Folding Pathway Mediated by an Intramolecular Chaperone

A FUNCTIONAL PEPTIDE CHAPERONE DESIGNED USING SEQUENCE DATABASES*

Yukihiro Yabuta, Ezhilkani Subbian, Catherine OiryDagger, and Ujwal Shinde§

From the Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239-3098

Received for publication, November 25, 2002, and in revised form, January 14, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha -beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT·subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subtilisin E is an alkaline serine protease isolated from Bacillus subtilis (1). In vivo this protein is produced as pre-pro-subtilisin (2), wherein the pre-region (signal peptide) facilitates protein secretion and the pro-region (propeptide) functions as an intramolecular chaperone that guides correct folding of subtilisin (2-6). Upon completion of folding, the propeptide is autoproteolytically removed (7). This is necessary because the propeptide is a potent inhibitor of subtilisin activity (4).

Propeptide-mediated folding mechanisms exist in several unrelated protease families. However, there is no sequence conservation in propeptide domains within these families (8). This functional conservation across unrelated protease families suggests that propeptides have evolved in multiple parallel pathways and may share a common mechanism of action (9). Subtilisin (2-6) and alpha -lytic protease (10-13) constitute the best-studied examples of propeptide-mediated protein folding. Bacterial subtilisins are models for the subtilase family that spans prokaryotes and eukaryotes. Amino acid sequence analysis of this family establishes that although the subtilisin domain is conserved, the cognate propeptides can vary significantly (8). Because propeptides perform functions different from the catalytic domains, they may be subjected to different mutational frequencies because of different functional constraints (14-16). However, given that they impart structural information to their catalytic domains (17-19), propeptides within one family could adopt similar structural scaffolds despite digressions in their polypeptide sequences. This makes propeptides attractive models for protein redesign and for understanding the relation between sequence and structure.

To establish minimum sequence requirements for a functional propeptide, in this study we have designed and developed a peptide chaperone using information databases on the subtilase family. This family is ideal because enormous structural and functional information on individual members is already available (8). The peptide sequence (ProD)1 was designed through a decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment. The DNA sequence coding for ProD was synthesized and subsequently expressed in Escherichia coli, and the resulting peptide was purified. Despite a difference of 5 pH units between their isoelectric points (pI) and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed two-dimensional structure and the distribution of hydrophobic residues within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha -beta conformation as an isolated peptide. The CD structure of ProD·subtilisin was found to be similar to ProWT·subtilisin. Hence, despite low sequence identity and opposite net charge, ProD and ProWT adopt similar structural scaffolds. Our results suggest conserved structural scaffolds and hydrophobicity, but not absolute charge, dictates propeptide function.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Algorithm for Designing a Peptide Chaperone-- The PSI-BLAST (position-specific iterated basic local alignment search tool) program (20) was used to obtain a multiple sequence alignment of proteins that are similar to ProWT (77 residues) using a threshold E-value of 0.8. This alignment was treated as a matrix and utilized to obtain the percentage of every individual amino acid at every position in the 77-residue polypeptide. The matrix contained 176 homologues of subtilisin. The computed percentage was employed in a decision-based algorithm that searched for the following criteria. If "X" is the most abundant amino acid residue and "Y" is the second most abundant residue at a particular position in the sequence, then residue "X" is always chosen over Y UNLESS (frequency(X) <=  A* frequency(Y)) AND residue X is present in ProWT, where "A" represents a "cutoff factor."

In the present analysis A = 2 and no two residues at any position display identical frequencies. The aim of the algorithm was to establish the absolute minimum sequence requirement within divergent propeptide sequences. The computed sequence ProD, which was designed to digress from ProWT, displays 16% sequence identity and opposite net charge (Fig. 1A).

Gene Synthesis of ProD-- The nucleotide sequence corresponding to ProD was obtained using the BACK-TRANSLATE module from the Genetics Computer Group Wisconsin Package program (21) and was based on the E. coli codon usage table. The nucleotide sequence was synthesized using a combination of DNA synthesis and PCR. The product was cloned into pET11a under the control of the T7-promoter (4), and the nucleotide sequence was verified on an ABI-310 DNA sequencer.

