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Disabling the folding catalyst is the last critical step in -lytic protease folding

¹ Graduate Group in Biophysics,
² Howard Hughes Medical Institute, and
³ Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94143-2240, USA

Reprint requests to: David A. Agard, Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California at San Francisco, 600 16th Street, Room S412, San Francisco, CA 94143-2240, USA; e-mail: agard{at}msg.ucsf.edu; fax: (415) 476-1902.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Alpha-Lytic protease (LP) is an extracellular bacterial pro-protease marked by extraordinary conformational rigidity and a highly cooperative barrier to unfolding. Although these properties successfully limit its proteolytic destruction, thereby extending the functional lifetime of the protease, they come at the expense of foldability (t_1/2 = 1800 yr) and thermodynamic stability (native LP is less stable than the unfolded species). Efficient folding has required the coevolution of a large N-terminal pro region (Pro) that rapidly catalyzes LP folding (t_1/2 = 23 sec) and shifts the thermodynamic equilibrium in favor of folded protease through tight native-state binding. Release of active LP from this stabilizing, but strongly inhibitory, complex requires the proteolytic destruction of Pro. LP is capable of initiating Pro degradation via cleavage of a flexible loop within the Pro C-terminal domain. This single cleavage event abolishes Pro catalysis while maintaining strong native-state binding. Thus, the loop acts as an Achilles’ heel by which the Pro foldase machinery can be safely dismantled, preventing Pro-catalyzed unfolding, without compromising LP native-state stability. Once the loop is cleaved, Pro is rapidly degraded, releasing active LP.

Keywords: -Lytic protease; protein folding; pro region; secondary cleavage; degradation

Abbreviations: LP, -lytic protease • Pro, wild-type pro region • Int, the intermediate state of LP • Nat, the native state of LP • Pro•Nat, complex of Pro with native of LP • TEV, tobacco etch virus protease • ProTEVloop, Pro with the disordered loop replaced with a TEV protease recognition site • CD, circular dichroism

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03389704.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Secreted by the soil bacterium Lysobacter enzymogenes, Alpha-Lytic protease (LP) is a chymotrypsin-like serine protease (Whitaker 1970; Brayer et al. 1979) that has evolved to resist degradation in harsh, proteolytic environments (Jaswal et al. 2002). LP achieves a remarkable level of kinetic stability via a large and highly cooperative unfolding barrier of 26 kcal/mole (t_1/2 = 1.2 yr), which prolongs the functional lifetime of the protease despite the thermodynamic metastability of the LP native state (Nat) (Sohl et al. 1998; Jaswal et al. 2002; Truhlar et al. 2003). Although this kinetic stability effectively suppresses proteolytic sensitivity, it appears to come at the heavy cost of an extremely high folding barrier (30 kcal/mole; Sohl et al. 1998). Under native conditions, LP folds to an on-pathway molten-globule intermediate (Int) incapable of converting to the native protease on a biologically feasible timescale (t_1/2 = 1800 yr). Efficient folding of LP is only realized in the presence of its requisite Pro (Silen et al. 1989; Baker et al. 1992; Sohl et al. 1998).

Like most extracellular bacterial proteases, LP is synthesized with a covalent N-terminal pro region extension (Baker et al. 1993). Whether supplied as an attached peptide sequence, as in the naturally occurring protein, or as a separate polypeptide, Pro is essential for the proper folding of LP (Silen and Agard 1989; Silen et al. 1989). Detailed examination of LP’s Pro-mediated folding mechanism revealed that Pro acts as a folding catalyst, preferentially stabilizing the folding transition state and thereby accelerating the folding rate 3x10⁹-fold (Sohl et al. 1998). Recent findings, in combination with earlier studies, indicate a model of Pro-catalyzed folding in which binding of both Pro N- and C-terminal domains serves to arrange key structural elements of the LP C-terminal domain, allowing the remainder of the LP N and C domains to dock and fold, thus completing the nascent active site (Peters et al. 1998; Sauter et al. 1998; Cunningham et al. 2002; Cunningham and Agard 2003). Intramolecular processing of the Pro-LP junction separates the protease from its pro region, with the newly formed LP N terminus repositioning to its native conformation and the Pro C-terminal tail remaining bound to the active site in an inhibitory manner (Sohl et al. 1997; Sauter et al. 1998). Indeed, Pro maintains a tight association with native LP, burying >4000 Ų in the Pro•Nat interface (Sauter et al. 1998). In this way, Pro further promotes LP folding by driving the thermodynamic equilibrium in favor of the Pro•Nat complex.

