Shreya Ghosh,Vitali Tugarinov,G. Marius Clore
Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-β42 aggregation and fibril formation
substoichiometric inhibition,amyloid fibril formation,kinetics,global fitting,NMR spectroscopy
Significance The yeast chaperone Hsp104 inhibits fibrillization of amyloidogenic polypeptides in a paradoxically substoichiometric manner. We monitored the disappearance of amyloid-β42 monomers during aggregation by NMR, and developed a kinetic model that quantitatively accounts for the experimental data over a wide range of Hsp104 concentrations. At high amyloid-β42 concentrations, aggregation involves a branched mechanism: irreversible on-pathway fibrillization, and a reversible off-pathway leading to nonfibrillar oligomers that are too large to be observed directly by NMR but too small to be imaged. Hsp104 binds with nanomolar affinity to sparsely populated amyloid-β42 nuclei, generated by primary and secondary nucleation, providing a general mechanism for complete inhibition of on-pathway fibrilization at substoichiometric ratios of Hsp104 to amyloid-β42 monomers, while only slightly impacting off-pathway oligomerization.
Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-β42 aggregation and fibril formation

The yeast chaperone Hsp104 inhibits fibrillization of amyloidogenic polypeptides in a paradoxically substoichiometric manner. We monitored the disappearance of amyloid-β42 monomers during aggregation by NMR, and developed a kinetic model that quantitatively accounts for the experimental data over a wide range of Hsp104 concentrations. At high amyloid-β42 concentrations, aggregation involves a branched mechanism: irreversible on-pathway fibrillization, and a reversible off-pathway leading to nonfibrillar oligomers that are too large to be observed directly by NMR but too small to be imaged. Hsp104 binds with nanomolar affinity to sparsely populated amyloid-β42 nuclei, generated by primary and secondary nucleation, providing a general mechanism for complete inhibition of on-pathway fibrilization at substoichiometric ratios of Hsp104 to amyloid-β42 monomers, while only slightly impacting off-pathway oligomerization.

Protein misfolding and aggregation are associated with a large number of diseases, including amyloidoses and a variety of neurodegenerative conditions, including Alzheimer’s disease (1, 2). To mitigate the effects of protein aggregation and in some instances to restore aggregated proteins to their native structure, prokaryotic and eukaryotic organisms have developed a complex machinery of molecular chaperones (3–5). Many chaperones play an important role in ensuring correct folding of native polypeptides and preventing aggregation of misfolded proteins (6–11). A special class of chaperones known as disaggregases act on protein aggregates, separate them into polypeptides and eventually refold them to their native state (12–15). While there are several examples of disaggregases that can structurally remodel disordered protein aggregates, only a few are purported to remodel highly ordered amyloid fibrils. One such chaperone, and the subject of the current work, is the yeast chaperone Hsp104, a member of the Hsp100/Clp family of AAA+ (ATPases Associated with various cellular Activities) translocases (16). Amyloids are unbranched protein aggregates/fibrils characterized by the presence of cross-β sheet secondary structure (17, 18). Fibrils formed by amyloid-β (Aβ) peptides are the main constituent of amyloid plaques found in the brains of individuals with Alzheimer’s disease, one of the most common causes of senile dementia (19). Of the several Aβ peptides generated by cleavage of the amyloid precursor protein, Aβ42 is the most aggregation prone (20, 21). Several chaperones have been shown to inhibit or delay the formation of Aβ42 fibrils (22–26). Once formed, however, mature Aβ42 fibrils are highly ordered and difficult to disaggregate. The active form of the Hsp104 chaperone is a ~600 kDa homo-hexamer of identical subunits, enclosing a large solvent-filled channel (27–31) through which substrates are translocated (32). Substrate recognition occurs via the N-terminal domain (33). Hsp104 has been shown to prevent the aggregation of native client proteins with and without co-chaperones or adenosine triphosphate (ATP) (22, 34–36), and in the case of various amyloids, to inhibit fibril formation in an apparently substoichiometric manner (22, 37–39). Although Hsp104 homologs are present in prokaryotes and eukaryotes, Hsp104 is the only known member of the Hsp100/Clp family that is thought to be able to disaggregate mature amyloid fibrils (38, 40, 41), but this has been subject to dispute (22). Here we provide a quantitative NMR analysis of the kinetic mechanism whereby Hsp104 inhibits fibrillization of Aβ42, even at substoichiometric ratios, by monitoring and global fitting the time courses of disappearance of monomeric Aβ42 in the absence and presence of variable concentrations of Hsp104 by means of serially recorded 1H-15N band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC) spectra (42, 43). We show that at high concentrations of Aβ42 (~50 μM) aggregation occurs via a branching mechanism entailing an irreversible productive pathway leading to mature amyloid fibrils, and a reversible off-pathway resulting in the formation of oligomers/aggregates that do not possess cross-β sheet structure. Hsp104 acts by binding tightly and reversibly to sparsely populated, on-pathway Aβ42 nuclei capable of chain elongation, thereby shutting down on-pathway fibril formation, while only minimally impacting off-pathway oligomerization. This kinetic mechanism resolves the observation of apparent substoichiometric inhibition of fibril formation since the concentration of Hsp104 exceeds that of Aβ42 nuclei for most of the aggregation time course.