Expression and Purification of ProD and S221C-subtilisin-- E. coli BL21(DE3) cells were transformed with plasmids pET-Pro-S221C-subtilisin and pET-ProD to obtain S221C-subtilisin and ProD, respectively. Cells were grown in M9 medium supplemented with 50 µg/ml ampicillin (22). At an absorbance of 0.6 A600 nm, the culture was rapidly cooled to 12 °C. Adding isopropyl beta -D-thiogalactopyranoside to a final concentration of 1 mM induced protein expression. After 16 h at 12 °C, the cells were harvested as described earlier (4). Under these conditions Pro·S221C-subtilisin and ProD remain in the soluble fraction. The samples were then centrifuged at 20,000 × g to remove the insoluble cell debris. The supernatant that contained Pro·S221C-subtilisin was then incubated at 4 °C to allow the degradation of the inhibitory ProWT through cellular proteases. The cell lysates were dialyzed overnight (using a 3-kDa cutoff) against 100 vol of 50 mM Tris-HCl, pH 8.6, and 1 mM CaCl2. The proteins were loaded onto a HighQ column (BioRad) equilibrated with 50 mM Tris-HCl, pH 8.6, and then eluted using a 0-1 M NaCl gradient. Fractions containing the desired proteins were pooled together and dialyzed against 50 mM Tris-HCl, pH 7.0, containing 0.5 M ammonium sulfate, 2 mM CaCl2 and concentrated by ultra-filtration using a 3-kDa cutoff membrane. Concentrated ProD was further purified using a Superdex-75 gel-filtration column (Amersham Biosciences). Both ProD and S221C-subtilisin were purified using the above approach. The only difference was that a Superdex-200 gel-filtration column was used to obtain mature S221C-subtilisin.

Trans Folding of Denatured Subtilisin-- 6 M guanidine hydrochloride denatured subtilisin (0.9 µM) was mixed with varying concentrations of ProD (0-36 µM) to a final volume of 100 µl. ProWT was used as a control. The mixture was dialyzed for 16 h against 50 mM MES, pH 6.5, containing 0.5 M ammonium sulfate and 1 mM CaCl2. A 20-µl aliquot of the renaturation mixture was treated with ~7.0 units/ml of TPCK-treated trypsin for 1 h to degrade the excess of propeptide. This is necessary because the propeptide can also inhibit subtilisin activity (3). Enzyme activity was measured using N-suc-AAPF-pNA as a synthetic substrate (23). TPCK-treated trypsin does not cleave the synthetic substrate and hence does not interfere with the activity assay.

Inhibition of Subtilisin E Activity-- Active subtilisin (65 nM) was rapidly added to the protease assay buffer that contained 0.5 µM synthetic substrate and 0.9 µM of ProD and ProWT. Protease activity was monitored as described earlier (4, 22, 23).

Complex Formation between ProD and S221C-Subtilisin-- ProD and ProWT (6.4 µM) were added to an equimolar amount of mature S221C-subtilisin in the folding buffer (50 mM MES, pH 6.5, 0.5 M ammonium sulfate, 2 mM CaCl2, and 1 mM beta -mercaptoethanol). The mixtures were incubated overnight at 4 °C to allow complex formation.

Trypsin-mediated Trans Degradation of ProD and ProWT Complexes with S221C-subtilisin-- The trans complex was obtained as discussed above. The ProD- and ProWT·S221C-subtilisin complexes (50 nM) were incubated with TPCK-treated trypsin (2.0 units/ml), and aliquots were removed at different time intervals. The reaction was terminated through trichloroacetic acid precipitation as described earlier (4), and the aliquots were separated using SDS-PAGE. The amount of residual propeptide was estimated using quantitative gel-scanning densitometry.

Circular Dichroism and Fluorescence Measurements-- CD measurements were performed on an automated AVIV 215 spectrometer at 25 °C. Protein concentration was maintained between 0.05-0.2 mg/ml (4). Spectra were taken between 190-260 nm using a 1-mm path length cuvette (Fig. 2C) and represent averages of three independent scans. For the fluorescence measurements, the denatured and folded ProD (7.0 µM) were excited at 295 nm, and the emission spectra from 310 to 410 nm were recorded as described earlier (23).

