI) was added until no more precipitate was formed (0.2-0.4% final concentration, depending on the individual preparation), and the pH was adjusted to 8.9 using dilute KOH. After centrifugation at 19,000 x g, the sediment was discarded, and 1.2 volumes of saturated ammonium sulfate were added to the supernatant. After incubation on ice for 15 min, the sample was centrifuged at 4 °C/19,000 x g for 5 min, the supernatant was removed completely in a brief second centrifugation step and discarded, and the sediment was dissolved in 80 mM potassium phosphate, pH 7.5, 5% glycerol, 2 mM DTT. The sample was then incubated at 30 °C for 20 min to reduce the amount of spindles carrying extensions, and then 500 µl of sample was loaded on a Superose 6 column, using 80 mM potassium phosphate, pH 7.5, 5% glycerol, 2 mM DTT as running buffer at a flow rate of 0.4 ml/min. Fractions eluting between 6.8 and 10 ml were collected and stored at room temperature. The peak fraction was collected between 7.2 and 8.8 ml. For storage of more than 2 days at room temperature, 3 mM sodium azide (final concentration) was added. The protein concentration in the eluate was typically 0.7 to 1 mg/ml.

Guanidine Titration—To TPP II purified as described above, 6 M GdnHCl was added dropwise to a final concentration of 550 mM during 20 min at 25 °C. The sample was then separated on a Superose 6 column as described above.

Chemical Cross-linking—To PEI-treated crude extract, 1 M triethanolamine/phosphate buffer, pH 8.5, was added to a final concentration of 100 mM, and the pH was adjusted to pH 8.3. The sample was kept on ice, and then the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate (Pierce) dissolved in Milli-Q grade water (Millipore) to a concentration of 50 mM was added in five increments during 10 min to a final concentration of 2.5 mM. After another 5 min, 20 mM Tris/Cl, pH 8.5, was added to bind excess reagent, and the samples were prepared for SDS-PAGE (see above).

Determination of Kinetic Constants—For the determination of Km, samples were incubated in the presence of 0.005 to 2 mM AAF-AMC, 5% Me2SO, 100 mM potassium phosphate, pH 7.5, 1 mM DTT, 0.1 mM bestatin, at 30 °C for up to 2 min, and reactions were terminated as described above. For determination of Vmax, the protein concentration was determined using the Bradford-assay (Bio-Rad) or by densitometric quantitation of Coomassie-stained bands on SDS-acrylamide gels.

Electron Microscopy—For negative staining, carbon-coated copper grids were incubated for 1 min on a drop of protein sample solution and then consecutively transferred to two drops of 40 mM ammonium sulfate, pH 7.5, for washing. Staining was performed by incubation on a drop of 2% uranyl acetate for 1 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Recombinant Expression and Purification of TPP II—Compared with eukaryotic sources, the expression of TPP II in E. coli has the advantage of high yields of recombinant protein. Because the expression yield of soluble protein significantly depended on the conditions of growth and induction, we optimized the conditions with respect to growth medium, temperature, incubation time, and concentration of inducing agent (isopropyl-beta-D-thiogalactopyranoside (IPTG)). At the temperature of 30 °C a significant fraction of protein was present as insoluble precipitate in overexpressing E. coli cells. The reduction of temperature and of IPTG concentration improved the yield of soluble protein. We found that 18 °C/0.1 mM IPTG resulted in optimal expression with minimal insoluble TPP II. Under these conditions, an induction time of 20 h is required to obtain full specific enzymatic activity of TPP II in the crude lysate.


Figure 1
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  		FIGURE 1.
Purification of recombinant TPP II from Escherichia coli. A, SDS-PAGE; lane 1, marker; lane 2, crude lysate; lane 3, TPP II-enriched second lysate; lane 4, supernatant after polyethyleneimine treatment; lane 5, ammonium sulfate precipitate; lane 6, complete high molecular mass peak (7-9 ml elution volume) of Superose 6 eluate. The same activity of TPP II was loaded in each lane. The TPP II preparation was homogeneous as verified by mass spectrometry. The 150-kDa protein lacked three N-terminal amino acids. The 115-kDa band is attributable to nicking of a C-terminal loop, which has no impact on the quaternary structure. A similar partial cleavage was also observed with human (21) and Arabidopsis (23) TPP II. B, SEC on Superose 6. Dashed line: sample as in A, lane 4; D, disassembly products. Solid line: ammonium sulfate precipitate; S(+): spindles plus extended complexes; S: spindles. E. coli proteins elute at 14-20 ml. C, electron micrograph of negatively stained particles of the S(+) fraction in B. The inset shows selected extended complexes. Arrows denote some extended complexes. D, electron micrograph of the spindle fraction (S). E, native TPP II purified from Drosophila eggs.