The kinetics of fibril formation of 5 μM Aβ42 in the presence of 2 to 12 μM (in monomer units) hexameric Hsp104 (SI Appendix, Fig. S1) was monitored by ThT fluorescence at 37 °C (Fig. 1). ThT fluorescence increases upon interaction with β-sheet structure (44, 45), a characteristic of amyloid fibrils (46). In the absence of Hsp104, a significant increase in ThT fluorescence is observed with a lag phase characteristic of fibril formation (47) and a half-time of ~5 h. Upon addition of as little as 2 μM Hsp104 (monomer concentration), no increase in fluorescence over a period of 40 h is observed indicating that fibril formation is completely blocked even at substoichiometric ratios of Hsp104 to Aβ42. These results demonstrate the intrinsic activity of Hsp104 in preventing Aβ42 fibrillization in the absence of ATP or co-chaperones, consistent with previous reports (22).

Whereas ThT fluorescence monitors the appearance or disappearance of fibrils characterized by β-sheet structure, NMR monitors the disappearance of NMR visible monomers as the NMR signals of higher-order oligomers, protofibrils and fibrils are broadened beyond detection (43). The approach employed here is to serially record a set of 2D 1H-15N SOFAST-HMQC spectra over a period of 66 h and monitor the time dependence of cross-peak intensities and volumes, as described previously for the aggregation of the huntingtin exon 1 protein (43). This particular experiment is spin-lattice relaxation time (T1) optimized, thereby permitting rapid recovery of longitudinal magnetization with short interscan delays, affording high-quality 1H-15N correlation spectra to be obtained in a reasonably short period of time on dilute samples (42, 48). Here the initial concentration of monomeric Aβ42 was 50 μM and good signal-to-noise 2D spectra were obtained in 20 min. While the ThT fluorescence experiments reported in Fig. 1 were conducted at 37 °C, a lower temperature had to be used for the NMR experiments as the 1H-15N cross-peaks were broadened at 37 °C owing to amide proton exchange with water. We used 20 °C which provided an optimal balance between spectral quality and aggregation time. At much lower temperatures, such as 5 °C, aggregation of 50 μM Aβ42 is very slow and occurs over a period of weeks (49). Fig. 2 displays the time courses of Aβ42 monomer decay as a function of Hsp104 concentration. Examples of the 2D 1H-15N SOFAST-HMQC spectra are provided in SI Appendix, Fig. S2. The data are displayed as the averages over 21 well-resolved residues spanning residues 5 to 42 (SI Appendix, Fig. S3). The time courses for the decay of the average cross-peak intensity and volume are superimposable (Fig. 2A), indicating that there are no observable submillisecond to millisecond pre-nucleation exchange processes involving sparsely populated species with significantly different 1HN/15N chemical shifts from the monomer, as was the case with the huntingtin exon-1 protein (43). Cross-peak volumes are determined only by the concentration of observable monomer, whereas cross-peak intensities are not only determined by concentration but also by linewidth if exchange processes are present that are fast or intermediate on the chemical shift time scale. In the case of the huntingtin exon-1 protein, for example, submillisecond pre-nucleation exchange between the