Molecular Modeling-- Homology modeling was performed using LOOK, Version 2.0. The sequence alignment was performed using the BestFit module of the Genetics Computer Group software (21), and the alignment was imported into LOOK. A structural model for ProD·S221C-subtilisin was built, and the energy minimization was carried out using the GROMOS 43B1 force field using the Swiss-Model software, Version 3.7 (25). Because the ProWT and ProD contain more than 36% charged residues, the electrostatic potentials were computed using coulomb interactions by taking into account the charged residues and assuming a pH of 7.0 (25). The electrostatic charges were mapped to the molecular surface (Fig. 4B).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Criteria for Designing and Selecting ProD-- The algorithm described under "Experimental Procedures" was used to obtain a matrix that was analyzed using the following criteria. If "X" is the most abundant amino acid residue and "Y" is the second most abundant residue at a particular position in the sequence, then residue "X" is always chosen over Y UNLESS (frequency(X) <=  A*frequency(Y)) AND residue X is present in ProWT, where "A" represents a "cutoff factor." Fig. 1D depicts the relation between the cutoff factor "A" and the sequence identity of the computed sequences to ProWT. It is evident from Fig. 1D that as the cutoff factor increases the percent identity decreases according to two different slopes. In the present analysis the value of the cutoff factor was selected as 2 because the transition in slopes occurs when A = 2. If for example, frequency(X) = 70%, then frequency(Y) <=  30%; then according to our criterion X was selected because (frequency(X) >=  2* frequency(Y)). If, however, frequency(X) = 60% and frequency(Y) = 40% AND residue X was present in ProWT, then residue Y was selected over X because (frequency(X) <=  2 * frequency(Y)). However, if frequency(X) = 60% and frequency(Y) = 40% AND residue X was NOT present in ProWT, then residue X was selected in the computer-generated sequence. Although it is possible that the frequencies of X or Y at any position may be identical, during the analysis of 176 subtilisin homologues this scenario did not arise when A = 2. Because no two residues at any position displayed identical X or Y frequencies, this algorithm provided us with a unique sequence for ProD (Fig. 1A).


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Fig. 1.   Design criteria and computational characterization of proD. A, primary sequence alignment between the subtilisin propeptide (ProWT) and the redesigned propeptide (ProD). Identical residues (black background), highly conserved substitutions (gray background), and less conserved substitutions (boldface) were identified using the program BestFit (21). The secondary structure of ProWT (obtained from the x-ray structure) when it forms a complex with subtilisin is depicted above the sequence alignment; the secondary structure computed using PREDICT-PROTEIN (34) is depicted below the sequence alignment. C, E, and H denote coils, beta -sheets, and alpha -helices, respectively. The predicted structure in boldface represents predictions that do not match with the secondary structure obtained from the crystallized complex of ProWT·subtilisin. Motifs N1 and N2 represent the conserved domains within the subtilase family (18). Asterisks denote residues that constitute the hydrophobic core within the propeptide. B, the difference in the amino acid composition between ProWT and ProD. Positive values represent those residues that are in excess in ProWT; negative values indicate residues that are in excess in ProD. C, hydropathy plot comparison between ProD (black line) and ProWT (gray line). D, change in sequence identity of the computed peptide with ProWT as a function of Cutoff Factor (A).

ProD Displays Low Sequence Identity with ProWT-- Fig. 1A compares the primary sequence of ProD with ProWT. N1 and N2 represent motifs that were identified using closely related subtilases and appear to be fairly well conserved (26). The sequence identity in Motif N1 and the C-terminal part of Motif N2 is significant, and these motifs are located in beta 1 and alpha 2 -beta 4, as seen from the x-ray crystallographic structure of ProWT (28). NMR structure analysis suggests that beta 1 and alpha 2-beta 4 display conformational rigidity and may represent potential folding nucleation sites in ProWT (27). Hence, motifs that nucleate the folding process appear to be conserved in ProD. When the difference in the total number of individual amino acids between the two propeptides is examined (Fig. 1B), it is clear that ProWT (pI = 9.76) has an excess of basic residues, whereas ProD (pI = 4.52) is rich in acidic amino acids. However, the hydrophobic patterns within these two peptides are similar (Fig. 1C), and the residues that constitute the buried hydrophobic core in ProS and ProD are always non-polar (Fig. 1A). Properties of ProD and ProWT are summarized in Table I.