 
SDS-PAGE of the cytosolic fraction (Fig. 1A) revealed that the 150-kDa protein was strongly overexpressed in E. coli. Because a major fraction of TPP II was not present in spindle form but as incompletely assembled large oligomers, a procedure was developed that permitted the purification of intact spindle complexes at high yield and conformational homogeneity. Because TPP II largely associates with cell debris in highly concentrated E. coli lysates, a significant enrichment of TPP II was achieved by passing the sediment through a second cycle of disruption and using only the supernatant of the second sedimentation (Fig. 1A, lane 3). Treatment with PEI at pH 8.9 caused TPP II to dissociate into small oligomers, mainly tetramers. Ensuing ammonium sulfate precipitation (see "Experimental Procedures") reconstituted the TPP II holo-complex, which was efficiently separated from E. coli proteins by SEC on Superose 6 (Fig. 1B). The purity of the TPP II preparation (Fig. 1A, lane 6) was confirmed by mass spectrometric analysis of faint bands visible in overloaded lanes (data not shown), which demonstrated these to be TPP II fragments, probably generated by proteolytic nicking.


Figure 2
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  		FIGURE 2.
Chemical cross-linking of partially dissociated TPP II complex. A, separation of PEI/pH 8.9-dissociated and cross-linked sample on Superose 6 and SDS-PAGE of fractions. The migration position of a trimer is indicated in brackets. M, monomer. B, calibration of the SDS-acrylamide gel in A. The position of a trimer is indicated with an empty circle. C, rechromatography of the peak fraction in A on Superose 6. D, native acrylamide gel; lane 1, peak fraction from C, lane 2, marker.

 
The elution peak of the holo-complex at 7.5 ml, corresponding to an apparent molecular mass of ~10-20 MDa, indicated that a significant fraction of the complex was present as particles larger than spindles. Electron microscopy revealed the presence of spindles having extensions of one or both of their component strands and single strands in addition to spindles (Fig. 1C). Upon incubation of the peak fraction collected at 7.2-8.0 ml at room temperature for at least 3 days, most of the material was converted to spindles. The fraction collected at 8.0-8.8 ml (Fig. 1D) consists essentially of spindles that are morphologically indistinguishable from the native protein complex isolated from Drosophila (Fig. 1E). The formation of extended particles is attributable to the extremely high concentration of TPP II in the ammonium sulfate precipitate. Whereas the concentration was ~10 mg/ml in the sample loaded on SEC, it was reduced to ~1 mg/ml in the peak fraction. Size equilibration of TPP II oligomers is a very slow process, particularly at relatively high concentration, but the upper marginal concentration for the stability of non-extended spindles may be roughly estimated to be 1-3 mg/ml.

The protein expressed in soluble form amounted to ~7.5% of total E. coli soluble protein. For comparison, the native protein makes up ~0.05% of soluble protein in Drosophila egg lysate. The yield of purified TPP II was 15-20 mg/liter of growth medium, or 1.5-2 mg/g of cells. In contrast, the yield of purified native TPP II was only ~0.005 mg/g of Drosophila eggs, which is comparable to other eukaryotic sources.3

Enzymological Characterization of Recombinant TPP II—In addition to the morphology, the enzymological properties of native and recombinant TPP II were compared, using AAF-AMC as substrate. The Km was determined to be 0.44 ± 0.05 mM for the recombinant and 0.47 ± 0.07 mM for the native enzyme. The Vmax values were 22300 ± 880 pmol x µg-1 x min-1 and 21800 ± 730 pmol x µg-1 x min-1, respectively. The inability to cleave succinyl-AAF-AMC (specific activity, <0.1% of exopeptidase activity) indicates the absence of contaminating nonspecific endoproteinase activities in the recombinant enzyme preparations.

Building Blocks of the TPP II Complex—It was suggested for human TPP II that dimers are the building blocks of the holo-complex (27). Indeed, the three-dimensional structure determined by cryo-electron microscopy shows the dimer to be the repeating structural unit of a TPP II strand, and it was suggested that assembly proceeds via successive concatenation of dimers into strands (24). However, intermediates of TPP II ranging in size between dimers and the holo-complex have not as yet been investigated.