As the strongest known inhibitor of LP (K_i = 0.32 nM), Pro binding greatly stabilizes the folded protease (Peters et al. 1998); however, active LP must eventually break free of this inhibitory complex in order to fulfill its biological role of degrading other soil microorganisms and providing nutrients to its host. Intermolecular cleavage of secondary cleavage sites within Pro by LP or other exogenous proteases is presumed to lead to the extracellular destruction of Pro (Sauter et al. 1998; Cunningham et al. 2002). Pro degradation not only releases active LP, but perhaps more important, it prevents the Pro folding catalyst from accelerating the LP unfolding reaction as well.

This paper investigates this final, but critical, step of the Pro-catalyzed LP folding reaction: the necessary destruction of Pro that liberates native, active protease and safeguards LP from catalyzed unfolding. Here we show that LP-mediated proteolysis of its Pro folding catalyst occurs via a secondary cleavage event within a disordered loop located in the Pro C-terminal domain. In addition to enabling the rapid destruction of Pro, secondary cleavage in this loop significantly and selectively weakens Pro stabilization of the folding transition state, thus effectively eliminating Pro’s ability to catalyze LP unfolding.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Degradation of Pro by LP
During the course of Pro-catalyzed LP folding reactions, the production of active LP results in Pro degradation. N-terminal sequencing and MALDI mass spectrometry have been used to characterize this proteolysis of Pro. Coomassie-stained SDS-PAGE shows three major Pro degradation bands present in the folding reactions, in addition to the band of uncleaved Pro starting material (Fig. 1). N-terminal sequencing of all four bands revealed the same sequence (PADQV), which corresponds to the N-terminal sequence of Pro, minus the initial methionine that is presumably removed by the host methionine aminopeptidase. The full-length Pro sequence is therefore P2-T168 (sequential numbering), as previously observed in earlier studies of Pro (Sauter et al. 1998).

View larger version (33K): [in this window] [in a new window]   Figure 1. Identification of Pro degradation fragments created by LP proteolysis. Pro (20 µM) refolds Int (2 µM) to native LP, which in turn degrades Pro through a series of fragments identified using MALDI mass spectrometry and N-terminal sequencing (Table 1). Initial cleavage occurs within a flexible loop located in the Pro C-terminal domain. Pro alone shows no degradation under the same conditions.

View larger version (18K): [in this window] [in a new window]   Figure 2. A time course of Pro degradation by LP. The relative amounts of Pro and Pro fragments created during the catalyzed folding of Int (2 µM) by Pro (20 µM) are shown plotted as a function of time. The cleavage of full-length Pro (circles) occurs at approximately the same rate as the appearance of the 12.6-kD (squares), 9.32-kD (triangles), and 7.37-kD (diamonds) degradation fragments, as determined by single exponential fits of the data (0.63 ± 0.046 min-1, 0.81 ± 0.15 min-1, 0.94 ± 0.26 min-1, and 0.78 ± 0.16 min-1, respectively). Direct addition of native LP resulted in vastly increased cleavage rates; yet, the extent of Pro degradation was only dependent on the final concentration of native LP present (data not shown).

Introduction of the reengineered loop decreases Pro stability, but does not significantly alter Pro function (Table 2). As observed by far UV CD, TEV proteolysis of the loop further destabilizes ProTEVloop, yet structure is restored on binding to native LP (data not shown). The similarities between the intact and cleaved ProTEVloop•Nat complex CD spectra indicate that although the ProTEVloop_cut fragments are no longer covalently attached, they both associate with LP in the native-state complex in a manner analogous to intact ProTEVloop.

View larger version (22K): [in this window] [in a new window]   Figure 3. ProTEVloop inhibition of native LP. Increasing amounts of ProTEVloopuncut (filled circles) and ProTEVloopcut (open circles) reduce the proteolytic activity of 6 nM and 0.25 nM native LP, respectively. ProTEVloopuncut displays tight-binding inhibition of LP (Ki = 0.26 ± 0.071 nM) similar to that of wild-type Pro (Table 2). Loop cleavage results in an approximately fourfold loss in affinity for native LP (Ki = 1.4 ± 0.097 nM), with ProTEVloopcut acting as a simple competitive inhibitor instead of a tight-binding inhibitor.

