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A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein
Article
A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein Graphical Abstract
Authors Ryo P. Honda, Ming Xu, Kei-ichi Yamaguchi, Heinrich Roder, Kazuo Kuwata
Correspondence [email protected] (K.K.), [email protected] (H.R.)
In Brief It is currently unclear whether and how unimolecular protein folding pathways are related to aggregation. Honda et al. reveal that a native-like state serves as a branching point between the folding and aggregation pathways of the mouse prion protein.
Highlights d
Prion protein folding and unfolding kinetics were studied using ultra-rapid mixing
d
The equilibrium A-state was shown to correspond to a late folding intermediate
d
A previous study suggested that the A-state is a monomeric precursor of aggregates
d
The A-state is a branching point between the folding and aggregation pathways
Honda et al., 2015, Structure 23, 1–8 September 1, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.str.2015.07.001
Please cite this article in press as: Honda et al., A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.07.001
Structure
Article A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein Ryo P. Honda,1 Ming Xu,2 Kei-ichi Yamaguchi,3 Heinrich Roder,2,4,* and Kazuo Kuwata3,5,* 1Department
of Molecular Pathobiochemistry, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu 501-1193, Japan Chase Cancer Center, Philadelphia, PA 19111, USA 3United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Yanagido 1-1, Gifu 501-1194, Japan 4Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA 5Department of Gene Development, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan *Correspondence: [email protected] (K.K.), [email protected] (H.R.) http://dx.doi.org/10.1016/j.str.2015.07.001 2Fox
SUMMARY
Transient folding intermediates and/or partially unfolded equilibrium states are thought to play a key role in the formation of protein aggregates. However, there is only indirect evidence linking accumulation of folding intermediates to aggregation, and the underlying mechanism remains to be elucidated. Here, we show that a partially unfolded state of the prion protein accumulates both as a stable equilibrium state at acidic pH (A-state) and as a late folding intermediate. With a time resolution of approximately 60 ms, we systematically studied the kinetics of folding and unfolding, starting from various initial conditions including the U-, N-, and A-states. Quantitative modeling showed that the observed kinetic data are completely consistent with a sequential four-state mechanism where the A-state is a late folding intermediate. Combined with previous evidence linking A-state accumulation to aggregation, the results indicate that this native-like state serves as a branching point between the folding and aggregation pathways.
INTRODUCTION Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals that are linked to the misfolding and aggregation of the prion protein (PrP) (Prusiner, 1982, 1998). According to the ‘‘protein-only’’ hypothesis, conversion of the cellular prion protein (PrPC) to its pathogenic isoform (PrPSc) is a crucial event in the pathogenesis of prion diseases (Prusiner, 1982, 1998). PrPC contains three a helices, helix 1 (H1), helix 2 (H2), and helix 3 (H3), as well as a small b sheet (Riek et al., 1996). H2 and H3 are connected by a disulfide bridge between Cys179 and Cys214. In contrast, PrPSc is mainly composed of b-sheet structures, as revealed by Fourier-transform infrared spectroscopy (Pan et al., 1993), and it has been reported that the infectious particles in prion diseases are oligo-
mers (Masel et al., 2005; Silveira et al., 2005). Thus, elucidating the mechanism by which the a helices of PrPC become the b sheets of PrPSc, a process termed b-conversion, as well as the structural basis for oligomerization, is critical for understanding and controlling prion pathogenesis. It is well known that acidic pH favors transformation of PrP into a variety of b-rich oligomers (Hornemann and Glockshuber, 1998; Jain and Udgaonkar, 2008; Morillas et al., 2001; Rezaei et al., 2002; Swietnicki et al., 1997). We recently reported the formation of an acid-induced partially unfolded state characterized by the molten globule-like structure (A-state) just prior to the formation of b-rich oligomers (Honda et al., 2014). The initial fraction of PrP in the A-state was strongly correlated with the rate of oligomerization at pH 2–5, suggesting that the A-state is directly involved in the oligomerization pathway. Previously, a folding intermediate state (I-state) was implicated as a precursor in the pathogenic conversion process because this state was stabilized by mutations observed in inherited prion diseases (Apetri et al., 2004, 2006). However, it is unclear whether and how the I- and A-states are related, and how the oligomerization pathway is connected to the folding pathway (Chiti et al., 2002; Jahn and Radford, 2005). The kinetic folding intermediates of several proteins have been shown to be structurally analogous to non-native equilibrium forms with features of a molten globule (Kuwajima, 1989; Ptitsyn et al., 1990), as demonstrated by techniques such as pulsed hydrogen/deuterium exchange, smallangle X-ray scattering, and time-resolved nuclear magnetic resonance (NMR) (Arai et al., 2002; Eliezer et al., 1995; Hughson et al., 1990; Jeng et al., 1990; Jennings and Wright, 1993; Raschke and Marqusee, 1997). Recent work on b2-microglobulin also demonstrated that formation of a folding intermediate containing a non-native prolyl cis-trans isomer precedes aggregate formation (Jahn et al., 2006; Mukaiyama et al., 2013a, 2013b). Although the folding mechanism of the PrP is significantly different to that of b2-microglobulin, it is essential to clarify the role of the A-state as a critical intermediate in both the folding reaction and oligomer formation. To investigate the kinetic role of the A-state, we observed the kinetics of folding and unfolding of PrP using various initial conditions favoring population of the native (N-), unfolded (U-), and A-states. An engineered tryptophan (Trp218) served as a probe for monitoring the conformational changes associated
Structure 23, 1–8, September 1, 2015 ª2015 Elsevier Ltd All rights reserved 1
Please cite this article in press as: Honda et al., A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.07.001
Figure 1. Structural and Thermodynamic Characteristics of the A-State (A–C) Far-UV CD (A), near-UV CD (B), and Trp-fluorescence (C) spectra for the N-state at pH 4.5 (red), the A-state at pH 2.0 (blue), and the U-state at pH 2.0 with 3 M urea (dotted black line), respectively. (D) Thermal unfolding of the N-state at pH 4.5 (red) and the A-state at pH 2.0 (blue) monitored via the change in mean residue ellipticity at 222 nm. The solid lines represent least-squared fits to van’t Hoff’s equation, where the equilibrium constant between two states is described by the exponential of (DHwf DST)/RT. DHwf (kcal/mol) and DS (cal/mol$K)] of N- and A-states are (42 ± 4 and 122 ± 11) and (16 ± 1 and 48 ± 2), respectively. (E) 13Ca-chemical shift indices of the N-state at pH 4.5 (red) and the A-state at pH 2.0 (blue). (F) 13Ca-chemical shift indices of the A-state plotted onto the 3D structure of mouse PrP(121–231) (PDB: 1AG2). The unassigned residues are shown in gray.