The presence of trimeric intermediates cannot be excluded a priori. Also, tetramers, the most abundant small complex (see Figs. 1B and 3 below), might dimerize into octamers as opposed to reacting with dimers to form hexamers. SEC cannot resolve trimers in the presence of dimers and tetramers. Therefore, to verify that assembly/disassembly intermediates of TPP II strands are multiples of two, we used chemical cross-linking in combination with SDS-PAGE to determine the population of these oligomeric states under conditions causing fragmentation of the TPP II complex. For this purpose, crude extract was subjected to partially dissociating PEI/pH 8.9 treatment as in the purification procedure but cross-linked with a lysine-specific bifunctional reagent (see "Experimental Procedures") and subjected to SEC and subsequently denaturing SDS-PAGE (Fig. 2A). The three major bands observed in the molecular mass range of 300-900 kDa were assigned to dimers, tetramers, and hexamers of TPP II, respectively (Fig. 2B). Trimers were only detected in trace amounts, and this was probably attributable to incomplete cross-linking of tetramers. Notably, octamers were not detected in significant amounts with respect to hexamers (Fig. 2A).

Rechromatography of the peak at 13.2 ml (12.8-13.6 ml, Fig. 2C) and native PAGE (Fig. 2D) yielded one band at an apparent molecular mass of ~600,000 Da, corresponding to the tetramer. Tetramers were likewise obtained by native PAGE of the purified holo-complex (data not shown), which confirms that, under dissociating conditions, tetramers are the most stable oligomeric form of Drosophila TPP II. In contrast, human TPP II migrated as a dimer in native PAGE (30).

Intermediates of assembly/disassembly were also generated by an alternative method: Titration of purified, recombinant TPP II with guanidine hydrochloride (GdnHCl) was used previously to disassemble all large oligomeric states of TPP II except the spindle-shaped holo-complex, showing its enhanced stability (24). Here, we used GdnHCl titration and subsequent separation on SEC to identify the oligomeric states of the disassembly products (Fig. 3A). As shown by electron microscopy, the high molecular mass fraction contains only spindles (Fig. 3B). In contrast, the breakdown products consisted essentially of hexamers, tetramers, and dimers (Fig. 3C), and the population of these states (Fig. 3A) was very similar to that of PEI/pH 8.9-induced decay (Fig. 1B). Thus, despite the relative stability of tetramers, the data in Figs. 2 and 3 confirm experimentally the idea of a repetitive, dimer-based subunit architecture.

The TPP II fragments generated by GdnHCl treatment were enzymatically active but showed 30-70% reduced specific activity. At >550 mM GdnHCl the proportion of dimers and monomers increased. Beyond ~650 mM GdnHCl, when dimers became the prevailing disassembly product, soluble but inactive aggregates eluting in the region of tetramers/hexamers were also formed (data not shown). Unlike all small and active oligomers, these aggregates did not slowly reassemble upon incubation at room temperature, indicating irreversible aggregation.

Assembly of TPP II in Vivo and in Cell Extract—To investigate the relationship between the oligomeric state of TPP II and its specific activity, conditions were required that would allow the preparation of samples containing functional TPP II in various oligomeric states. Because the disassembly of the TPP II holo-complex into dimers using GdnHCl unavoidably lead in part to irreversible denaturation, we sought to scavenge dimers during the process of assembly in expressing E. coli cells. We found that assembly of the holo-complex is a slow process, especially under the conditions we used for recombinant expression. To monitor the assembly of TPP II, aliquots of induced cultures were harvested at different times, and the cleared lysates were subjected to SEC on Superose 6. The following stages of assembly were discerned: 1-1.5 h: monomers assemble rapidly into dimers; 2 h: mainly dimers are present in the crude extract; 3-10 h: mainly tetramers are present; and 10-24 h: larger oligomers increasing in size are present. Thus, the crude cell extract harvested after 2 h of induction (termed "2-h cell extract" below) was a suitable source of dimers. Although the expression yield of TPP II after 2 h was still low (~0.1 mg/ml in crude lysate) compared with the final stage (~4 mg/ml), it roughly corresponded to the overall concentration present in Drosophila eggs (and likewise, in erythrocytes and hepatocytes),3 which was estimated to ~0.05 mg/ml. Although the cellular environment in the host is somewhat different, the high concentration of proteins and nucleic acids in a crude cell extract creates a crowded environment mimicking the natural milieu to some extent. This implies that 2-h cell extract is an adequate model system for studying TPP II assembly.


Figure 3
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  		FIGURE 3.
Dissociation of TPP II complexes in the presence of GdnHCl. Recombinant TPP II titrated with 550 mM GdnHCl was separated on Superose 6. A, Superose 6 chromatogram. S: spindles; D: disassembly products. Oligomeric states (hexamers to monomers) are denoted with arrows. B, electron micrograph of the spindle fraction (S). C, electron micrograph of disassembly products (Fraction D in A).