                              
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Table I
Properties of the isolated peptide chaperones

ProD Can Inhibit Subtilisin Activity and Chaperone Folding of Denatured Subtilisin-- It has been established that ProWT inhibits subtilisin through slow binding inhibition kinetics (29). Because the primary sequence and the amino acid composition between ProWT and ProD are significantly different, we examined whether ProD can inhibit the proteolysis of the synthetic substrate and whether this inhibition follows slow binding kinetics (Fig. 2A). From Fig. 2A, it is evident that in 10 min the activity of subtilisin (65 nM) drops ~12- and 135-fold in the presence of 0.9 µM of ProWT and ProD, respectively. Hence under these conditions, ProD is an ~10-fold better inhibitor than ProWT and does not demonstrate slow binding inhibition. At lower concentrations, however, ProD demonstrates slow binding inhibition (Fig. 2A, inset), whereas the same concentration of ProWT is not inhibitory (data not shown). We next examined whether ProD can chaperone folding of subtilisin in a trans folding reaction. Denatured subtilisin was folded using ProD and ProS as a chaperone, and the subtilisin activity that was recovered was measured (see "Experimental Procedures"). Fig. 2B demonstrates that ProWT and ProD can both chaperone folding of subtilisin in trans, and the activity recovered increases with the chaperone concentration. When a 40-fold excess of the chaperone was used, ProWT and ProD recovered ~40 and 10 units of activity, respectively. Hence, under these conditions ProD is ~25% as efficient as ProWT in folding subtilisin (Table I).


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Fig. 2.   Biochemical characterization of ProD. A, inhibition of subtilisin activity as a function of time. Subtilisin (65 nM) rapidly degrades N-suc-AAPF-pNA to produce p-NA, which can be monitored spectroscopically (black line without symbols). The ProWT (open circles and triangles represent 0.8 and 1.2 µM of ProWT, respectively) can inhibit proteolysis of the substrate, and this inhibition follows slow binding kinetics. However, 0.8 (filled circles) and 1.2 µM (filled triangles) of ProD almost completely blocks subtilisin activity. Inset depicts slow binding inhibition of subtilisin by low concentrations of ProD (inverted triangles, 0.45 µM; filled diamonds, 0.30 µM). B, units of subtilisin activity recovered by refolding denatured subtilisin E using ProD (filled circles) and ProWT (open circles) as peptide chaperones. Refolding was carried out as described under "Experimental Procedures," and the amount of denatured subtilisin was maintained at 0.9 µM, whereas the concentrations of ProD and ProWT were varied from 0 to 36 µM. C, kinetics of trypsin mediated degradation of ProD (filled circles) and ProWT (open circles) from their cognate inhibition complexes with S221C-subtilisin. Amount of residual ProD and ProWT were estimated from SDS-PAGE using quantitative gel-scanning densitometry as described under "Experimental Procedures." D, SDS-PAGE analysis of a trans refolding reaction. Mature subtilisin (band A) was refolded using ProWT (band C) and ProD (band D) as described under "Experimental Procedures." The subtilisin-propeptides (1:20 ratio) (lanes 2 and 5) were dialyzed against refolding buffer. After 16 h, subtilisin folded by ProWT and ProD (lanes 3 and 6) were treated with trypsin (band B) as described under "Experimental Procedures." Trypsin degrades the excess of ProWT and ProD within 1 h (lanes 4 and 7) to give active subtilisin.