with folding and unfolding on the submillisecond timescale, using an ultra-rapid continuous-flow mixing technique with a dead time of approximately 60 ms (Roder et al., 2004, 2006; Shastry et al., 1998). Our results indicate that the A-state is clearly distinct from the I-state. Quantitative analysis of the folding and unfolding kinetics suggested that the A-state corresponds to a late, native-like intermediate in the folding of PrP, and that the A-state is a key intermediate located at a bifurcation point between the monomeric folding and oligomerization aggregation pathways. RESULTS Structural and Thermodynamic Characteristics of the A-State As in previous kinetic studies on the human PrP fragment 90–231 (Apetri et al., 2006; Apetri and Surewicz, 2002), we worked with a W145H/Y218W mutant of the highly homologous mouse protein, which contains a single Trp residue in the folded domain. By lowering the pH from 4.5 to 2.0, the native state of PrP (N-state) was transformed into an acid-induced partially unfolded state (A-state), which is characterized by a moderate change in secondary structure (Figure 1A) and a partially disrupted tertiary structure (Figure 1B), as revealed by far- and near-UV circular dichroism (CD), respectively. The Trp-fluorescence emission spectrum of the A-state coincides with that of the N-state (Fig-
ure 1C), indicating that the A-state maintains native-like tertiary structure in the region surrounding the tryptophan residue (see also Figure 1F). The A-state is thermodynamically characterized by a low enthalpy and entropy change upon unfolding (16 kcal/mol and 48 cal/mol$K for DHwf and DS, respectively) compared with the N-state (42 kcal/mol and 122 cal/mol$K for DHwf and DS, respectively) (Figure 1D). The 13Ca-chemical shift indices at pH 2.0 obtained by sequentially assigning the peaks in the NMR spectrum (Figure S1) exhibit values near zero in the S1H1-H2 segment (amino acid residues 129–160) (Figures 1E and 1F). This shows that this segment is almost completely unfolded in the A-state (Wishart et al., 1992), which is consistent with previous hydrogen/deuterium exchange results (Honda et al., 2014). In contrast, most of the H2-H3 segment remains unassigned due to severe line broadening (Figure S1), which suggests that this region fluctuates between some folded and unfolded structures on the millisecond timescale. Thus, the N-terminal S1-H1-S2 segment appears to be extensively disordered or completely unfolded in the A-state, while the C-terminal H2-H3 segment retains a marginally stable structure (Honda et al., 2014). Accumulation of a Kinetic Intermediate State during Folding We examined the folding and unfolding kinetics of the W145H/ Y218W variant of mouse PrP(90–231) at pH 4.5 in the presence of various concentrations of urea (0–7.0 M) starting from
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Please cite this article in press as: Honda et al., A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.07.001
Figure 2. Folding and Unfolding Kinetics (A) Representative kinetic traces for folding of mouse PrP at pH 4.5 in the presence of 0.5–2.5 M urea, starting from conditions favoring the U-state (pH 2.0, 3.0 M urea). (B) Representative traces for unfolding at pH 4.5 in the presence of 5.0–7.0 M urea, starting from conditions favoring the N-state (pH 4.8, 2.0 M urea). The solid lines represent least-squared fits to a single exponential function. (C) Chevron plots in the absence (filled circles) or presence (empty squares) of 0.17 M Na2SO4. The solid and dotted lines represent curves generated by assuming a three-state scheme (Table S1).
the U-state at pH 2.0 and 3.0 M urea, as well as from the N-state at pH 4.8 and 2.0 M urea. To resolve the kinetics of folding of PrP, which occurs on the millisecond timescale or faster, even at 5 C (Apetri et al., 2006; Apetri and Surewicz, 2002), we combined ultra-rapid mixing (Shastry et al., 1998) with conventional stopped-flow measurements. The refolding and unfolding time courses are both well described by a single exponential function over the time range from approximately 100 ms to 30 ms (Figures 2A and 2B). However, the log(rate) versus urea concentration (chevron) plot strongly deviates from a linear dependence on urea concentration below approximately 2 M (Figure 2C). This non-linearity, known as ‘‘rollover,’’ is generally attributed to the accumulation of a folding intermediate within the dead time. Furthermore, the rollover was more prominent in the presence of 0.17 M Na2SO4 (Figure 2C and Table S1), which generally stabilizes folding intermediates (Gorski et al., 2001; Jahn et al., 2006; Khorasanizadeh et al., 1996). Thus, an early kinetic intermediate, which we refer to as the I-state, accumulates during the refolding of PrP from strongly denaturing conditions. Unfortunately, without direct observation of the fast U/I transition, we cannot determine whether the I-state lies on a direct path from U to N (Capaldi et al., 2001). However, the main purpose of this study is to elucidate the kinetic role of the A-state, and the following kinetic analysis was found to be independent of the kinetic role of the I-state (data not shown). Therefore, for simplicity, we assume that the I-state is an on-pathway intermediate. The Folding/Unfolding Kinetics of the A-State Next, we examined the folding/unfolding kinetics starting from Astate conditions at pH 2.0. Final conditions were the same as those in experiments starting from the N- and U-states (pH 4.5 in the presence of 0–7 M urea). It should be noted that the folding kinetics of the A-state below 2.0 M urea is not accompanied by a prominent fluorescence change (Figure 3A), in contrast to the folding experiment shown in Figure 2A, indicating that the Astate does not correspond to the kinetic intermediate I. In addition, the A-state can probably reach the N-state without going through the I-state. Otherwise, its folding rate would be limited by the major kinetic barrier of the I/N transition and we should
observe a phase with a rate constant of approximately 3,000 s1 (Figures 2A and 2C). This also indicates that the A-state lies on the native side of the major kinetic barrier for the I/N folding transition. Interestingly, the folding/unfolding kinetics of the Astate above 3.0 M urea exhibit two distinct phases with rate constants of approximately 5,000 and 500 s1, respectively (Figures 3A and 3B). The fast phase was not observed in Figure 2B, while the slow phase closely matches that observed in the previous unfolding experiments (Figure 3B). This indicates that the A-state partially shares a common unfolding pathway with the N-state. Evidence for the A-State as a Late Folding Intermediate Next, we investigated which folding scheme best describes the folding/unfolding kinetics of the A-state. As we have shown that A and I are distinct states (Figures 3A and 3B), the minimal mechanism is a four-state scheme. We initially tested different linear four-state schemes, such as U4I4A4N or U4A4I4N. Theoretically, there are 16 distinct linear schemes (Figure S2), but 11 of them can be ruled out by assuming that the I-state is an on-pathway intermediate (black in Figure S2). Furthermore, based on the folding/unfolding kinetics of the A-state (Figures 3A and 3B), we were able to place the A-state beyond the kinetic barrier of the I/N transition, which rules out three of the five remaining schemes. As a result, only two schemes remained: U4I4A4N, where the A-state corresponds to a late folding intermediate, and U4I4N4A, where the A-state corresponds to an off-pathway intermediate (blue in Figure S2). The latter scheme, however, can also be ruled out by taking into account the amplitude plot (Figure 3C). In the U4I4N4A scheme, the fast and slow phases observed during the unfolding of the A-state are interpreted as the A/N and N/I transitions, respectively. Therefore, a major fraction of PrP is expected to form the N-state just after the fast phase. However, the fluorescence intensity just after the fast phase, which is approximately described by the sum of the fluorescence intensity at infinity and the kinetic amplitudes of the slow phase, FN + As (downward triangles in Figure 3C), was much higher than the fluorescence of the N-state or the native baseline (inset, Figure 3C). On the other hand, the U4I4A4N scheme fully reproduces the folding/unfolding kinetics of the A-state, including the rate constants and kinetic
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Figure 3. Kinetics of Folding and Unfolding of the A-State (A) Representative folding/unfolding kinetic traces at pH 4.5 in the presence of 0–7.0 M urea starting from conditions favoring the A-state (pH 2.0). The solid lines represent least-squared fits to a double exponential function: fðtÞ=fðAÞ = Af expð lf tÞ + As expð ls tÞ + fN : (Equation 1) (B) Chevron plot. Filled circles and empty triangles represent lf and ls in Equation 1, respectively. For comparison, the apparent rate constants in Figure 2C are shown as empty squares. The solid curve represents the curve reproduced by assuming the U4I4A4N scheme (Table 1). (C) Amplitude plot. Circles, downward triangles, and upward triangles represent fN, (fN + As), and (fN + As + Af) from Equation 1, respectively. The solid curve represents the curve reproduced by assuming the U4I4A4N scheme (Table 1). The fluorescence intensity of the U-, I-, A-, and N-states at 0 M urea is 1.01, 1.30, 1.05, and 1.05, respectively. The slope against urea concentration (M1) of the U-, I-, A-, and N-states is 0.23, 0.10, 0.05, and 0.05, respectively. (Inset) Equilibrium urea titration curve obtained under the same conditions. The dotted line represents the baseline N-state. (D) Free-energy diagram. The activation free energies of the transitions, Ea = RTln(k/A0), were calculated from kIA and kAN (Table 1) values using a pre-exponential factor A0 = 106 (s1). The double dagger symbols represent the transition states. Bars represent fitting errors (SD).