 
The 2-h cell extract was adjusted to 0.1 mg/ml TPP II and kept at 0 °C (see "Experimental Procedures"). Whereas oligomerization was completely arrested at 0 °C, the holo-complex was formed upon incubation above ~10 °C, and the rate of complex formation increased strongly with temperature up to 30 °C. Above 32 °C, TPP II was destabilized. The activity profiles of chromatograms obtained at 30 °C and different incubation times show the distribution of activity over the oligomeric states and reflect different stages of assembly (Fig. 4A): after 10 min of incubation the enzymatic activity is distributed over a broad range of oligomeric states, with a maximum at 6- to 10-mers. At 20 min, the activity is distributed almost evenly between 6-mers and large complexes. After 40 min, the maximum of activity is found in the fraction containing the holo-complex (elution volume, 8-8.8 ml). Finally, at 180 min the total activity does not increase further, indicating the equilibrium state. Between 40 and 180 min, the overall activity increases ~1.5-fold, whereas the relative distribution of activities over the different oligomeric states is almost unchanged. Because the sum of values for each data set in Fig. 4A reflects the respective total activity, and because the total concentration of TPP II is constant, it is clear that assembly is accompanied by a strong increase in specific activity. Overall, there is a 12-fold increase of specific activity upon formation of the holo-complex from dimers. The Km for AAF-AMC, as determined with the cell extract, concomitantly changed from 0.95 for dimers to 0.45 for the spindle complex, showing an increased substrate affinity of oligomeric complexes. Scrutiny by electron microscopy verified that the TPP II fraction eluting at 8-8.8 ml contained spindles such as shown in Fig. 1.


Figure 4
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  		FIGURE 4.
Assembly of the TPP II complex from dimers in cell extract of E. coli cells harvested after 2 h of induction. A, kinetics of TPP II assembly in vitro as monitored by activity measurements. Cell extract was incubated at 30 °C for different periods of time and separated on Superose 6. The elution positions of dimers (2-mers) and spindles (S) are marked. The complete data set is representative for two independent experiments. B, time dependence of specific activity. Open squares, left hand axis, fraction eluting at 8 ml corresponding to spindles, solid squares, right hand axis, total cell extract. C, equilibrium activity profiles obtained after assembly at different TPP II concentrations. Solid line, solid squares, 0.030 mg/ml; dotted line, solid triangles, 0.010 mg/ml; dashed line, crossed circles, 0.003 mg/ml. D, disassembly of the TPP II complex after dilution to 0.001 mg/ml, as monitored via activity decrease. The initial value for the holo-complex was taken as 100%. Dark gray columns, cell extract containing the holo-complex (spindles) assembled after incubation at 30 °C for 3 h as in panel A. Light gray, cell extract diluted 1:10 before incubation at 30 °C and assembled into mainly 10-14mers (see panel C). Dilution was performed using 100 mM potassium phosphate, pH 7.5, 5% glycerol, 3 mM DTT, 0.05 mg/ml bovine serum albumin, and the samples were incubated at 30 °C.

 
From the 1.5-fold increase of total activity between 40 and 180 min, it follows that assembly is faster than activation under the prevailing conditions. Thus, at non-equilibrium (i.e. incubation time < 180 min) specific activity and oligomeric state appear not to be correlated. This was confirmed by determining the specific activity of the chromatographic fraction containing the complete spindles over time (Fig. 4B). Fractions eluting at 8-8.8 ml (spindle fraction) were assayed for activity and also separated on SDS-polyacrylamide gels to quantitate the 150-kDa bands densitometrically. The specific activity of large complexes thus obtained is initially on the level of the dimer and then increases steadily with time, concurrently with the overall activity.

According to the data in Fig. 4A, the assembly and activation of TPP II may be basically described as a cascade of two-step reactions of the kind,

FormulaREACTION 1
where (DD)+ is an assembled but not, or not fully, activated intermediate form, and DD* is the fully activated state. Whereas the first step (I), the addition of a dimer, is a bimolecular reaction that must be of second order, the second step (II), most probably a conformational change, is monomolecular and therefore of first order. As follows from the rate law, t1/2 (II) is independent of the concentration of reactant, whereas t1/2 (I) ~ 1/[D]0. As a consequence, with increasing [D]0, the activation will become increasingly rate-limiting.

From the kinetic data in Fig. 4B it follows that the overall reaction scheme leading to the holo-complex is not completely described by a linear sequence of type I/type II steps as sketched above: Activation can lag behind assembly and even complexes having the size of spindles but only the specific activity of dimers were detected initially (Fig. 4B). Therefore, consecutive assembly reactions (type I) may occur at any intermediate stage (Dn)+ without prior activation (type II).

Dependence of the Oligomeric State on TPP II Concentration—Because TPP II has a concatameric structure, its oligomer size should, according to the rate law, depend critically on its concentration. Thus, dilution of the cell extract before assembly was expected to cause a decrease of final strand length at equilibrium as well as to slow down equilibration of size and activity. This effect was confirmed experimentally, and the incubation time was consequently increased to 20 h for dilute samples to allow for complete equilibration.