predominant monomeric species and sparsely populated helical coiled-coil dimers and tetramers results in concentration-dependent exchange broadening; as a result the cross-peak intensities for residues involved in this exchange process initially increase as the concentration of monomer decreases resulting in a reduction in linewidth (43). The half-time for the complete disappearance of monomeric Aβ42, at an initial concentration of 50 μM, is ~20 h at 20 °C (Fig. 2 B, Left). The time course is multiphasic and characteristic of the kinetics of fibril formation (46). It should also be noted that the time courses of decay for the 1HN/15N cross-peaks for the 21 well-resolved residues (SI Appendix, Fig. S3) are identical within experimental error, indicating that the formation of NMR invisible aggregates (large oligomers, protofibrils and fibrils) is concerted over the length of the polypeptide. The addition of either 0.05 or 0.1 μM Hsp104 prolongs the initial slow decay in cross-peak intensity and increases the overall half-time for complete disappearance of NMR signal to ~28 and 38 h, respectively (Fig. 2 B, Left). Upon addition of 1 μM Hsp104, however, ~72% of the monomer Aβ42 signal is still visible at 66 h, and the decay curve appears to be linear (Fig. 2 B, Right). As the concentration of Hsp104 is increased further, the decrease in the normalized cross-peak intensity becomes increasingly biphasic with a fast initial phase, and a slow, almost linear second phase (Fig. 2 B, Right). Paradoxically as the concentration of Hsp104 is increased beyond 1 μM, the amplitude of the initial phase is increased and the remaining monomer signal at 66 h is actually decreased such that at 75 μM Hsp104 only ~43% of the detectable monomeric signal remains. It is also important to note that the decay in normalized cross-peak intensity at concentrations of Hsp104 ≥1 μM is only attributable to a decrease in the concentration of NMR visible, monomeric Aβ42 as the linewidths remain unchanged during the course of the experiment. A complete quantitative description of the effect of Hsp104 on the kinetics of disappearance of Aβ42 monomer is presented later in the paper.

Upon completion of the 1H-15N SOFAST-HMQC experiments after 66 h at 20 °C, aliquots of each NMR sample were taken and characterized by AFM and negative stain EM (Fig. 3). The samples of Aβ42 alone and in the presence of either 0.05 or 0.1 μM Hsp104 are characterized by the presence of an extensive network of amyloid fibrils (Fig. 3, AFM/EM panels 1 to 3), consistent with the complete disappearance of monomeric Aβ42 observed by NMR (Fig. 2 B, Left). As the concentration of Hsp104 is increased to 1 μM or higher, globular Hsp104 particles (comparable to those seen for a sample of Hsp104 alone, Fig. 3, Bottom panels) together with a few thin, short filamentous Aβ42 structures are seen (Fig. 3, AFM/EM panels 4 to 7). The density of the latter clearly decreases as the concentration of Hsp104 is increased. Thus, at higher Hsp104 concentrations (≥1 μM) the decay in the monomeric Aβ42 NMR signal (Fig. 2 B, Right), in conjunction with the absence of ThT reactivity (Fig. 1), is largely attributable to the formation of nonspecific Aβ42 aggregates that are too large to be directly observable by NMR but too small to be seen by either AFM or negative stain EM.