amplitudes (Figures 3B and 3C, Table 1). This analysis assumed that the A-state shares common fluorescence properties with the N-state, namely 1.05 of the fluorescence intensity at 0 M urea and 0.05 of the slope versus urea concentration (Figure 3C), consistent with the well-organized tertiary structure of the A-state (Figures 1B and 1C). Hence, we concluded that the PrP folding is described by the U4I4A4N scheme, i.e., the A-state corresponds to a late folding intermediate. The fact that we observed only one intermediate state in our first set of folding/unfolding experiments (Figures 2A–2C) is not inconsistent with this conclusion, since the four-state scheme predicts only one exponential phase with measurable amplitude within the approximately 100-ms time resolution of our measurements, as observed (Figures 4A and 4B). A three-state scheme can also reproduce the chevron plot (data not shown). Since the A-state lies beyond the rate-limiting step in both folding and unfolding experiments (Table 1), its maximum population is only 23% and 11% during folding and unfolding, respectively. The fluorescence properties of the A-state closely correspond to those of the N- state (Table 1), which further hampered detection of the A-state. Thus, the four-state scheme is fully consistent with all our kinetic data, including those in Figures 2A–2C.
Table 1. Kinetic Parameters for the Four-State Scheme (U4I4A4N) KUIa (MUI b) kIAc (mIAd) 29 (0.80) a
kAI (mAI)
Equilibrium constant. m value (kcal/mol$M). c Elementary rate constant (s1). d Kinetic m value (kcal/mol$M). b
kAN (mAN)
kNA (mNA)
4,176 (0.03) 1,298 (0.08) 8,222 (0.14) 331 (0.07)
It should be noted that we cannot definitively rule out more complicated schemes, such as five-state schemes and branched four-state schemes (Radford et al., 1992). Pulsed hydrogen/deuterium exchange and/or the quenched-flow experiments would be beneficial to rule out these more complicated schemes, but the short lifetime of the kinetic intermediate states (Figures 2A and 3A) and the high tendency of PrP to aggregate at alkaline pH make such studies very challenging. However, the quality of the fits in Figure 3 indicates that the U4I4A4N scheme is the simplest mechanism that describes the folding/unfolding kinetics of mouse PrP. DISCUSSION In this study we investigated the kinetic role of the A-state of mouse PrP, an acid-induced partially unfolded state with some of the characteristics of a molten globule recently identified as a possible pre-oligomeric state (Honda et al., 2014). By examining the kinetics of folding and unfolding under various initial and final conditions, we showed that the A-state corresponds to a late folding intermediate, suggesting that oligomerization starts from this partially unfolded state, which contains a native-like H2-H3 hairpin (Figure 5). Under physiological conditions, late folding intermediates are expected to be more highly populated than early intermediates. They are, therefore, more likely to lead to formation of pathogenic oligomers (Brockwell and Radford, 2007). Thus, the A-state may be a key intermediate in both folding and self-association, giving rise to the formation of b-rich oligomers. A number of studies have demonstrated that lowering pH increases the rate constant of b-rich oligomer formation (Hornemann and Glockshuber, 1998; Jain and Udgaonkar,
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Figure 4. Folding and Unfolding Kinetics Predicted by the U4I4A4N Scheme The folding kinetics at 0 M urea (A) and the unfolding kinetics at 6 M urea (B) were calculated by solving the four-state mechanism using the parameters in Table 1 and the legend of Figure 3. The upper and lower panels represent the timedependent fluorescence change and the fraction of each state, respectively.
2008; Morillas et al., 2001; Rezaei et al., 2002; Swietnicki et al., 1997). For instance, for mouse PrP we observed rates of 0.0389 min1 and 15.7 min1 at pH 4.5 and 2.0, respectively (Honda et al., 2014). Acceleration of the aggregation process at acidic pH may be attributed to an increase in the equilibrium population of the A-state. From the kinetic parameters of PrP folding presented in this report, the equilibrium population of the A-state at pH 4.5 was calculated to be 3.8% (Table 1), whereas at pH 2.0 almost 100% of the protein forms the A-state. This correlation between aggregation rate and A-form population is consistent with the hypothesis that the A-state is located at the branching point between folding and aggregation. Since there is no evidence for infectivity of the b-rich PrP oligomers formed at acidic pH, it remains unclear whether or not the A-state is actually involved in the pathogenic conversion into PrPSc. However, recent studies have revealed that mutations linked to prion diseases destabilize H1 and accelerate the formation of b-rich oligomers (Singh and Udgaonkar, 2015). They further suggest that the RNA and POPG (1-palmitoyl-2-
Figure 5. A Schematic Free-Energy Landscape for Folding and Oligomerization of PrP In this scheme, unfolded PrPC (U) folds into the N structure via two intermediate states: the I- and A-states (black arrow). Oligomerization and aggregation starts from a late folding intermediate, the A-state (red arrow). The A-state is a key intermediate from which the folding and oligomerization pathways diverge. F, Q1, and Q2 represent the Gibbs free energy, the first reaction coordinate, and the second reaction coordinate, respectively.
oleoylphosphatidylglycerol) molecules that facilitate the pathogenic conversion (Wang et al., 2010) also destabilize H1 (Miller et al., 2013). The conformational characteristics of the A-state suggest that partial unfolding of the S1-H1-S2 region may trigger further conformational changes at the C terminus of H2 and in the H2-H3 loop region (Kuwata et al., 2007; Miller et al., 2013; Singh et al., 2014), which facilitate intermolecular interactions by exposing the hydrophobic residues at the interface between the H2 and H3 helices. Furthermore, anti-prion compounds (‘‘medical chaperones’’) intercalate between the H1 and H2-H3 loops and stabilize PrP (Kamatari et al., 2013; Kuwata et al., 2007). Thus, H1 destabilization and formation of the A-state (Figure 1E) may be related to the pathogenesis of prion diseases. Since the initial process of b-rich oligomer formation is similar to that of PrPSc formation, it is reasonable to assume that the A-state corresponds to the initial monomeric precursor in the pathogenic conversion or PrP* (Cohen et al., 1994). In a recent ultra-fast mixing study of the folding/unfolding kinetics of sheep PrP, Chen et al. (2011) observed accumulation of an intermediate state during unfolding that corresponds to the A-state observed in the present study. This unfolding intermediate was stabilized by mutations related to inherited prion disease (Chen et al., 2011), suggesting that the kinetic intermediate detected is involved in pathogenesis. This sheep PrP unfolding intermediate is characterized by native-like fluorescence properties and compactness, which is consistent with our current observations (Figure 1C and Table 1), as well as the structural characteristics observed for the equilibrium A-state of mouse PrP (Honda et al., 2014). Previous kinetic studies identified an early intermediate in the folding of human PrP (I-state in our terminology) that was proposed to act as a monomeric precursor to b-rich oligomers. This proposal was based on the fact that the intermediate is stabilized by mutations linked to prion diseases (Apetri et al., 2004, 2006) and by conditions known to promote aggregation, such as low pH (Apetri et al., 2006; Apetri and Surewicz, 2002). In terms of its kinetic and structural properties, the I-state is clearly distinct from the A-state (Figures 3A and 3B). Given the high degree of sequence conservation among the three PrP variants and the similarity of the chevron plots for the rate-limiting folding/unfolding phases (Apetri et al., 2006; Chen et al., 2011), it is likely that they share a common folding mechanism, such as the sequential four-state mechanism shown in Figure 5. The observed species-dependent differences in kinetic behavior on the submillisecond timescale can be explained in terms of relatively subtle changes in the free-energy profiles among the three
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PrP variants (cf. Figure 3D). For example, the fact that the fast phase seen during the unfolding of mouse and sheep PrPs is absent during the unfolding of human PrP can be explained by a modest shift in the relative free energies of the two barriers, resulting in a decrease in transient accumulation of the A-state. By modeling the folding/unfolding kinetics of mouse PrP using alternative kinetic schemes, we were able to show that the A-state lies on a direct path from the unfolded ensemble to the N-state (Figure 5). Combined with the structural properties of the A-state (Figures 1E and 1F), this finding implies that during the folding of mouse PrP, formation of the H2-H3 helical hairpin precedes structure acquisition in other regions, including H1 and the small b sheet. This conclusion is consistent with the relatively high hydrophobicity of the H2-H3 segment, as well as the presence of a disulfide bond connecting H2 and H3. A similar scenario was proposed for human PrP, based on temperature jump experiments coupled with F-value analyses, which suggested that the hydrophobic core of the H2-H3 segment is formed during the rate-limiting step in folding (Hart et al., 2009).