At a concentration of 0.1 mg/ml, TPP II assembles completely into spindles (Fig. 4A). To determine the dependence of the oligomeric state on the TPP II concentration at equilibrium, 2-h cell extract was diluted with phosphate buffer and subsequently incubated at 30 °C for 20 h (Fig. 4C). At 0.030 mg/ml, spindles were still stable. At 0.010 mg/ml the maximum of activity after assembly was observed at an oligomer size of 10-14. At 0.003 mg/ml, assembly only reached the stage of tetramers to hexamers. The total specific activity corresponding to the area under the curve concomitantly decreased from 100% at 0.030 mg/ml to 85% at 0.010 mg/ml and 66% at 0.003 mg/ml, respectively, showing a decrease of specific activity with decreasing oligomer size. Because TPP II was fully active and assembled at 0.030 mg/ml, the spindle-shaped holo-complex may be expected to be stable under physiological conditions (~0.050 mg/ml, see above).

From the data shown in Fig. 4C it follows that the TPP II holo-complex should decay below a concentration of ~0.03 mg/ml. However, the experimental verification revealed that the pairing of strands into spindles did not only confer thermodynamic but also significant kinetic stabilization (Fig. 4D): when diluted to a TPP II concentration of 0.001 mg/ml, spindles retained full activity over 24 h, indicating structural integrity. In contrast, incompletely assembled complexes (mainly 10- to 14-mers) obtained by assembly of dimers at 0.010 mg/ml lost ~30% of their initial activity, indicating disassembly (see Fig. 4C) after dilution to the same protein concentration. Because the initial activity of the 10- to 14-mer fraction was only 85% of that of the holo-complex, the final activity was ~60% of the holo-complex activity and thus compares to the value reached by dimers assembling into mainly tetramers (Fig. 4C).

Dependence of the Specific Activity on the Oligomeric State— We have shown earlier that TPP II can assemble into single strands and form spindles once the stage of the decamer of dimers (or higher) is reached (24). Thus, the strands do not dimerize at an early stage and, consequently, the elution volumes in SEC may be taken to reflect the actual linear oligomeric states. Although spindles can hardly be separated from bows, i.e. their constituent single strands, on SEC, this has no effect on the determination of the specific activity versus size relationship, because the single-stranded mutant was found to have a specific activity indistinguishable from that of spindles (24).

The data in Fig. 4C indicate that the specific activity of TPP II increases along with oligomer size. To establish a correlation between oligomer size and specific activity, a TPP II concentration was used where all oligomeric states were populated at equilibrium. To ensure the complete absence of contaminating higher molecular mass complexes present in small amounts in the 2-h cell extract, the dimer fraction (Fig. 4A, tincubation = 0 min, telution = 13.6-14.4 ml) was incubated at 30 °C to induce assembly. As shown by SDS-PAGE following SEC, only small amounts of large oligomers were formed at equilibrium, owing to the 11-fold dilution caused by the chromatographic separation of the cell extract (Fig. 5, top, large box). From the low molecular mass end of the elution profile, starting with the dimer, the specific activity of TPP II increased hyperbolically with oligomer size from right to left. Whereas dimers only possessed a low specific activity, tetramers were ~50% activated.

We generally observed that, under non-equilibrium conditions, the specific activities always ranged between the low specific activity of dimers and the respective equilibrium values for different oligomeric states. This indicates that assembly may skip activation steps but not vice versa. For the tetramer of TPP II, a specific activity of 12 700 pmol x µg-1 x min-1 was never exceeded at equilibrium for assembly as well as disassembly under different conditions. This supports the idea that there is a correlation between the oligomeric state of TPP II and its specific activity, although the equilibration time may be as long as 20 h.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Concentration Determines Size Determines Activity—In this work we have investigated the relationship between enzyme concentration, oligomeric state, and activity for the TPP II complex whose architecture and activation is, to our knowledge, fundamentally different from all large proteolytic complexes described thus far. Whereas the addition or removal of one subunit in the large ring-shaped protein complexes such as the proteasome is strongly disfavored energetically, this only entails relatively small free energy changes in the linear concatameric structure of TPP II. Consequently, its size is strongly concentration-dependent and not discrete. This is illustrated by the fact that a concentration change from 0.010 to 0.003 mg/ml caused a decrease in oligomer size from ~10- to 14-mers to 4- to 6-mers (see Fig. 4C). Conversely, an increase in concentration from 0.010 to 0.030 mg/ml caused a doubling to tripling of oligomer size. Thus, the critical concentration range determining the oligomeric state of TPP II is only one power of ten. The fact that discrete 40-mers are nevertheless observed as the main oligomeric state over a concentration range of approximately two powers of ten (0.03 to roughly 3 mg/ml) shows that a substantial amount of free energy is gained through spindle formation by strand pairing. This is illustrated by a rough energetic consideration: From electron micrographs of a preparation containing 0.25 mg/ml TPP II it was estimated that the ratio of spindles over the constituent bows was at least 100:1.