We next examined the kinetics of trans degradation of ProD and ProWT as complexes with Ser221C-subtilisin by TPCK-treated trypsin. Fig. 2C depicts the kinetics measured using quantitative gel-scanning densitometry as described under "Experimental Procedures." The S221C-subtilisin is proteolytically inactive and is unable to degrade the inhibitory propeptides. This enables one to monitor the kinetics of trans degradation of ProD and ProWT, in the absence of autodegradation. The data were fitted to a single exponential rate equation, and rate constants for degradation of ProD and ProWT were estimated as 0.09 and 0.31 min-1, respectively. This suggests that in a complex with S221C-subtilisin, ProD is degraded ~3-fold slower than ProWT, which may be attributed to increased structure in the isolated ProD, higher affinity for subtilisin, and fewer cleavage sites for trypsin. To examine whether lower activity recovered by folding mediated by ProD occurs because of incomplete degradation of ProD, an SDS-PAGE analysis of the refolding reaction before and after digestion by TPCK-treated trypsin was performed. Fig. 2D establishes that both ProD and ProWT (at 20-fold molar excess) are completely degraded by 1 h of trypsin digestion. Moreover, the amount of active subtilisin obtained after refolding and trypsin treatment of the ProD folding reaction (Fig. 2D, lane 7) is lower when compared with that of the ProWT reaction (Fig. 2D, lane 4). It is also important to note that active subtilisin produced during folding can also degrade ProD and ProWT. However, allowing the folded subtilisin to degrade the excess of ProD and ProWT does not significantly improve the units of activity recovered (data not shown). Hence, the data presented in Fig. 2D suggest that ProD folds subtilisin less efficiently than ProWT.

ProD Is Structured as an Isolated Peptide and Forms a Complex with Subtilisin-- Because ProD can function both as a chaperone and an inhibitor of subtilisin, we next attempted to analyze the secondary structure of ProD in its isolated form and as a complex with S221C-subtilisin using CD spectroscopy. Equimolar concentrations of ProD and ProWT were added to S221C-subtilisin, and the samples were incubated overnight. The S221C substitution at the active site lowers the proteolytic activity by 10,000-fold and is insufficient to degrade the inhibitory propeptide (28). Far-UV CD spectroscopy establishes that unlike ProWT, which is completely unstructured (3), ProD adopts a well defined alpha -beta conformation (Fig. 3A). Deconvolution of this spectrum suggests that ProD contains 20% alpha -helices and 26% beta -sheets. Hence, as shown in Equation 1, the equilibrium (Keq) between the folded (Profolded) and unfolded states (Prounfolded), which normally favors the unfolded state in ProWT (5), appears to have shifted toward the folded state in the case of ProD,


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Fig. 3.   Biophysical characterization of ProD. A, CD spectroscopy of ProD (thick black) and ProWT (gray) alone (dotted) and as stoichiometric complexes (solid) with mature S221C-subtilisin (thin black line). The difference spectra between ProD·S221C-subtilisin and mature subtilisin alone give the spectra of ProD (black dashes) in a complex with subtilisin. The gray dashed line represents the structure of ProWT when complexed with S221C-subtilisin obtained using the difference spectra. B, tryptophan fluorescence spectra of denatured (dotted line) and folded ProD (dashed line) excited at 295 nm. Arrows indicate the fluorescence maxima. C, near-UV CD spectra for denatured (dotted line) and folded ProD (dashed line). Denatured (thick solid line) and folded (thin solid line) subtilisin is used as a control for folded and unfolded protein.


<UP>Pro<SUB>Unfolded </SUB> </UP><AR><R><C>k<SUB><UP>f</UP></SUB></C></R><R><C><UP>⇌</UP></C></R><R><C>k<SUB><UP>u</UP></SUB></C></R></AR><UP>  Pro<SUB>Folded</SUB></UP> (Eq. 1)
where Keq = kf/ku.

However, the CD spectra of the complexes of ProD and ProWT with S221C-subtilisin appear to be similar. Because the x-ray structure of the ProWT·S221C-subtilisin complex establishes that the conformation of S221C-subtilisin does not change through its interactions with ProWT (28), Fig. 3A suggests that the structure of ProD as a complex with S221C-subtilisin is similar to that of ProWT.

ProD contains one tryptophan, four tyrosine, and five phenylalanine residues. Because phenylalanine, tyrosine, and tryptophan contribute to the fluorescence of a protein and are useful probes for monitoring the solvent accessibility of the side chains, we next examined the intrinsic fluorescence to detect conformational differences between the folded and denatured states of ProD. The protein was excited at 295, and the emission spectra were recorded (Fig. 3B). ProD shows an emission maximum at 352 and 349 nm for denatured and folded ProD, respectively. The shift in maxima to a lower wavelength (blue shift) suggests that the environment around the tryptophan side chain becomes non-polar upon folding and is consistent with a conformational change within the protein. The 10% decrease in fluorescence intensity of folded ProD may occur because of quenching of the neighboring aromatic residues (Fig. 4, A-C).