Folding/unfolding was initiated by mixing the initial solutions with a folding/unfolding buffer (pH approximately 4.5, 50 mM sodium acetate with 0–7 M urea in the presence or absence of 0.2 M Na2SO4) using a mixing ratio of 1:5. This procedure allowed us to observe the refolding/unfolding kinetics at pH 4.5 as a function of urea concentration. The kinetics were monitored via the change in fluorescence at 308–420 nm (excitation at 297 nm) using both the stopped-flow and ultra-rapid mixing instruments (Roder et al., 2004, 2006; Shastry et al., 1998; Xu et al., 2012). The dead time of the ultra-rapid mixer was determined to be 56 ± 5 ms by measuring the quenching of N-acetyltryptophanamide fluorescence by N-bromosuccinimide (Roder et al., 2004, 2006; Xu et al., 2012). The fluorescence traces independently obtained using the stopped-flow and ultra-rapid mixing apparatus were globally fitted to a single or double exponential function with common values of the apparent rate constants and kinetic amplitudes. The fluorescence intensities were normalized to those measured under the initial conditions. Protein concentrations of 5 and 10 mM were utilized for the stopped-flow and ultra-rapid mixing measurements, respectively. The family of kinetic traces for folding/unfolding of the N- and A-states of mouse PrP was modeled globally, assuming a four-state sequential scheme (U4I4A4N). Elementary rate constants and kinetic m values obtained by numeric solution of the differential equations describing this scheme (Xu et al., 2012) are provided in Supplemental Experimental Procedures.
EXPERIMENTAL PROCEDURES
SUPPLEMENTAL INFORMATION
Protein Preparation A plasmid encoding mouse PrP, amino acid residues 90–231 (mouse PrP(90– 231)), with an N-terminal 63 His tag, was prepared according to a previously reported protocol (Kuwata et al., 2007). Two mutations (W145H/Y218W) were introduced into mouse PrP(90–231) utilizing the PrimeSTAR Mutagenesis Basal Kit (Takara Bio). Mouse PrP(90–231,W145H/Y218W) was expressed and purified as previously described (Kuwata et al., 2007).
Supplemental Information includes Supplemental Experimental Procedures, two figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.str.2015.07.001.
Equilibrium Measurements Solutions of mouse PrP(90–231,W145H/Y218W) were prepared under N-state (pH 4.5, 20 mM sodium acetate), A-state (pH 2.0, 20 mM glycine-HCl), and U-state (pH 2.0, 20 mM Gly-HCl, 3 M urea) conditions. The protein concentrations were 10, 70, and 10 mM for far-UV CD, near-UV CD, and fluorescence measurements, respectively. CD spectra were acquired at 20 C according to a previously described protocol (Honda et al., 2014). Fluorescence emission spectra were acquired on an RF-5300PC spectrofluorophotometer (Shimadzu) at 20 C using an excitation wavelength of 298 nm. Thermal unfolding of the N- or A-state was monitored by the change of mean residue ellipticity at 222 nm by increasing the temperature up to 95 C at a rate of 2 C$min1. More than 90% of the mean residue ellipticity was recovered after the thermal denaturation experiment (data not shown). For NMR studies of the A-state, 13C,15N uniformly labeled mouse PrP(90– 231,W145H/Y218W) was dissolved with the A-state buffer containing 5% D2O to a final concentration of 0.5 mM, and a series of 3D NMR spectra (1H-15N HSQC, HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCBCA, and HN(CO)CBCA) were acquired. A relatively higher temperature, 32 C, at which approximately 80% of the secondary structure of A-state was retained, was utilized to improve the quality of the NMR spectra (Eliezer et al., 1997) (Figure 1D). The NMR peaks were assigned by the sequential method (Ikura et al., 1990; Kay et al., 1990) using NMRpipe (Delaglio et al., 1995) and KUJIRA (Kobayashi et al., 2007) software. Secondary chemical shifts were calculated by subtracting the chemical shifts of the NMR peaks from those of random coils that were predicted using a freely available program coded by SBiNLab (Kjaergaard et al., 2011). For NMR studies of the N-state, mouse PrP(90–231, W145H/Y218W) was dissolved in native buffer containing 5% of D2O to a final concentration of 0.5 mM. A series of 3D NMR spectra (1H-15N HSQC, HNCO, HN(CA)CO, HNCA, and HN(CO)CA) were acquired at 12 C and analyzed as described above. Kinetic Measurements and Modeling The kinetics of folding and unfolding starting from various conditions that favor the N- (pH 4.8, 20 mM sodium acetate, 2 M urea), U- (pH 2.0, 20 mM Gly-HCl, 3 M urea), and A-states (pH 2.0, 20 mM Gly-HCl) were examined at 5 C.
ACKNOWLEDGMENTS We thank Yumiko Okuda and the Life Science Research Center in Gifu University for assistance with the experiments. This work was supported by grants from the Ministry of Health, Labor, and Welfare of Japan (Research on Measures for Intractable Diseases [Prion Disease and Slow Virus Infections, and Development of Low Molecular Weight Medical Chaperone Therapeutics for Prion Diseases]) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants-in Aid for Scientific Research, and X-ray Free Electron Laser [XFEL] Program). Funding was also received from NSF grant MCB-1412378 and NIH grant R01 GM056250 (to H.R.) and an NIH P30 grant CA06927 (to the Fox Chase Cancer Center). Received: April 16, 2015 Revised: June 8, 2015 Accepted: July 1, 2015 Published: August 6, 2015 REFERENCES Apetri, A.C., and Surewicz, W.K. (2002). Kinetic intermediate in the folding of human prion protein. J. Biol. Chem. 277, 44589–44592. Apetri, A.C., Surewicz, K., and Surewicz, W.K. (2004). The effect of diseaseassociated mutations on the folding pathway of human prion protein. J. Biol. Chem. 279, 18008–18014. Apetri, A.C., Maki, K., Roder, H., and Surewicz, W.K. (2006). Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments. J. Am. Chem. Soc. 128, 11673–11678. Arai, M., Ito, K., Inobe, T., Nakao, M., Maki, K., Kamagata, K., Kihara, H., Amemiya, Y., and Kuwajima, K. (2002). Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering. J. Mol. Biol. 321, 121–132. Brockwell, D.J., and Radford, S.E. (2007). Intermediates: ubiquitous species on folding energy landscapes? Curr. Opin. Struct. Biol. 17, 30–37. Capaldi, A.P., Shastry, M.C.R., Kleanthous, C., Roder, H., and Radford, S.E. (2001). Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nat. Struct. Biol. 8, 68–72.