Figure 5
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  		FIGURE 5.
Size dependence of specific activity at equilibrium. The dimer fraction of 2-h crude extract was incubated at 30 °C for 20 h and separated on Superose 6. The specific activities of different fractions were determined using densitometric evaluation of 150-kDa bands obtained after SDS-PAGE (top left, large box). The specific activity of the dimer fraction (top right, small box) was determined separately before incubation. The columns correspond to the SEC fractions. The lower scale at the bottom indicates the elution position of different oligomers as calculated from a calibration curve.

 
Because {Delta}G0assn =-RT ln[Spindles]/[Bows]2, and [Spindles] = 4.2 x 10-8 M, the free energy gain for spindle formation from two single bows would be at least -15.4 kcal/mol, which is of the same order of magnitude as the stability of a typical protein toward denaturation. Thus, it is only the binding energy of strand pairing into spindles that provides a discrete structure of TPP II at an enzyme concentration of ~0.050 mg/ml such as calculated for Drosophila eggs. This is in accordance with the observation that TPP II is present completely as spindles in Drosophila egg lysate (24, 28), as also confirmed by cross-linking studies of lysates.4 However, in the absence of molecular crowding (32) in vitro and at dilutions of TPP II to much less than 0.030 mg/ml, such as occurring during purification procedures, concentration and time are critical parameters. This may explain why previously published electron microscopic images and size-exclusion chromatograms of purified TPP II from different sources reveal a significant degree of fragmentation (15, 27, 29, 30, 33).

The stability of spindles may be attributable to a dimerization motif called double clamp (24) at each end of the holo-complex. The interaction is reciprocal, thus involving four dimeric subunits at each spindle pole. In addition to the thermodynamic stability, such a clamp can also account for the remarkable kinetic stability of TPP II observed here: As shown by the slow assembly of dimers into single strands (Fig. 4A) the activation energy for strand extension is rather high. Clamping of strands at both ends in a spindle complex may be expected to further destabilize the transition state by hampering conformational movements and the "snapping-in" of the dimers into the interdigitated strand.

The crucial role of the TPP II concentration was confirmed using a supposedly non-assembling, inactive mutant: in a previous report it was concluded that the mutation G252R discovered with human TPP II prevents the assembly of dimeric subunits into the native complex. The isolated protein had no activity, and attempts to associate this material into the active complex were unsuccessful (33). We hypothesized that the mutation G252R lowers the association constants for assembly, which should be compensatable by increased concentration of TPP II. Therefore, we introduced the corresponding mutation G260R into recombinant Drosophila TPP II. After precipitation with ammonium sulfate and subsequent SEC (see above) we observed spindles that possessed the same specific activity and appearance on electron microscopic images as the wild-type complex. The same result was obtained with the human G252R variant overexpressed in E. coli (data not shown). The concentration of TPP II from human cell culture (33) may be inferred from volume activity data and the specific activity for human TPP II (13 300 pmol x µg-1 x min-1)tobe in the range of 0.1 to 0.5 mg/ml. This is sufficient for the assembly of the wild type but not the destabilized mutant protein complex. However, the concentration reached during ammonium sulfate precipitation of the recombinant Drosophila TPP II mutant (>10 mg/ml) compensated for the reduced Kassn in the G260R mutant. We conclude that, unlike previously suggested (33), the mutation close to the active site His-264 impairs TPP II activity only indirectly through the prevention of assembly by lowering Kassn.


Figure 6
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  		FIGURE 6.
Proposed model for assembly and activation of the TPP II holo-complex. A, Left: single strand (cryo-electron microscopy structure, see also Ref. 24). Two adjacent dimers are highlighted in color. I, and II, two alternate arrangements of active sites (white boxes) corresponding to different conceivable modes of activation. B, comparison of the specific activity data from Fig. 5 with a function derived from the model proposed. The activation status of monomers in selected oligomeric states is indicated; white, basal activity; black, fully activated. Filled squares, data calculated according to P = 100(N - 2)/N + 2Pbasal/N. Empty squares, experimental data. The maximum value from Fig. 5 was set to 91% (as resulting from the formula for N = 20) to allow a comparison of theoretically predicted and experimental data. C, model scheme integrating kinetics and equilibrium situation. For simplicity, only the initial stage of a highly complex scheme is shown. White, basal activity; gray, activation-competent intermediate state; black, activated state. It is assumed that assembly involving the formation of an interface between two dimers, as highlighted in color in panel A, is a prerequisite for activation but assembly is not dependent on previous activation. Both, assembly and activation reactions may compete, as indicated for the tetramer. Non-activated species may be activated later in the course of assembly, as indicated exemplarily by the dashed arrow. At equilibrium, all species are fully activated (compare with B), but the final oligomer size distribution is concentration-dependent. For simplicity, only forward reactions are depicted.

