Biochemistry 2010, 49, 365–371 365 DOI: 10.1021/bi9014055
The Prosegment Catalyzes Pepsin Folding to a Kinetically Trapped Native State† Derek R. Dee‡ and Rickey Y. Yada*,‡,§ ‡
Biophysics Interdepartmental Group and §Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received August 12, 2009; Revised Manuscript Received December 8, 2009 ABSTRACT: Investigations of irreversible protein unfolding often assume that alterations to the unfolded state,
rather than the nature of the native state itself, are the cause of the irreversibility. However, the present study describes a less common explanation for the irreversible denaturation of pepsin, a zymogen-derived aspartic peptidase. The presence of a large folding barrier combined with the thermodynamically metastable nature of the native state, the formation of which depends on a separate prosegment (PS) domain, is the source of the irreversibility. Pepsin is unable to refold to the native state upon return from denaturing conditions due to a large folding barrier (24.6 kcal/mol) and instead forms a thermodynamically stable, yet inactive, refolded state. The native state is kinetically stabilized by an unfolding activation energy of 24.5 kcal/mol, comparable to the folding barrier, indicating that native pepsin exists as a thermodynamically metastable state. However, in the presence of the PS, the native state becomes thermodynamically stable, and the PS catalyzes pepsin folding by stabilizing the folding transition state by 14.7 kcal/mol. Once folded, the PS is removed, and the native conformation exists as a kinetically trapped state. Thus, while PS-guided folding is thermodynamically driven, without the PS the pepsin energy landscape is dominated by kinetic barriers rather than by free energy differences between native and denatured states. As pepsin is the archetype of a broad class of aspartic peptidases of similar structure and function, and many require their PS for correct folding, these results suggest that the occurrence of native states optimized for kinetic rather than thermodynamic stability may be a common feature of protein design. It is not unusual to observe that a protein is irreversibly unfolded; for example, this is often reported to be the case in DSC studies (1-3). However, the basis for such irreversibility may not be trivial (i.e., due to irreversible modification of the unfolded state) but may reveal fundamental elements of protein folding and stabilization mechanisms. Such is the case for R-lytic peptidase (RLP),1 in which the native state was found to be less thermodynamically stable than the denatured and unfolded states (4). Another such example is pepsin. As a zymogen-derived protein, pepsin is initially produced at neutral pH as an inactive precursor form, termed pepsinogen, which contains an N-terminal, 44 amino acid prosegment (PS) domain. With increasing acidity, below pH 5, the PS is removed by hydrolysis (5) to result in mature, active pepsin (Mw 34.6). Native pepsin (Np) is stable under acidic pH and has a bilobal conformation with two catalytic aspartic residues on either side of the active site cleft, formed between the N- and C-terminal domains (Figure 1). In pepsinogen, the PS is located inside this active site cleft. Curiously, while pepsinogen can be reversibly unfolded, pepsin is irreversibly denatured at neutral pH and does not refold to the native, active form upon acidification. For this reason native pepsin is known to be irreversibly denatured, a process that has been the subject of numerous studies (6-12). Recent studies have revealed unusual aspects of pepsin denaturation by comparing the structures and stabilities of native, † The financial support of the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs Program is gratefully acknowledged. *Corresponding author. Tel: 519-824-4120 ext 58915. Fax: 519-824-6631. E-mail:
[email protected]. 1 Abbreviations: PS, prosegment; PSp, synthetic prosegment peptide; Np, native pepsin; Rp, refolded pepsin; RLP, R-lytic peptidase; SGPB, Streptomyces griseus peptidase B; GdnHCl, guanidine hydrochloride.