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A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein Graphical Abstract
Authors Ryo P. Honda, Ming Xu, Kei-ichi Yamaguchi, Heinrich Roder, Kazuo Kuwata
Correspondence [email protected] (K.K.), [email protected] (H.R.)
In Brief It is currently unclear whether and how unimolecular protein folding pathways are related to aggregation. Honda et al. reveal that a native-like state serves as a branching point between the folding and aggregation pathways of the mouse prion protein.
Highlights d
Prion protein folding and unfolding kinetics were studied using ultra-rapid mixing
d
The equilibrium A-state was shown to correspond to a late folding intermediate
d
A previous study suggested that the A-state is a monomeric precursor of aggregates
d
The A-state is a branching point between the folding and aggregation pathways
Honda et al., 2015, Structure 23, 1–8 September 1, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.str.2015.07.001
Please cite this article in press as: Honda et al., A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.07.001
Structure
Article A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein Ryo P. Honda,1 Ming Xu,2 Kei-ichi Yamaguchi,3 Heinrich Roder,2,4,* and Kazuo Kuwata3,5,* 1Department
of Molecular Pathobiochemistry, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu 501-1193, Japan Chase Cancer Center, Philadelphia, PA 19111, USA 3United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Yanagido 1-1, Gifu 501-1194, Japan 4Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA 5Department of Gene Development, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan *Correspondence: [email protected] (K.K.), [email protected] (H.R.) http://dx.doi.org/10.1016/j.str.2015.07.001 2Fox
SUMMARY
Transient folding intermediates and/or partially unfolded equilibrium states are thought to play a key role in the formation of protein aggregates. However, there is only indirect evidence linking accumulation of folding intermediates to aggregation, and the underlying mechanism remains to be elucidated. Here, we show that a partially unfolded state of the prion protein accumulates both as a stable equilibrium state at acidic pH (A-state) and as a late folding intermediate. With a time resolution of approximately 60 ms, we systematically studied the kinetics of folding and unfolding, starting from various initial conditions including the U-, N-, and A-states. Quantitative modeling showed that the observed kinetic data are completely consistent with a sequential four-state mechanism where the A-state is a late folding intermediate. Combined with previous evidence linking A-state accumulation to aggregation, the results indicate that this native-like state serves as a branching point between the folding and aggregation pathways.
INTRODUCTION Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals that are linked to the misfolding and aggregation of the prion protein (PrP) (Prusiner, 1982, 1998). According to the ‘‘protein-only’’ hypothesis, conversion of the cellular prion protein (PrPC) to its pathogenic isoform (PrPSc) is a crucial event in the pathogenesis of prion diseases (Prusiner, 1982, 1998). PrPC contains three a helices, helix 1 (H1), helix 2 (H2), and helix 3 (H3), as well as a small b sheet (Riek et al., 1996). H2 and H3 are connected by a disulfide bridge between Cys179 and Cys214. In contrast, PrPSc is mainly composed of b-sheet structures, as revealed by Fourier-transform infrared spectroscopy (Pan et al., 1993), and it has been reported that the infectious particles in prion diseases are oligo-
mers (Masel et al., 2005; Silveira et al., 2005). Thus, elucidating the mechanism by which the a helices of PrPC become the b sheets of PrPSc, a process termed b-conversion, as well as the structural basis for oligomerization, is critical for understanding and controlling prion pathogenesis. It is well known that acidic pH favors transformation of PrP into a variety of b-rich oligomers (Hornemann and Glockshuber, 1998; Jain and Udgaonkar, 2008; Morillas et al., 2001; Rezaei et al., 2002; Swietnicki et al., 1997). We recently reported the formation of an acid-induced partially unfolded state characterized by the molten globule-like structure (A-state) just prior to the formation of b-rich oligomers (Honda et al., 2014). The initial fraction of PrP in the A-state was strongly correlated with the rate of oligomerization at pH 2–5, suggesting that the A-state is directly involved in the oligomerization pathway. Previously, a folding intermediate state (I-state) was implicated as a precursor in the pathogenic conversion process because this state was stabilized by mutations observed in inherited prion diseases (Apetri et al., 2004, 2006). However, it is unclear whether and how the I- and A-states are related, and how the oligomerization pathway is connected to the folding pathway (Chiti et al., 2002; Jahn and Radford, 2005). The kinetic folding intermediates of several proteins have been shown to be structurally analogous to non-native equilibrium forms with features of a molten globule (Kuwajima, 1989; Ptitsyn et al., 1990), as demonstrated by techniques such as pulsed hydrogen/deuterium exchange, smallangle X-ray scattering, and time-resolved nuclear magnetic resonance (NMR) (Arai et al., 2002; Eliezer et al., 1995; Hughson et al., 1990; Jeng et al., 1990; Jennings and Wright, 1993; Raschke and Marqusee, 1997). Recent work on b2-microglobulin also demonstrated that formation of a folding intermediate containing a non-native prolyl cis-trans isomer precedes aggregate formation (Jahn et al., 2006; Mukaiyama et al., 2013a, 2013b). Although the folding mechanism of the PrP is significantly different to that of b2-microglobulin, it is essential to clarify the role of the A-state as a critical intermediate in both the folding reaction and oligomer formation. To investigate the kinetic role of the A-state, we observed the kinetics of folding and unfolding of PrP using various initial conditions favoring population of the native (N-), unfolded (U-), and A-states. An engineered tryptophan (Trp218) served as a probe for monitoring the conformational changes associated
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Please cite this article in press as: Honda et al., A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.07.001
Figure 1. Structural and Thermodynamic Characteristics of the A-State (A–C) Far-UV CD (A), near-UV CD (B), and Trp-fluorescence (C) spectra for the N-state at pH 4.5 (red), the A-state at pH 2.0 (blue), and the U-state at pH 2.0 with 3 M urea (dotted black line), respectively. (D) Thermal unfolding of the N-state at pH 4.5 (red) and the A-state at pH 2.0 (blue) monitored via the change in mean residue ellipticity at 222 nm. The solid lines represent least-squared fits to van’t Hoff’s equation, where the equilibrium constant between two states is described by the exponential of (DHwf DST)/RT. DHwf (kcal/mol) and DS (cal/mol$K)] of N- and A-states are (42 ± 4 and 122 ± 11) and (16 ± 1 and 48 ± 2), respectively. (E) 13Ca-chemical shift indices of the N-state at pH 4.5 (red) and the A-state at pH 2.0 (blue). (F) 13Ca-chemical shift indices of the A-state plotted onto the 3D structure of mouse PrP(121–231) (PDB: 1AG2). The unassigned residues are shown in gray.