 
A Model Explaining the Size Dependence of Specific Activity— Our experimental findings provide some insights into the mechanism of assembly and activation of TPP II. Here we present a global scheme that is compatible with the observed size/activity dependence: The formation of new interfaces between dimeric subunits is crucial for activation. The specific activity of tetramers (~50% of full activity) shows that only one dimer out of two, or two monomers out of four, are activated. A plausible reason may be derived from Fig. 6A: according to our previous studies with MBP-tagged TPP II, the N-terminal domain harboring the active site is probably situated within the globular domains that form the inner backbone of the strands (24). When viewed from the side that is oriented toward the neighboring strand in the TPP II model, the two monomers forming a dimer appear to be connected diagonally (see Fig. 6A). A consequence of this arrangement is that the adjoining monomers belonging to consecutive dimers form two interfaces, one parallel to the strand axis and one orthogonal to it. Assuming that activation occurs through a contact-induced conformational change at or near the active site, one can distinguish between two different arrangements (Fig. 6A) as follows. Type I: juxtaposition of two active sites belonging to two adjoining dimers along the strand axis. This is possible in view of the established 2-fold symmetrical arrangement of the (horizontally) facing monomers. Type II: vertical contact formation of active sites with the non-identical surfaces on the neighboring dimer. An activation motif resembling Type I is found in thiamindiphosphate-dependent enzymes such as pyruvate decarboxylase (34), pyruvate dehydrogenase (35), and transketolase (36). In the dimeric HIV-1-protease, the two active-site aspartates are contributed by the two monomeric subunits (37).

In contrast, in the active dimer of the human cytomegalovirus protease HCMV, the active sites are located at opposite sides. Each active site is thought to be complemented by the other subunit to create a binding site for the substrate assembly protein (40 kDa) (38, 39). However, in all these and a number of other cases the constituent subunits are inactive, whereas TPP II dimers still have a specific activity exceeding that of the 20 S proteasome toward its standard artificial substrate by more than one power of ten (15).

Our model of TPP II activation implies that there are always two unpaired monomers, one at each end, that are not activated. Accordingly, the share of subunits forming interfaces with neighboring subunits that lead to their activation increases to 90% in the 20-mer, which corresponds to one component strand of a spindle. At equilibrium, this mode of activation may be expressed by the function, P = 100(N - 2)/N + 2Pbasal/N, where P is the relative specific activity given in percent, Pbasal is 8%, and N is the number of monomeric subunits. A comparison of the specific activities for different oligomeric states determined at equilibrium (Fig. 5) with the function derived from this model reveals a good agreement of theory and experiment (Fig. 6B). Whether the terminal contacts of strands leading to assembly into spindles possibly activates the lone terminal monomers, thus endowing the complex with 100% instead of 91% activity, cannot be determined using the present analytical system, because homogeneous single bows cannot be prepared. Nevertheless, single bows (20-mers) are at least close to fully activated.

The model of activation through dimerization of active sites suggests an alternative interpretation of the 8% "basal" dimer activity: a small fraction of monomers might associate such that the two active sites are juxtaposed to form a 100% active complex (Fig. 6A, Type I), whereas the dimers formed as highlighted in the TPP II structure (Fig. 6A) are inactive. According to this hypothesis, the activity of monomers should be zero, and the activity determined would reflect the relative concentrations of both dimeric forms. Because monomers are rather short-lived intermediates of TPP II-complex assembly, they cannot be isolated on SEC without assembly into dimers or even larger complexes. Thus, whether the observed specific activity of dimers is 8% throughout, or on the average, remains an open question. However, the fact that we determined a Km of ~2-fold for dimers compared with spindles (see above) supports the idea that dimers indeed have a basal activity.

As mentioned above, the path of activation need not proceed linearly via alternating assembly and activation steps. A scheme representing the initial stage of assembly and activation is proposed in Fig. 6C. With growing concentration of TPP II, the bimolecular assembly reaction becomes increasingly favored over monomolecular activation, leading to preference of the vertical reaction path. The bypass of activation of the tetramer by formation of a partially activated hexamer from a non-activated tetramer (Fig. 6C) is meant to be exemplary for the skipping of activation steps. Because assembly does not depend on previous activation, and because all non-activated states are finally activated, a plethora of states may thus be populated transiently throughout the assembly/activation period, as indicated by the presence of full-size oligomers having from basal to full activity (Fig. 4B).