The simplest model that fully accounts for the time course of monomer decay for 50 µM Aβ42 (in the absence of Hsp104) at 20 °C is depicted in Fig. 4A and consists of a branched mechanism: an irreversible on-pathway leading to the formation of mature fibrils described by primary nucleation, saturating elongation, and secondary nucleation (46, 50), and a reversible off-pathway leading to the formation of NMR invisible (and ThT nonreactive) oligomers/aggregates (O′). This model is described by three ordinary differential equations: where M(t) is the (mass) concentration of mature fibrils (in monomer units); P(t) the (total) number concentration of nuclei (extendable ends); O′(t) the concentration of “off-pathway” NMR unobservable oligomeric species that cannot convert directly to fibrils; and m(t), the concentration of Aβ42 monomers given by m(t) = mtot – M(t) - O′(t), where mtot is the total monomer concentration at the start of aggregation (t = 0). nc, n2 and np are the orders of primary nucleation, secondary nucleation and oligomer O′ formation, respectively, and their respective optimal (integer) values were found to be 2, 1 and 2. Neither on-pathway nucleation nor off-pathway oligomerization strictly obey the tenets of classical nucleation theory; that is the reaction order of these processes are not necessarily related to the physical size of the nuclei P or off-pathway oligomers O′, and may therefore be viewed as a “coarse-grained” representation (51) of mechanistically more complicated, multi-step processes. kn is the primary nucleation rate constant (in units of M−1h−1); k2, the secondary nucleation rate constant (M−1h−1); k+, the rate constant for chain elongation (M−1h−1); Kr, the Michaelis constant for saturating elongation (in units of concentration, M) that accounts for retardation of chain elongation at high initial concentrations of Aβ42 [if Kr = m(t), the elongation is retarded twofold]; kp, the rate constant describing formation (nucleation) of the unobservable off-pathway oligomers O′ (M−1h−1); and kd, the dissociation rate constant of oligomers O′ back to monomers (h−1). It should be noted that in the above formulation, the time dependence of m(t) is expressed in terms of mtot, M(t) and O′(t), and hence Eq. 1.3 is not fully coupled to Eqs. 1.1 and 1.2. Consequently, in the later parts of the decays when M(t) and O′(t) are large, m(t) can become transiently negative. Under these conditions, the values of the reaction orders nc, n2 and np cannot be optimized since exponentiation of transiently negative values of m(t) by noninteger powers results in complex numbers. This problem can be circumvented by an alternative but equivalent representation in which an additional differential equation is added to explicitly express the time evolution of m(t) (SI Appendix, Eq. S1); this ensures that all the differential equations are fully coupled to one another. The resulting optimized noninteger values for the reaction orders are the same, within the 5 to 95% confidence limits, to the integer values for the reaction orders determined above by trial and error (SI Appendix). The calculated normalized NMR signal intensity, Icalc, is given by: where M(t) and O′(t) are obtained by numerical integration of Eqs. 1.1–1.3; A0 is a scaling factor that accounts for inaccuracies in the normalization of peak heights (and typically optimizes to ~0.99), and α is a scaling factor (~0.985) that accounts for inaccuracies in the measurement of very low (close to zero) NMR cross-peak intensities and corresponds to the normalized intensity at t = 0 minus the average intensity of the noise. The initial conditions are given by m(0) = mtot; P(0) = 0; M(0) = 0, and O′(0) = 0. The kinetic model of aggregation depicted pictorially in Fig. 4A and mathematically by Eqs. 1.1–1.3 is different from that employed previously for Aβ42 at much lower concentrations (≤6 µM) by Knowles and co-workers (50) in three respects. In order of importance, these are as follows. a) The kinetic scheme includes the addition of a reversible process for the formation of off-pathway oligomers O′ (Eq. 1.3) which cannot be converted to fibrils directly but first have to be converted back to monomers. This process is formally equivalent to primary nucleation with order np = 2. Eq. 1.3 is coupled to Eqs. 1.1 and 1.2 through the reduction in the monomer concentration m(t). b) Fibril elongation is retarded by the denominator in Eq. 1.2 following the law of “saturating” elongation (46). This occurs predominantly at high Aβ42 monomer concentrations and might arise from a monomer of Aβ42 binding nonproductively to an extendable end, thereby blocking further elongation of the fibril by addition of Aβ42 monomers. c) The order for secondary nucleation (n2 = 1 in the second term in Eq. 1.1) is lower than that for primary nucleation (nc = 2). Secondary nucleation is dependent on the concentration of fibrils, M(t), and, hence, assumes some interactions between Aβ42 monomers and fibrils as described for the case of Aβ42 aggregation in multiple instances by Knowles and co-workers (46, 47, 50, 52–56). When aggregation is unseeded, that is P(0) = 0, the target function (Experimental Procedures, Eq. 5) used to minimize the sum of squares between the experimental and calculated (Eq. 2) time courses of aggregation is characterized by a global minimum region comprising a very large number of local minima depending on the starting values used for k+, k2 and kn (46). As a result, the values