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Fig. 4.   A, ribbon diagram depicting x-ray structure of the ProWT· subtilisin complex (left) and the homology model for ProD·subtilisin (right). B, the electrostatic potential mapped to the molecular surfaces of the two complexes. Red depicts negative charges. Blue indicates positive charges. C, the side chains that contribute to the hydrophobic core in ProWT and ProD. The extended C terminus interacts with the substrate binding loops to inhibit subtilisin activity.

We next measured the tertiary structure of ProD under denaturing and non-denaturing conditions using near-UV CD spectroscopy. The near-UV CD profiles of denatured and non-denatured ProD differ only marginally and suggest that ProD may not possess a well defined tertiary. However, the lack of a tertiary structure in small peptides in not unusual (30).

Molecular Model of ProD-- From Fig. 2, A-D and Fig. 3, A-C, it is evident that (i) ProD functions as an inhibitor and a chaperone of subtilisin similar to ProWT, and (ii) ProD and ProS adopt similar conformations as complexes with S221C-subtilisin. Hence we attempted to build a homology model of ProD·S221C-subtilisin (Fig. 4A) using the x-ray structure of the ProWT·subtilisin complex (28) as described under "Experimental Procedures." The goal of this model is to obtain the spatial orientation of residues within ProD and ProWT, because such models can guide mutagenesis experiments and hypotheses about structure-function relationships (24). From this model it is evident that the distribution of the electrostatic potential around the complexes formed by ProD and ProWT is remarkably different (Fig. 4B). Whereas ProD appears to be completely negative (acidic), ProWT displays a strongly positive electrostatic potential. This is consistent with the finding that ProD contains a large number of acidic amino acids and ProWT displays a bias for basic residues (Table I; Fig. 1A). However, it is interesting to note that the hydrophobic core within ProD and ProWT appears to be maintained (Figs. 1A and 4C). Hydrophobic interactions can make significant contributions to folding and stability of proteins. The average hydrophobicity of ProD is greater than ProWT. Furthermore, the computed free energy for the transfer of the 10-residue hydrophobic core (Fig. 4C) from an aqueous to a hydrophobic environment stabilizes ProD by ~2.5 kcal (Table I).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have used the subtilase family as a model to analyze the relation between sequence, charge, structure, and function within propeptides. An indigenously developed algorithm provided a novel peptide, ProD, that displays only 16% sequence identity and has a pI 5 pH units lower than ProWT (Table I). However, the overall hydrophobic patterns, the computed secondary structures, and the potential folding nucleation motifs (N1 and N2) appear to be conserved in ProD (Fig. 1, A-C). These properties seem to be important for function because, despite low sequence identity, ProD and ProWT can function as peptide chaperones and potent protease inhibitors (Fig. 2, A and B). However, ProD is a less efficient chaperone and a more potent inhibitor than ProWT. This confirms that the chaperone and inhibitory functions are not obligatorily related (22). The differences in the amino acid composition are also reflected in the secondary structures of the isolated ProWT and ProD measured using CD spectroscopy (Fig. 3A). Unlike ProWT, ProD adopts a well defined alpha -beta conformation as an isolated peptide and can form a stoichiometric complex with mature subtilisin. The CD spectra of these two complexes are similar and suggest that ProD and ProWT adopt similar structural scaffolds. Based on our results, we propose that the chaperoning activity of the propeptide of the subtilisin family appears to be encoded in the ability to adopt a specific conformation.

It is important to note that although the sequence similarity between ProWT and ProD is low, the sequence similarity of the residues that form interface with subtilisin (as judged by three-dimensional structure 1SCJ) is at least 70%. As a result, one would expect the net binding energy of ProWT and ProD to subtilisin to be very similar. However, in the case of ProWT its binding to subtilisin is coupled to its folding. Hence a part of the interaction energy will be spent on ProWT folding, thereby lowering the ProWT-subtilisin affinity (or inhibition constant). The ProD is already folded; it does not need to expend as much energy on its folding when compared with ProWT. This in turn may result in a higher affinity and hence a 10-fold tighter inhibition constant.