View larger version (19K): [in this window] [in a new window]   Figure 4. The effect of loop cleavage on the catalyzed folding of LP. (A) A full enzymatic profile of ProTEVloopuncut-catalyzed LP folding, in which catalyzed folding rate constants are plotted as a function of ProTEVloopuncut concentration and the data are fit to a variant of the Michaelis-Menten equation, gives kinetic constants (kcat and KM) within error of those for wild-type Pro (Table 2). (B) The fraction of Int (0.8 µM) refolded to native LP, both in the absence of catalyst (filled squares) and in the presence of either 3.75 µM intact (filled circles) or TEV-cleaved (open circles) ProTEVloop, is plotted as a function of time. The resulting progress curves illustrate that although ProTEVloopcut accelerates folding (kobs = 4.2x10-5 ± 0.29x10-5 min-1) over that of the uncatalyzed reaction (inset), this rate of refolding is substantially slower than the ProTEVloopuncut-catalyzed folding reaction (kobs = 0.37 ± 0.036 min-1). (C) The addition of increasing amounts of ProTEVloopcut to wild-type Pro-catalyzed refolding reactions retards initial refolding rates, as ProTEVloopcut competes for Int binding (Ki = 73 ± 13 µM), but cannot catalyze refolding to native LP (see Table 2).

Because the extremely slow rate of ProTEVloop_cut-catalyzed folding is only a minor component of the overall refolding reaction under these conditions, it is impossible to accurately determine K_M and k_cat values for the TEV-cleaved variant from a full enzymatic profile as was done for ProTEVloop_uncut (see Fig. 4A). Yet, it is mechanistically quite important to discern whether loop cleavage specifically debilitates catalysis (k_cat), or greatly weakens initial binding to Int (K_M). Therefore, the effect of loop cleavage on Int binding was measured through the ability of ProTEVloop_cut to compete with wild-type Pro-catalyzed LP refolding. In Figure 4C, we see that addition of ProTEVloop_cut to wild-type Pro-catalyzed LP refolding reactions does not enhance, but instead, slows the initial refolding rate, as ProTEVloop_cut appears to effectively compete with wild-type Pro for Int binding, but is then unable to catalyze folding to Nat. Although aggregation of ProTEVloop_cut at concentrations higher than 20 µM limits this analysis, a fit of the data to the competitive inhibitor variant of the Michaelis-Menten equation estimates ProTEVloop_cut affinity for Int to be ~70 µM (see Table 2). Thus, Pro binding to Int is only weakened approximately fourfold by secondary cleavage, making the apparent 10⁷-fold ProTEVloop_cut folding defect primarily a k_cat effect.

Kinetic modeling of ProTEVloop-catalyzed folding
Returning to the ProTEVloop_cut-catalyzed folding reaction shown in Figure 4B, we now know that LP folding is catalyzed by trace amounts of ProTEVloop_uncut because loop cleavage effectively abolishes ProTEVloop catalysis. Yet, although the observed rate of LP refolding correlates with the concentration of residual ProTEVloop_uncut present in the reaction, neither the refolding rate, nor the amount of LP refolded, agree with values predicted from the ProTEVloop_uncut kinetic constants (see Table 2). Having determined these rate and affinity constants for both ProTEVloop variants, we used these kinetic parameters to model ProTEVloop-catalyzed folding, using the Berkeley Madonna modeling and analysis software (http://www.berkeleymadonna.com), to better understand this anomalous behavior. Comparisons of direct measurements and simulated data from the model (Fig. 5) reveal that, as expected, refolding of Int by substoichiometric quantities of uncut ProTEVloop is dominated by singleturnover kinetics, in which the tight association of the ProTEVloop_uncut•Nat complex prevents multiple rounds of refolding catalysis. However, when micromolar concentrations of TEV-cleaved ProTEVloop are also present, competition for native LP binding appears to free intact ProTEVloop_uncut to refold multiple Int molecules, consistent with experimental observations. Under these conditions, the LP refolding rate is not dependent on the rate of ProTEVloop_uncut catalysis, but instead on the rate at which ProTEVloop_uncut is released from its complex with native LP.