with folding and unfolding on the submillisecond timescale, using an ultra-rapid continuous-flow mixing technique with a dead time of approximately 60 ms (Roder et al., 2004, 2006; Shastry et al., 1998). Our results indicate that the A-state is clearly distinct from the I-state. Quantitative analysis of the folding and unfolding kinetics suggested that the A-state corresponds to a late, native-like intermediate in the folding of PrP, and that the A-state is a key intermediate located at a bifurcation point between the monomeric folding and oligomerization aggregation pathways. RESULTS Structural and Thermodynamic Characteristics of the A-State As in previous kinetic studies on the human PrP fragment 90–231 (Apetri et al., 2006; Apetri and Surewicz, 2002), we worked with a W145H/Y218W mutant of the highly homologous mouse protein, which contains a single Trp residue in the folded domain. By lowering the pH from 4.5 to 2.0, the native state of PrP (N-state) was transformed into an acid-induced partially unfolded state (A-state), which is characterized by a moderate change in secondary structure (Figure 1A) and a partially disrupted tertiary structure (Figure 1B), as revealed by far- and near-UV circular dichroism (CD), respectively. The Trp-fluorescence emission spectrum of the A-state coincides with that of the N-state (Fig-
ure 1C), indicating that the A-state maintains native-like tertiary structure in the region surrounding the tryptophan residue (see also Figure 1F). The A-state is thermodynamically characterized by a low enthalpy and entropy change upon unfolding (16 kcal/mol and 48 cal/mol$K for DHwf and DS, respectively) compared with the N-state (42 kcal/mol and 122 cal/mol$K for DHwf and DS, respectively) (Figure 1D). The 13Ca-chemical shift indices at pH 2.0 obtained by sequentially assigning the peaks in the NMR spectrum (Figure S1) exhibit values near zero in the S1H1-H2 segment (amino acid residues 129–160) (Figures 1E and 1F). This shows that this segment is almost completely unfolded in the A-state (Wishart et al., 1992), which is consistent with previous hydrogen/deuterium exchange results (Honda et al., 2014). In contrast, most of the H2-H3 segment remains unassigned due to severe line broadening (Figure S1), which suggests that this region fluctuates between some folded and unfolded structures on the millisecond timescale. Thus, the N-terminal S1-H1-S2 segment appears to be extensively disordered or completely unfolded in the A-state, while the C-terminal H2-H3 segment retains a marginally stable structure (Honda et al., 2014). Accumulation of a Kinetic Intermediate State during Folding We examined the folding and unfolding kinetics of the W145H/ Y218W variant of mouse PrP(90–231) at pH 4.5 in the presence of various concentrations of urea (0–7.0 M) starting from
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Please cite this article in press as: Honda et al., A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.07.001
Figure 2. Folding and Unfolding Kinetics (A) Representative kinetic traces for folding of mouse PrP at pH 4.5 in the presence of 0.5–2.5 M urea, starting from conditions favoring the U-state (pH 2.0, 3.0 M urea). (B) Representative traces for unfolding at pH 4.5 in the presence of 5.0–7.0 M urea, starting from conditions favoring the N-state (pH 4.8, 2.0 M urea). The solid lines represent least-squared fits to a single exponential function. (C) Chevron plots in the absence (filled circles) or presence (empty squares) of 0.17 M Na2SO4. The solid and dotted lines represent curves generated by assuming a three-state scheme (Table S1).
the U-state at pH 2.0 and 3.0 M urea, as well as from the N-state at pH 4.8 and 2.0 M urea. To resolve the kinetics of folding of PrP, which occurs on the millisecond timescale or faster, even at 5 C (Apetri et al., 2006; Apetri and Surewicz, 2002), we combined ultra-rapid mixing (Shastry et al., 1998) with conventional stopped-flow measurements. The refolding and unfolding time courses are both well described by a single exponential function over the time range from approximately 100 ms to 30 ms (Figures 2A and 2B). However, the log(rate) versus urea concentration (chevron) plot strongly deviates from a linear dependence on urea concentration below approximately 2 M (Figure 2C). This non-linearity, known as ‘‘rollover,’’ is generally attributed to the accumulation of a folding intermediate within the dead time. Furthermore, the rollover was more prominent in the presence of 0.17 M Na2SO4 (Figure 2C and Table S1), which generally stabilizes folding intermediates (Gorski et al., 2001; Jahn et al., 2006; Khorasanizadeh et al., 1996). Thus, an early kinetic intermediate, which we refer to as the I-state, accumulates during the refolding of PrP from strongly denaturing conditions. Unfortunately, without direct observation of the fast U/I transition, we cannot determine whether the I-state lies on a direct path from U to N (Capaldi et al., 2001). However, the main purpose of this study is to elucidate the kinetic role of the A-state, and the following kinetic analysis was found to be independent of the kinetic role of the I-state (data not shown). Therefore, for simplicity, we assume that the I-state is an on-pathway intermediate. The Folding/Unfolding Kinetics of the A-State Next, we examined the folding/unfolding kinetics starting from Astate conditions at pH 2.0. Final conditions were the same as those in experiments starting from the N- and U-states (pH 4.5 in the presence of 0–7 M urea). It should be noted that the folding kinetics of the A-state below 2.0 M urea is not accompanied by a prominent fluorescence change (Figure 3A), in contrast to the folding experiment shown in Figure 2A, indicating that the Astate does not correspond to the kinetic intermediate I. In addition, the A-state can probably reach the N-state without going through the I-state. Otherwise, its folding rate would be limited by the major kinetic barrier of the I/N transition and we should
observe a phase with a rate constant of approximately 3,000 s1 (Figures 2A and 2C). This also indicates that the A-state lies on the native side of the major kinetic barrier for the I/N folding transition. Interestingly, the folding/unfolding kinetics of the Astate above 3.0 M urea exhibit two distinct phases with rate constants of approximately 5,000 and 500 s1, respectively (Figures 3A and 3B). The fast phase was not observed in Figure 2B, while the slow phase closely matches that observed in the previous unfolding experiments (Figure 3B). This indicates that the A-state partially shares a common unfolding pathway with the N-state. Evidence for the A-State as a Late Folding Intermediate Next, we investigated which folding scheme best describes the folding/unfolding kinetics of the A-state. As we have shown that A and I are distinct states (Figures 3A and 3B), the minimal mechanism is a four-state scheme. We initially tested different linear four-state schemes, such as U4I4A4N or U4A4I4N. Theoretically, there are 16 distinct linear schemes (Figure S2), but 11 of them can be ruled out by assuming that the I-state is an on-pathway intermediate (black in Figure S2). Furthermore, based on the folding/unfolding kinetics of the A-state (Figures 3A and 3B), we were able to place the A-state beyond the kinetic barrier of the I/N transition, which rules out three of the five remaining schemes. As a result, only two schemes remained: U4I4A4N, where the A-state corresponds to a late folding intermediate, and U4I4N4A, where the A-state corresponds to an off-pathway intermediate (blue in Figure S2). The latter scheme, however, can also be ruled out by taking into account the amplitude plot (Figure 3C). In the U4I4N4A scheme, the fast and slow phases observed during the unfolding of the A-state are interpreted as the A/N and N/I transitions, respectively. Therefore, a major fraction of PrP is expected to form the N-state just after the fast phase. However, the fluorescence intensity just after the fast phase, which is approximately described by the sum of the fluorescence intensity at infinity and the kinetic amplitudes of the slow phase, FN + As (downward triangles in Figure 3C), was much higher than the fluorescence of the N-state or the native baseline (inset, Figure 3C). On the other hand, the U4I4A4N scheme fully reproduces the folding/unfolding kinetics of the A-state, including the rate constants and kinetic
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Figure 3. Kinetics of Folding and Unfolding of the A-State (A) Representative folding/unfolding kinetic traces at pH 4.5 in the presence of 0–7.0 M urea starting from conditions favoring the A-state (pH 2.0). The solid lines represent least-squared fits to a double exponential function: fðtÞ=fðAÞ = Af expð lf tÞ + As expð ls tÞ + fN : (Equation 1) (B) Chevron plot. Filled circles and empty triangles represent lf and ls in Equation 1, respectively. For comparison, the apparent rate constants in Figure 2C are shown as empty squares. The solid curve represents the curve reproduced by assuming the U4I4A4N scheme (Table 1). (C) Amplitude plot. Circles, downward triangles, and upward triangles represent fN, (fN + As), and (fN + As + Af) from Equation 1, respectively. The solid curve represents the curve reproduced by assuming the U4I4A4N scheme (Table 1). The fluorescence intensity of the U-, I-, A-, and N-states at 0 M urea is 1.01, 1.30, 1.05, and 1.05, respectively. The slope against urea concentration (M1) of the U-, I-, A-, and N-states is 0.23, 0.10, 0.05, and 0.05, respectively. (Inset) Equilibrium urea titration curve obtained under the same conditions. The dotted line represents the baseline N-state. (D) Free-energy diagram. The activation free energies of the transitions, Ea = RTln(k/A0), were calculated from kIA and kAN (Table 1) values using a pre-exponential factor A0 = 106 (s1). The double dagger symbols represent the transition states. Bars represent fitting errors (SD).