alkaline denatured, and acid refolded porcine pepsin (Rp) (13). The stabilities and low-resolution secondary and tertiary structures of these states were characterized using differential scanning calorimetry, small-angle neutron scattering, and circular dichroism. Rp was shown to have secondary and tertiary structures intermediate between the alkaline denatured and native forms but was found to be thermodynamically more stable relative to Np, as evidenced by a reversible unfolding transition with a higher melting midpoint (Tm). These data, therefore, strongly suggested that the irreversibility of pepsin denaturation was due to the native state existing as a thermodynamically metastable form. The above finding brings to question how native pepsin is originally folded and stabilized. While it has been qualitatively understood that the PS is important for correct folding of aspartic peptidases, only for the serine peptidases RLP and Streptomyces griseus peptidase B (SGPB) (4, 14-17) and subtilisin’s E and BPN0 (18-20), have there been quantitative studies to understand the requirement for and mechanism of the PS in folding. In the case of RLP and SGPB, the PS has been shown to actively catalyze folding from the denatured state to the native state by lowering the free energy barrier to folding, enhancing the folding rate by as much as 3 109 for RLP and 400 times for SGPB (17). Once the PS is removed, the resulting native states are kinetically trapped, characterized by very slow unfolding and refolding rates, indicating a large energy barrier to unfolding/ refolding in the absence of the PS. The PS was shown to act as a foldase for each of these enzymes; i.e., rather than preventing off-pathway folding events, as in the case of a chaperone, the PS directly catalyzes folding to the native state. Based on the finding that Rp was more stable than Np (13), it was hypothesized that the PS of pepsin functions in a similar manner. To examine this hypothesis, the folding/unfolding kinetics of pepsin were
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Biochemistry, Vol. 49, No. 2, 2010
FIGURE 1: Crystal structures of (A) pepsinogen (PDB: 2PSG) and (B) pepsin (PDB: 3PEP). The prosegment (PS, blue) forms part of a six-stranded β-sheet plate and occludes the active site cleft in pepsinogen. After removal of the PS, residues 1-12 (orange) undergo a conformational change, with the mature N-terminus moving over 40 A˚ to form a strand within the six-stranded β-sheet plate, thus replacing the strand once formed by the PS. The catalytic Asp32 and Asp215 residues are shown in red.
measured and compared with the folding and interaction of pepsin when combined with a synthetic PS peptide (PSp). It was observed that the PS catalyzes the folding of pepsin and, once the PS is removed, a kinetically trapped native state is obtained. This finding explains the basis for pepsin irreversible denaturation and provides insight into how the PS facilitates correct folding of native pepsin. EXPERIMENTAL PROCEDURES Materials. High-purity porcine pepsin A (EC 3.4.23.1) was purchased from Sigma (St. Louis, MO) and used without further purification. The peptide substrates KPAEFF(NO2)AL and MOCAc-APAKFFRLK(Dnp)-NH2 were purchased from Sigma Genosys (Oakville, Ontario, Canada) and from Peptide Institute, Inc. (Osaka, Japan), respectively. The synthetic 44-residue PSp with C-terminal amidation (LVKVPLVRKKSLRQNLIKNGKLKDFLKTHKHNPASKYFPEAAAL) was purchased from CanPeptide Inc. (Pointe-Claire, Quebec, Canada) and was more than 95% pure as judged by LC-MS. Protein stocks were prepared by mass (w/v), with concentrations determined by absorbance at 280 nm, using extinction coefficients of 52830 M-1 cm-1
Dee and Yada for pepsin and 1490 M-1cm-1 for prosegment, both of which were estimated using the ProtParam tool (21). Pepsin binding, folding, and unfolding experiments were carried out at pH 5.3 (20 mM acetic acid/NaOH buffer), as pepsin has been shown to display reduced proteolysis above pH 5 (22, 23). Refolded Pepsin Sample Preparation. Alkaline denatured pepsin was prepared by making a 20 mg/mL solution in 30 mM NaOH, with a final pH of 8, and allowed to equilibrate for at least 10 min. Refolded pepsin (Rp) was then prepared by diluting an aliquot of the alkaline denatured protein to 0.35 mg/mL in 20 mM acetic acid/NaOH buffer with a final pH of 5.3. Uncatalyzed Pepsin Folding. Rp samples were prepared (0.35 mg/mL) and incubated on ice (0 °C) in 20 mM acetic acid/ NaOH buffer, pH 5.3, containing 100 mM NaCl. Aliquots were withdrawn at intervals from 0 to 4 days and assayed for hydrolytic activity using a sensitive fluorescence-based assay (detection limit