View larger version (17K): [in this window] [in a new window]   Figure 5. Modeling of ProTEVloop-catalyzed LP refolding demonstrates multiple turnover. The fraction of Int (0.8 µM) refolded to native LP by 24 nM ProTEVloopuncut, alone (filled circles) and in the presence of 9 µM ProTEVloopcut (open circles), is plotted as a function of time with data generated from kinetic simulations of modeled ProTEVloop-catalyzed folding reactions (solid lines) for both refolding scenarios. The kinetic model is consistent with the observed folding behaviors, including the increase in LP folding seen on addition of excess, but catalytically inactive, ProTEVloopcut. Competition for native LP binding appears to allow ProTEVloopuncut to catalyze multiple rounds of LP folding.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

As demonstrated by the kinetic analysis of intact and cleaved ProTEVloop-catalyzed LP folding reactions, loop cleavage also asserts a dramatic and selective effect on transition-state stabilization. Whereas loop cleavage nearly abolishes stabilization of the folding transition state, the affinity of ProTEVloop_cut for either Int or Nat ground states is only reduced approximately fourfold compared with uncleaved ProTEVloop, or WT Pro. This minor reduction in affinity for Int and native LP likely arises from the instability of ProTEVloop_cut, as binding energy must be diverted to the restructuring of unfolded ProTEVloop_cut.

In all previous LP refolding studies, Pro has behaved as a single-turnover catalyst, with tight native-state binding preventing Pro from being released to fold multiple Int molecules. Interestingly, in refolding reactions containing both forms of ProTEVloop, the strong native-state binding of the catalytically inactive ProTEVloop_cut enables it to compete with intact ProTEVloop for binding to native LP, freeing the potent ProTEVloop_uncut catalyst to facilitate multiple rounds of folding. Kinetic modeling of these reactions agrees well with the observed refolding behaviors, which provide the first evidence of multiple turnover in pro region-mediated refolding, further supporting the idea that pro regions function as true folding catalysts.

Most important, differential binding of Pro to the native state versus the folding transition state upon loop cleavage secures the success of the LP folding reaction despite the thermodynamic instability of the folded product. The single cleavage event within the flexible Pro loop effectively disables Pro catalysis while maintaining a strong affinity for the native protease, thereby ensuring native-state stabilization while simultaneously neutralizing Pro’s catalytic activity. This highly selective and ordered deactivation of the Pro folding catalyst prevents native LP from undergoing catalyzed unfolding to the thermodynamically favored Int and unfolded states. Thus, as with many aspects of LP folding and stability, the mechanism of Pro destruction also appears to be highly optimized. Once Pro is destroyed, the LP folding pathway is completed, leaving the protease kinetically trapped in its active conformation by a large and extremely cooperative unfolding barrier that imparts LP with a remarkable resistance to proteolysis.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
ProTEVloop construct
Silent mutations were introduced into the Pro gene (pT7XmaPro plasmid; Sohl et al. 1997) using PCR and QuickChange mutagenesis to create two unique restriction sites, MfeI and SnaBI (pT7XmaProMS). Pro loop residues 107–115 were replaced with the TEV protease recognition sequence (ENLYFQG) by ligating a synthetic oligonucleotide cassette into the pT7XmaProMS plasmid digested with MfeI and SnaBI. All oligonucleotides were synthesized by Invitrogen and mutations were verified by sequencing.

Protein expression and purification
Wild-type Pro and ProTEVloop were purified as described (Derman and Agard 2000), with a portion of wild-type Pro further purified by hydrophobic interaction chromatography (Sauter et al. 1998). Both Pro variants contain an additional N-terminal proline residue, a cloning artifact that does not affect the behavior of Pro (Peters et al. 1998). Wild-type LP was prepared according to published protocols (Hunkapiller et al. 1973; Mace and Agard 1995) and a portion was denatured as described (Derman and Agard 2000).