Because propeptide domains appear to have emerged through convergent evolution (9), this raises another important question. Are there common structural features that define propeptide function in different protein families? To examine this possibility we compared the structures of the propeptides from subtilisin (PDB identifier, 1SCJ), alpha -lytic protease (1P02), and carboxypeptidase B (1KWM). Fig. 5A suggests that all three proteins display similar structural scaffolds despite there being no sequence and structural similarities between their cognate protease domains (31). Interestingly, potent subtilisin inhibitors (chymotrypsin inhibitor II (2SNI), streptomyces subtilisin inhibitor (3SSI), and eglin C (1CSE)) that have no known chaperone function also display similar structural scaffolds without any sequence similarity (Fig. 5B). Does this structural similarity imply a common evolutionary domain? In other words, have propeptides emerged from a primordial protease inhibitor? The conservation of the structural scaffold in ProD, a chaperone designed to digress from the ProWT sequence while maintaining function, suggests that the ability to adopt a specific alpha -beta conformation along with certain specific interactions appears to be crucial for propeptide function. These functions are to chaperone and to inhibit the protease domain. We suggest that the structural scaffold may contribute to the inhibitory function and certain residue specific interactions may be important for its chaperone function. Such specific interactions may be provided by motifs N1 and N2 in the subtilase family and may vary between families. Although the structural similarities are striking in Fig. 5, A and B, it is also possible that they are purely co-incidental.


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Fig. 5.   The ribbon diagrams for (A) propeptides of subtilisin, alpha -lytic protease, and carboxypeptidase B and (B) ProWT, chymotrypsin inhibitor II, streptomyces subtilisin inhibitor, and eglin C.

The functional conservation in ProD, although having a difference of 5 pH units, suggests that charge per se may not be important for propeptide function. So why is there a large bias for charge in propeptide domains? Also, if the propeptides within a family adopt similar structures and perform similar functions, why do the primary sequences of propeptide diverge faster than their cognate catalytic domains? One possibility is that the charged N terminus increases the solubility of the precursor protein. Another possibility is that the environment in which these proteins are required to become active influences sequence digressions. For example, subtilisin is secreted out by B. subtilis for the purpose of scavenging food, whereas mammalian cells secrete the eukaryotic subtilisin homologue furin into the trans Golgi network for processing precursor proteins (32). Although the catalytic domains of bacterial subtilisin and their eukaryotic counterparts are similar and robustly stable (8), the propeptides in the above example are removed in dramatically different environments (4). We propose that the amino acid differences between their propeptides allow these proteins to respond to different environments to modulate activation precision of proteases. Another noteworthy point is that our work raises questions regarding the lower limit for sequence identity that is acceptable for two proteins to perform similar functions.

    ACKNOWLEDGEMENT

We thank the Medical Research Foundation of Oregon for support.

    FOOTNOTES

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

Dagger Present address: Laboratoire des Aminoacides Peptides et Proteins, Faculté de pharmacie, 15 Av Charles Flahault, BP 14491, 34093 Montpellier, France.

§ To whom correspondence should be addressed. Tel.: 503-494-8683; Fax: 503-494-8393; E-mail: shindeu@ohsu.edu.

Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M212003200

    ABBREVIATIONS

The abbreviations used are: ProD, propeptide designed using databases; ProWT, propeptide of subtilisin E; MES, 4-morpholineethane-sulfonic acid; pI, isoelectric point; N-suc-AAPF-pNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; TPCK, n-tosyl-L-phenylalanine chloromethyl ketone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Wong, S. L., and Doi, R. H. (1986) J. Biol. Chem. 261, 10176-10181[Abstract/Free Full Text]
2. Zhu, X., Ohta, Y., Jordan, F., and Inouye, M. (1989) Nature 339, 483-484[CrossRef][Medline] [Order article via Infotrieve]
3. Shinde, U. P., Li, Y., Chatterjee, S., and Inouye, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6924-6928[Abstract]
4. Yabuta, Y., Takagi, H., Inouye, M., and Shinde, U. P. (2001) J. Biol. Chem. 276, 44427-44433[Abstract/Free Full Text]
5. Ruan, B., Hoskins, J., and Bryan, P. A. (1999) Biochemistry 38, 8562-8571[CrossRef][Medline] [Order article via Infotrieve]
6. Bryan, P., Wang, L., Hoskins, J., Ruvinov, S., Strausberg, S., Alexander, P., Almog, O., Gilliland, G., and Gallagher, T. (1995) Biochemistry 34, 10310-10318[Medline] [Order article via Infotrieve]
7. Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864[Abstract/Free Full Text]
8. Shinde, U. P., and Inouye, M. (2000) Semin. Cell Dev. Biol. 11, 35-44[CrossRef][Medline] [Order article via Infotrieve]
9. Eder, J., and Fersht, A. (1995) Mol. Microbiol. 16, 609-614[Medline] [Order article via Infotrieve]
10. Silen, J. L., and Agard, D. A. (1989) Nature 341, 362-364
11. Baker, D., Sohl, J. L., and Agard, D. A. (1992) Nature 356, 263-265[CrossRef][Medline] [Order article via Infotrieve]
12. Jaswal, S., Sohl, J. L., Davis, J. H., and Agard, D. A. (2002) Nature 415, 343-346[CrossRef][Medline] [Order article via Infotrieve]
13. Sohl, J. L., Jaswal, S. S., and Agard, D. A. (1998) Nature 395, 817-819[CrossRef][Medline] [Order article via Infotrieve]
14. Miyata, T., and Yasunaga, T. (1980) J. Mol. Evol. 16, 23-36[Medline] [Order article via Infotrieve]
15. Li, W.-H., Lu, C.-C., and Wu, C.-I. (1985) in Molecular Evolutionary Genetics (MacIntyre, R. J., ed) , pp. 1-94, Plenum Press, New York
16. Li, W.-H., Wu, C.-I., and Lu, C.-C. (1985) Mol. Biol. Evol. 2, 150-174[Abstract]
17. Shinde, U. P., Liu, J. J., and Inouye, M. (1997) Nature 389, 520-522[CrossRef][Medline] [Order article via Infotrieve]
18. Shinde, U. P., Fu, X., and Inouye, M. (1999) J. Biol. Chem. 274, 15615-15621[Abstract/Free Full Text]
19. Muller, L., Cameron, A., Fortenberry, Y., Apletalina, E. V., and Lindberg, I. (2000) J. Biol. Chem. 275, 39213-39222[Abstract/Free Full Text]
20. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
21. Genetics Computer Group, Program Manual for the Wisconsin Package, Version 9.1, Genetics Computer Group, Madison, WI
22. Fu, X. F., Inouye, M., and Shinde, U. P. (2000) J. Biol. Chem. 275, 16871-16878[Abstract/Free Full Text]
23. Marie-Claire, C., Yabuta, Y., Suefuji, K., Matsuzawa, H., and Shinde, U. (2001) J. Mol. Biol. 305, 151-165[CrossRef][Medline] [Order article via Infotrieve]
24. Baker, D., and Sali, A. (2001) Science 294, 93-96[Abstract/Free Full Text]
25. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723[Medline] [Order article via Infotrieve]
26. Shinde, U. P., and Inouye, M. (1993) Trends Biochem. Sci. 18, 442-446[CrossRef][Medline] [Order article via Infotrieve]
27. Buevich, A. V., Shinde, U., Inouye, M., and Baum, J. (2001) J. Biomol. NMR 20, 233-249[CrossRef][Medline] [Order article via Infotrieve]
28. Jain, S., Shinde, U., Li, Y., Inouye, M., and Berman, H. (1998) J. Mol. Biol. 284, 137-144[CrossRef][Medline] [Order article via Infotrieve]
29. Li, Y., Hu, Z., Jordan, F., and Inouye, M. (1995) J. Biol. Chem. 270, 25127-25132[Abstract/Free Full Text]
30. Greenfield, N., and Fasman, G. D. (1996) Biochemistry 8, 4108-4116
31. Baker, D. (1998) Nat. Struct. Biol. 5, 1021-1024[CrossRef][Medline] [Order article via Infotrieve]
32. Anderson, E. D., Molloy, S. S., Jean, F., Fei, H., Shimamura, S., and Thomas, G. (2002) J. Biol. Chem. 277, 12879-12890[Abstract/Free Full Text]
33. Bigelow, C. C. (1967) J. Theor. Biol. 16, 187-211[Medline] [Order article via Infotrieve]
34. Rost, B. (1996) Methods Enzymol. 266, 525-539[CrossRef][Medline] [Order article via Infotrieve]


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