of the individual rate constants k+, kn and k2 cannot be determined uniquely. The subset of physically plausible solutions, however, is constrained by the dominance of the elongation rate (determined by 2k+) over those of the nucleation steps (determined by kn and k2), so that the ratio M/P of the final concentration of mature fibrils (M) to nuclei (P) is an approximate measure of the average length of mature fibrils (in units of length extension per monomer). The best fit of Eqs. 1.1–1.3 and 2 to the time course for the decay in NMR signal (monomeric Aβ42) intensity is shown by the black continuous line in Fig. 4 B, Left. The normalized χ2 of the fit is 0.8 and no systematic deviations between observed and calculated time courses are apparent. The calculated M/P ratio at ~66 h, the time at the end of the experiment, is ~800 corresponding to a fibril length of ~0.4 μm [given a length extension of ~5 Å per monomer (57)], consistent with the AFM and EM images shown in the first set of AFM/EM panels in Fig. 3. When saturation of chain elongation is eliminated by setting the Michaelis constant Kr to a very high value such that [Kr + m(t)]/Kr ≈ 1, the normalized χ2 is increased to 1.7 (green curve in Fig. 4B) and clear systematic deviations are observed in the two expanded regions; removing off-pathway oligomerization by setting the association rate constant kp to 0 results in a normalized χ2 of 9.1 with very large systematic deviations throughout (Fig. 4B, red curve); and finally setting both Kr → ∞ and kp to 0 increases the normalized χ2 still further to 20.3 (Fig. 4B, blue curve). Fig. 4C shows the calculated time courses for the fractional contributions of primary and secondary nucleation. Similar to Aβ42 aggregation at much lower initial concentrations (50), the kinetics of nucleation is dominated by secondary nucleation after ~7.5 h (when a critical amount of fibrils is produced). As might be expected for Aβ42 at high concentrations, the elongation step is significantly retarded—especially so at the start of fibrillization (where the ratio of m(t)/Kr >> 1; see Fig. 4 C, Inset). The kinetic model for Aβ42 aggregation described by Eqs. 1.1–1.3 is applicable to even higher Aβ42 concentrations as shown by the fit to the 80 μM Aβ42 in SI Appendix, Fig. S4. However, at 80 μM Aβ42, the order of primary nucleation (nc) is reduced from 2 to 1 (and consequently a different value for the primary nucleation rate constant kn) implying that some mechanistic changes take place in the primary nucleation step at higher (>50 χM) initial concentrations of Aβ42.

To investigate whether Hsp104 binds to monomeric Aβ42 we conducted 15N lifetime line-broadening (15N–ΔR2) measurements on 50 μM 15N-labeled Aβ42 in the presence of 65 μM Hsp104 (monomer concentration) at 5 °C. ΔR2 measured as the difference in the values of the backbone amide 15N transverse relaxation rate (15N–R2) of the NMR observable species (monomeric Aβ42) in the presence and absence of a high molecular weight partner (~600 kDa hexameric Hsp104) arises from exchange between the free, low molecular weight, fast tumbling (NMR visible) and bound, high molecular weight, slow tumbling (NMR invisible) species (49, 58). The choice of temperature was dictated by the need to ensure that Aβ42 remained monomeric during the experimental measurements (49). The 15N-R2 profiles at 600 and 800 MHz in the absence of Hsp104 are typical of an intrinsically disordered peptide (Fig. 5 A, Top). The 15N-ΔR2 profiles display two distinct regions with high 15N-ΔR2 values between residues 17 to 21 and 38 to 41 indicative of immobilization in the Hsp104 bound state. These two regions, which comprise stretches of hydrophobic amino acids, have previously been shown by 15N-ΔR2 measurements to be involved in binding to the chaperonin GroEL (24, 59). The exchange regime and the population of the bound high molecular weight state can be ascertained from the ratio at 800 and 600 MHz spectrometer fields (58). When the population of the bound species is low (< ~15%), this ratio is given by 1.2α, where, kex is the exchange rate given by the sum of the dissociation rate constant koff and the apparent first-order association rate constant , and is the transverse relaxation rate in the bound state at spectrometer field i. When α → 1, and the ratio is equal to 1.2, the exchange is fast on the relaxation time scale and ; when α → 0, and the ratio is equal to ~ 1, the exchange is slow on the relaxation time scale and ; for values of α between 0 and 1, the exchange is intermediate on the relaxation time scale and kex is comparable to . The average ratio for 50 μM Aβ42 in the presence of 65 μM Hsp104 (monomer concentration) obtained from the slope of the correlation plot between the ΔR2 values at the two spectrometer fields is 1.15 (Fig. 5B), corresponding to α ~0.7. For a ~600 kDa protein at 5 °C, the expected 15N-R2 value is ~400 s−1 (59), from which one can deduce that kex ~ 1,300 s−1 and the population of the bound state, given by , is only ~2.5%, corresponding to an equilibrium dissociation constant of ~2.4 mM (with the concentration of Hsp104 expressed in terms of monomeric units). Thus, one can conclude that the interaction of monomeric Aβ42 with Hsp104 is very weak and the population of the monomeric bound state is so low that it can be neglected in terms of global quantitative kinetic analysis of the 1H-15N SOFAST-HMQC time courses for Aβ42 aggregation in the presence of Hsp104 (Fig. 2B and next section).