Identification and kinetic analysis of Pro degradation fragments
The production of Pro fragments during Pro-catalyzed LP folding (Sohl et al. 1998) was followed by SDS-PAGE. Protein bands were blotted onto PVDF membrane and visualized by nonacidic Coomassie Brilliant Blue stain, then excised and N-terminally sequenced by Dr. Chris Turck at the Howard Hughes Medical Institute at the University of California at San Francisco. Additional aliquots of the folding reaction were mixed 1 : 1 with 10 mg/mL sinnapinic acid, 60% acetonitrile, and 0.03% TFA and analyzed by MALDI mass spectrometry. Pro fragments with masses that matched the detected mass peaks and that were consistent with LP’s specificity profile (P1 residues in which k_cat/K_M >100; G, A, V, T, S, Q, M, C; Mace 1995) were identified using the PAWS freeware edition software version 8.1.1 (ProteoMetrics).

At various time points, aliquots from Pro-catalyzed refolding reactions were removed to reducing SDS sample buffer that was acidified with 1M HCl such that the resultant gel samples were at pH 5, effectively eliminating artifactual proteolysis during sample denaturation. Samples were run on 20% acrylamide Phast Gels (Pharmacia) and Coomassie stained, and the relative intensities of the protein bands were quantified and normalized within each lane using Kodak 1D Image Analysis Software version 3.5.2.

ProTEVloop cleavage by TEV protease
ProTEVloop was incubated with recombinant TEV protease (Invitrogen) at 4°C and 25°C, according to the manufacturer’s recommended cleavage conditions, until <1% of ProTEVloop remained uncut, as determined by silver-stained SDS-PAGE with uncleaved ProTEVloop standards. The HIS-tagged TEV protease was removed with Ni-NTA resin and ProTEVloop was exchanged into 20 mM potassium succinate (pH 5.6).

Stability measurements
ProTEVloop_uncut stability was analyzed by urea denaturation as described (Cunningham et al. 2002) and in the presence of 15% glycerol to better define the folded baseline. Determination of ProTEVloop_cut stability was limited to an upper estimate of G <0, as the cleaved mutant was unstructured in the absence of urea.

Inhibition measurements
Tight-binding inhibition of 6 nM LP by ProTEVloop_uncut was determined as described (Cunningham et al. 2002). A modified inhibition assay developed for unstable Pro mutants (Cunningham et al. 2002) was used to assess inhibition of 0.25 nM LP by ProTEVloop_cut.

ProTEVloop-catalyzed folding
ProTEVloop_uncut-mediated folding of LP was performed and analyzed as described (Derman and Agard 2000), except the amplitudes of the biphasic refolding curves were fixed at a consensus ratio of 65 : 35 (fast phase : slow phase).

The catalyzed folding of LP by ProTEVloop_cut was sufficiently slow as to require the use of a very sensitive thiobenzyl ester substrate assay (Sohl et al. 1998; Derman and Agard 2000). Int (1 µM) was incubated alone and in the presence of 2–17.5 µM ProTEVloop_cut and LP refolding was monitored as described (Cunningham et al. 2002). The resultant initial rates of refolding were fit to the linear portion of the exponential refolding curves to determine the k_obs magnitude for each refolding reaction.

Initial binding of ProTEVloop_cut to Int was determined through competition assays with wild-type Pro in which the initial rate of wild-type Pro-catalyzed LP refolding was observed in the presence of increasing amounts of ProTEVloop_cut and the data were fit to the competitive inhibitor variation of the Michaelis-Menten equation to extract a K_i,Int (~K_M) for ProTEVloop_cut. For all ProTEVloop_cut refolding reactions, the concentration of uncleaved ProTEVloop contaminant was quantified by gel densitometry of silver-stained SDS-PAGE of ProTEVloop_cut stocks and ProTEVloop_uncut standards.

Data analysis
Data analysis was performed using the Kaleidagraph version 3.08 (Synergy Software) unless otherwise specified. Modeling of ProTEVloop-mediated refolding reactions was done using the Berkeley Madonna version 8.15 kinetic simulation software (http://www.berkeleymadonna.com).

	Acknowledgments

 
We thank Dr. Luke Rice and Stephanie Truhlar for many helpful discussions and Peter Chien for his assistance with the PAWS analysis software. This work was supported by the Howard Hughes Medical Institute.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cunningham, E. L. Articles by Agard, D. A. Search for Related Content PubMed PubMed Citation Articles by Cunningham, E. L. Articles by Agard, D. A. Social Bookmarking                   What's this?