amplitudes (Figures 3B and 3C, Table 1). This analysis assumed that the A-state shares common fluorescence properties with the N-state, namely 1.05 of the fluorescence intensity at 0 M urea and 0.05 of the slope versus urea concentration (Figure 3C), consistent with the well-organized tertiary structure of the A-state (Figures 1B and 1C). Hence, we concluded that the PrP folding is described by the U4I4A4N scheme, i.e., the A-state corresponds to a late folding intermediate. The fact that we observed only one intermediate state in our first set of folding/unfolding experiments (Figures 2A–2C) is not inconsistent with this conclusion, since the four-state scheme predicts only one exponential phase with measurable amplitude within the approximately 100-ms time resolution of our measurements, as observed (Figures 4A and 4B). A three-state scheme can also reproduce the chevron plot (data not shown). Since the A-state lies beyond the rate-limiting step in both folding and unfolding experiments (Table 1), its maximum population is only 23% and 11% during folding and unfolding, respectively. The fluorescence properties of the A-state closely correspond to those of the N- state (Table 1), which further hampered detection of the A-state. Thus, the four-state scheme is fully consistent with all our kinetic data, including those in Figures 2A–2C.
Table 1. Kinetic Parameters for the Four-State Scheme (U4I4A4N) KUIa (MUI b) kIAc (mIAd) 29 (0.80) a
kAI (mAI)
Equilibrium constant. m value (kcal/mol$M). c Elementary rate constant (s1). d Kinetic m value (kcal/mol$M). b
kAN (mAN)
kNA (mNA)
4,176 (0.03) 1,298 (0.08) 8,222 (0.14) 331 (0.07)
It should be noted that we cannot definitively rule out more complicated schemes, such as five-state schemes and branched four-state schemes (Radford et al., 1992). Pulsed hydrogen/deuterium exchange and/or the quenched-flow experiments would be beneficial to rule out these more complicated schemes, but the short lifetime of the kinetic intermediate states (Figures 2A and 3A) and the high tendency of PrP to aggregate at alkaline pH make such studies very challenging. However, the quality of the fits in Figure 3 indicates that the U4I4A4N scheme is the simplest mechanism that describes the folding/unfolding kinetics of mouse PrP. DISCUSSION In this study we investigated the kinetic role of the A-state of mouse PrP, an acid-induced partially unfolded state with some of the characteristics of a molten globule recently identified as a possible pre-oligomeric state (Honda et al., 2014). By examining the kinetics of folding and unfolding under various initial and final conditions, we showed that the A-state corresponds to a late folding intermediate, suggesting that oligomerization starts from this partially unfolded state, which contains a native-like H2-H3 hairpin (Figure 5). Under physiological conditions, late folding intermediates are expected to be more highly populated than early intermediates. They are, therefore, more likely to lead to formation of pathogenic oligomers (Brockwell and Radford, 2007). Thus, the A-state may be a key intermediate in both folding and self-association, giving rise to the formation of b-rich oligomers. A number of studies have demonstrated that lowering pH increases the rate constant of b-rich oligomer formation (Hornemann and Glockshuber, 1998; Jain and Udgaonkar,
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Figure 4. Folding and Unfolding Kinetics Predicted by the U4I4A4N Scheme The folding kinetics at 0 M urea (A) and the unfolding kinetics at 6 M urea (B) were calculated by solving the four-state mechanism using the parameters in Table 1 and the legend of Figure 3. The upper and lower panels represent the timedependent fluorescence change and the fraction of each state, respectively.
2008; Morillas et al., 2001; Rezaei et al., 2002; Swietnicki et al., 1997). For instance, for mouse PrP we observed rates of 0.0389 min1 and 15.7 min1 at pH 4.5 and 2.0, respectively (Honda et al., 2014). Acceleration of the aggregation process at acidic pH may be attributed to an increase in the equilibrium population of the A-state. From the kinetic parameters of PrP folding presented in this report, the equilibrium population of the A-state at pH 4.5 was calculated to be 3.8% (Table 1), whereas at pH 2.0 almost 100% of the protein forms the A-state. This correlation between aggregation rate and A-form population is consistent with the hypothesis that the A-state is located at the branching point between folding and aggregation. Since there is no evidence for infectivity of the b-rich PrP oligomers formed at acidic pH, it remains unclear whether or not the A-state is actually involved in the pathogenic conversion into PrPSc. However, recent studies have revealed that mutations linked to prion diseases destabilize H1 and accelerate the formation of b-rich oligomers (Singh and Udgaonkar, 2015). They further suggest that the RNA and POPG (1-palmitoyl-2-
Figure 5. A Schematic Free-Energy Landscape for Folding and Oligomerization of PrP In this scheme, unfolded PrPC (U) folds into the N structure via two intermediate states: the I- and A-states (black arrow). Oligomerization and aggregation starts from a late folding intermediate, the A-state (red arrow). The A-state is a key intermediate from which the folding and oligomerization pathways diverge. F, Q1, and Q2 represent the Gibbs free energy, the first reaction coordinate, and the second reaction coordinate, respectively.
oleoylphosphatidylglycerol) molecules that facilitate the pathogenic conversion (Wang et al., 2010) also destabilize H1 (Miller et al., 2013). The conformational characteristics of the A-state suggest that partial unfolding of the S1-H1-S2 region may trigger further conformational changes at the C terminus of H2 and in the H2-H3 loop region (Kuwata et al., 2007; Miller et al., 2013; Singh et al., 2014), which facilitate intermolecular interactions by exposing the hydrophobic residues at the interface between the H2 and H3 helices. Furthermore, anti-prion compounds (‘‘medical chaperones’’) intercalate between the H1 and H2-H3 loops and stabilize PrP (Kamatari et al., 2013; Kuwata et al., 2007). Thus, H1 destabilization and formation of the A-state (Figure 1E) may be related to the pathogenesis of prion diseases. Since the initial process of b-rich oligomer formation is similar to that of PrPSc formation, it is reasonable to assume that the A-state corresponds to the initial monomeric precursor in the pathogenic conversion or PrP* (Cohen et al., 1994). In a recent ultra-fast mixing study of the folding/unfolding kinetics of sheep PrP, Chen et al. (2011) observed accumulation of an intermediate state during unfolding that corresponds to the A-state observed in the present study. This unfolding intermediate was stabilized by mutations related to inherited prion disease (Chen et al., 2011), suggesting that the kinetic intermediate detected is involved in pathogenesis. This sheep PrP unfolding intermediate is characterized by native-like fluorescence properties and compactness, which is consistent with our current observations (Figure 1C and Table 1), as well as the structural characteristics observed for the equilibrium A-state of mouse PrP (Honda et al., 2014). Previous kinetic studies identified an early intermediate in the folding of human PrP (I-state in our terminology) that was proposed to act as a monomeric precursor to b-rich oligomers. This proposal was based on the fact that the intermediate is stabilized by mutations linked to prion diseases (Apetri et al., 2004, 2006) and by conditions known to promote aggregation, such as low pH (Apetri et al., 2006; Apetri and Surewicz, 2002). In terms of its kinetic and structural properties, the I-state is clearly distinct from the A-state (Figures 3A and 3B). Given the high degree of sequence conservation among the three PrP variants and the similarity of the chevron plots for the rate-limiting folding/unfolding phases (Apetri et al., 2006; Chen et al., 2011), it is likely that they share a common folding mechanism, such as the sequential four-state mechanism shown in Figure 5. The observed species-dependent differences in kinetic behavior on the submillisecond timescale can be explained in terms of relatively subtle changes in the free-energy profiles among the three
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PrP variants (cf. Figure 3D). For example, the fact that the fast phase seen during the unfolding of mouse and sheep PrPs is absent during the unfolding of human PrP can be explained by a modest shift in the relative free energies of the two barriers, resulting in a decrease in transient accumulation of the A-state. By modeling the folding/unfolding kinetics of mouse PrP using alternative kinetic schemes, we were able to show that the A-state lies on a direct path from the unfolded ensemble to the N-state (Figure 5). Combined with the structural properties of the A-state (Figures 1E and 1F), this finding implies that during the folding of mouse PrP, formation of the H2-H3 helical hairpin precedes structure acquisition in other regions, including H1 and the small b sheet. This conclusion is consistent with the relatively high hydrophobicity of the H2-H3 segment, as well as the presence of a disulfide bond connecting H2 and H3. A similar scenario was proposed for human PrP, based on temperature jump experiments coupled with F-value analyses, which suggested that the hydrophobic core of the H2-H3 segment is formed during the rate-limiting step in folding (Hart et al., 2009).