Functional Implications of Stability and Size-dependent Activity—Based on the observation of a decrease of specific activity of TPP II dissociated under different non-natural conditions, the idea of assembly/disassembly of the TPP II holo-complex as a way of regulating enzymatic activity has been put forward and reiterated (2, 29, 30, 33). We consider this idea unlikely for the following reasons: (i) Assembly/disassembly and activation of TPP II is a reaction much too slow to be utilized by the cell as a response to environmental stimuli. In contrast, a rapid modulation of activity may occur via substrate concentration, according to Michaelis-Menten kinetics. (ii) Disassembly, at least in Drosophila, leads to mainly tetramers that are still ~50% active. (iii) In vitro, spindles of recombinant TPP II are thermodynamically stable at a concentration such as prevailing in Drosophila eggs. Moreover, because of their high molecular mass and subunit number, large oligomeric states are strongly stabilized in vivo by molecular crowding. For instance, the enhancing effect of molecular crowding on the self-association of bovine pancreatic trypsin inhibitor into a decamer is drastic: the addition of 14% dextran increased the equilibrium constant 5105-fold and the concentration of decamers 30-fold (40). Thus, the spindle structure of TPP II is most likely to be quite stable in vivo, which is corroborated by experimental data obtained with Drosophila (24).

Distinction from Functionally Related Proteins—To our knowledge the model of activity enhancement upon assembly presented here is unique. A common activation mechanism of enzymes is phosphorylation such as observed in the close functional relative thimet oligopeptidase (41). However, we can exclude this possibility for Drosophila TPP II, because the recombinant complex expressed in E. coli has the same specific activity as the native protein.

Tricorn, a functional relative of TPP II in some archaea,isthe only peptidase known to form assemblies similar in size, but in contrast to TPP II, the 720-kDa hexameric form appears to represent the "ground state," and icosahedral capsid formation does not enhance the activity (26). Other peptidases acting at the late stage of the proteolytic cascade are much smaller. These include thimet oligopeptidase, which is a 70-kDa monomer (41), the members of the prolyloligopeptidase family (~80 kDa), which includes acylaminopeptidase, dipeptidylpeptidase IV, oligopeptidase B, and prolyloligopeptidase (42), as well as TPP I (280 kDa hexamer), the lysosomal counterpart of TPP II (43).


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
This work shows that the TPP II holo-complex is indeed built of dimeric modules and that the specific activity of TPP II is correlated with its strand length in a hyperbolic fashion. We also demonstrate the pronounced concentration dependence of TPP II oligomer size, resulting in a mass action-dependent heterogeneity of single strand length. In contrast, within a concentration window of roughly 0.030-3 mg/ml, spindles are the strongly prevailing form of the TPP II complex in vitro. This is in accordance with the situation in Drosophila eggs, where the spindle-shaped holo-complex is the highly predominant, if not the exclusive form.

We conclude that, by locking two decamers of dimers into a relatively stable complex, strand pairing of TPP II substantially stabilizes a state of high specific activity, both kinetically and thermodynamically. However, whereas this is an "economical" reason for the organization of TPP II dimers into the spindle-shaped complex, we cannot as yet explain why this complex is so huge in the first place, being almost two orders of magnitude larger than the small, functionally related peptidases. Therefore, the possibilities of (i) structural relationships with functionally associated proteins, (ii) special requirements for compartmentalization and, finally, (iii) an impact of the unique structural organization on specificity and quantity of exopeptidase/endopeptidase activity need to be explored. The issues of activity and specificity will have to be addressed using native substrates.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 49-89-8578-2698; Fax: 49-89-8578-2641; E-mail: Peters{at}biochem.mpg.de.

2 The abbreviations used are: TPP II, tripeptidylpeptidase II; LB, Luria-Bertani medium; TB, Terrific Broth; IPTG, isopropyl-beta-D-thiogalactopyranoside; AAF-AMC, Ala-Ala-Phe-7-amino-4-methylcoumarin; DTT, dithiothreitol; SEC, size exclusion chromatography; PEI, polyethyleneimine; EM, electron microscopy; GdnHCl, guanidine hydrochloride. Back

3 J. Peters, unpublished data. Back

4 J. Peters, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Brigitte Kühlmorgen and Marietta Peters for excellent technical assistance. We are also grateful to Bing Jap and Peter Zwickl for reading the manuscript and to Allen Minton, Martin Beck, and Harald Engelhardt for discussions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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