Folding/unfolding was initiated by mixing the initial solutions with a folding/unfolding buffer (pH approximately 4.5, 50 mM sodium acetate with 0–7 M urea in the presence or absence of 0.2 M Na2SO4) using a mixing ratio of 1:5. This procedure allowed us to observe the refolding/unfolding kinetics at pH 4.5 as a function of urea concentration. The kinetics were monitored via the change in fluorescence at 308–420 nm (excitation at 297 nm) using both the stopped-flow and ultra-rapid mixing instruments (Roder et al., 2004, 2006; Shastry et al., 1998; Xu et al., 2012). The dead time of the ultra-rapid mixer was determined to be 56 ± 5 ms by measuring the quenching of N-acetyltryptophanamide fluorescence by N-bromosuccinimide (Roder et al., 2004, 2006; Xu et al., 2012). The fluorescence traces independently obtained using the stopped-flow and ultra-rapid mixing apparatus were globally fitted to a single or double exponential function with common values of the apparent rate constants and kinetic amplitudes. The fluorescence intensities were normalized to those measured under the initial conditions. Protein concentrations of 5 and 10 mM were utilized for the stopped-flow and ultra-rapid mixing measurements, respectively. The family of kinetic traces for folding/unfolding of the N- and A-states of mouse PrP was modeled globally, assuming a four-state sequential scheme (U4I4A4N). Elementary rate constants and kinetic m values obtained by numeric solution of the differential equations describing this scheme (Xu et al., 2012) are provided in Supplemental Experimental Procedures.
EXPERIMENTAL PROCEDURES
SUPPLEMENTAL INFORMATION
Protein Preparation A plasmid encoding mouse PrP, amino acid residues 90–231 (mouse PrP(90– 231)), with an N-terminal 63 His tag, was prepared according to a previously reported protocol (Kuwata et al., 2007). Two mutations (W145H/Y218W) were introduced into mouse PrP(90–231) utilizing the PrimeSTAR Mutagenesis Basal Kit (Takara Bio). Mouse PrP(90–231,W145H/Y218W) was expressed and purified as previously described (Kuwata et al., 2007).
Supplemental Information includes Supplemental Experimental Procedures, two figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.str.2015.07.001.
Equilibrium Measurements Solutions of mouse PrP(90–231,W145H/Y218W) were prepared under N-state (pH 4.5, 20 mM sodium acetate), A-state (pH 2.0, 20 mM glycine-HCl), and U-state (pH 2.0, 20 mM Gly-HCl, 3 M urea) conditions. The protein concentrations were 10, 70, and 10 mM for far-UV CD, near-UV CD, and fluorescence measurements, respectively. CD spectra were acquired at 20 C according to a previously described protocol (Honda et al., 2014). Fluorescence emission spectra were acquired on an RF-5300PC spectrofluorophotometer (Shimadzu) at 20 C using an excitation wavelength of 298 nm. Thermal unfolding of the N- or A-state was monitored by the change of mean residue ellipticity at 222 nm by increasing the temperature up to 95 C at a rate of 2 C$min1. More than 90% of the mean residue ellipticity was recovered after the thermal denaturation experiment (data not shown). For NMR studies of the A-state, 13C,15N uniformly labeled mouse PrP(90– 231,W145H/Y218W) was dissolved with the A-state buffer containing 5% D2O to a final concentration of 0.5 mM, and a series of 3D NMR spectra (1H-15N HSQC, HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCBCA, and HN(CO)CBCA) were acquired. A relatively higher temperature, 32 C, at which approximately 80% of the secondary structure of A-state was retained, was utilized to improve the quality of the NMR spectra (Eliezer et al., 1997) (Figure 1D). The NMR peaks were assigned by the sequential method (Ikura et al., 1990; Kay et al., 1990) using NMRpipe (Delaglio et al., 1995) and KUJIRA (Kobayashi et al., 2007) software. Secondary chemical shifts were calculated by subtracting the chemical shifts of the NMR peaks from those of random coils that were predicted using a freely available program coded by SBiNLab (Kjaergaard et al., 2011). For NMR studies of the N-state, mouse PrP(90–231, W145H/Y218W) was dissolved in native buffer containing 5% of D2O to a final concentration of 0.5 mM. A series of 3D NMR spectra (1H-15N HSQC, HNCO, HN(CA)CO, HNCA, and HN(CO)CA) were acquired at 12 C and analyzed as described above. Kinetic Measurements and Modeling The kinetics of folding and unfolding starting from various conditions that favor the N- (pH 4.8, 20 mM sodium acetate, 2 M urea), U- (pH 2.0, 20 mM Gly-HCl, 3 M urea), and A-states (pH 2.0, 20 mM Gly-HCl) were examined at 5 C.
ACKNOWLEDGMENTS We thank Yumiko Okuda and the Life Science Research Center in Gifu University for assistance with the experiments. This work was supported by grants from the Ministry of Health, Labor, and Welfare of Japan (Research on Measures for Intractable Diseases [Prion Disease and Slow Virus Infections, and Development of Low Molecular Weight Medical Chaperone Therapeutics for Prion Diseases]) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants-in Aid for Scientific Research, and X-ray Free Electron Laser [XFEL] Program). Funding was also received from NSF grant MCB-1412378 and NIH grant R01 GM056250 (to H.R.) and an NIH P30 grant CA06927 (to the Fox Chase Cancer Center). Received: April 16, 2015 Revised: June 8, 2015 Accepted: July 1, 2015 Published: August 6, 2015 REFERENCES Apetri, A.C., and Surewicz, W.K. (2002). Kinetic intermediate in the folding of human prion protein. J. Biol. Chem. 277, 44589–44592. Apetri, A.C., Surewicz, K., and Surewicz, W.K. (2004). The effect of diseaseassociated mutations on the folding pathway of human prion protein. J. Biol. Chem. 279, 18008–18014. Apetri, A.C., Maki, K., Roder, H., and Surewicz, W.K. (2006). Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments. J. Am. Chem. Soc. 128, 11673–11678. Arai, M., Ito, K., Inobe, T., Nakao, M., Maki, K., Kamagata, K., Kihara, H., Amemiya, Y., and Kuwajima, K. (2002). Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering. J. Mol. Biol. 321, 121–132. Brockwell, D.J., and Radford, S.E. (2007). Intermediates: ubiquitous species on folding energy landscapes? Curr. Opin. Struct. Biol. 17, 30–37. Capaldi, A.P., Shastry, M.C.R., Kleanthous, C., Roder, H., and Radford, S.E. (2001). Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nat. Struct. Biol. 8, 68–72.
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