The time courses for the decay of monomeric Aβ42 (50 μM) obtained from serially recorded 1H-15N SOFAST-HMQC experiments at 20 °C in the presence of varying concentrations of Hsp104 (ranging from 0.05 to 75 μM in monomer units; see Fig. 2B) can be fully accounted for by the introduction of a single additional step to the kinetic scheme for Aβ42 aggregation (depicted in Fig. 4A and described by Eqs. 1.1–1.3) that entails fast (on the time scale of Aβ42 aggregation), reversible binding of Hsp104 to the nuclei P generated by both primary and secondary nucleation (Fig. 6A). The differential equation (1.2) describing the time evolution of the mass concentration (in monomer units) of mature Aβ42 fibrils, M(t), is then replaced by: with the concentration of free nuclei, Pfree(t), given by: where P(t) is the total concentration of nuclei obtained by numerical integration of the system of coupled differential equations given by Eqs. 1.1, 1.3, and 3 (see also SI Appendix for an alternative, but equivalent formulation that makes use of six differential equations, SI Appendix, Eqs. S1.1–S1.6); [Hsp104] is the monomer concentration of Hsp104; and is the apparent equilibrium dissociation constant for the binding of Hsp104 to the nuclei P. (Note we refer to the equilibrium dissociation constant as “apparent” because the stoichiometry and other properties of the P–Hsp104 binding interaction are not known.) The rate constants for primary (kn) and secondary (k2) nucleation, and the dissociation rate constant (kd) for off-pathway oligomerization were treated as global optimized parameters; the rate constant (kp) for the formation of off-pathway oligomers (O′) was optimized separately for each concentration of Hsp104; and the rate constant (k+) for elongation and the Michaelis constant (Kr) for saturating elongation were distinguished only between the absence ([Hsp104] = 0) and presence ([Hsp104] > 0) of Hsp104. This “separation” of k+ and Kr was necessary to ensure the best fits of the highest possible quality (reducing the normalized χ2 of the fit by ~4-fold), and the impact and possible origin of this phenomenon is discussed below. The global best-fits to the experimental aggregation profiles obtained for 50 µM Aβ42 in the presence of varying amount of Hsp104 are shown in Fig. 6B, and the Inset (Left) shows the dependence of the rate constant kp for off-pathway oligomer formation as a function of Hsp104 concentration. The normalized χ2 of the overall fit (which includes the fit to the aggregation profile for 80 μM Aβ42 in the absence of Hsp104 shown in SI Appendix, Fig. S4) is 0.88. The treatment of kp as a separate parameter for each concentration of Hsp104 represents an ad hoc coarse-grained procedure since the mechanism whereby Hsp104 affects off-pathway oligomerization is not known. The empirical observation, however, is that Hsp104 initially slows down off-pathway oligomerization with kp rapidly decreasing from ~520 M−1h−1 in the absence of Hsp104 to a minimum of ~120 M−1h−1 at 1 μM Hsp104; as the concentration of Hsp104 is increased beyond 1 μM, the rate of off-pathway oligomerization is increased with kp reaching a value of ~500 M−1h−1 at 75 μM Hsp104, comparable to its value in the absence of Hsp104. As noted above, the elongation rate constant k+ and Michaelis retardation constant Kr were treated separately in the absence and presence of Hsp104 to ensure optimal fits to the data. The impact of the slightly different optimized values for these parameters in the absence and presence of Hsp104 (see Fig. 6B legend) on the time evolution of the chain elongation retardation factor (given by the ratio of the rates of mature fibril formation, dM(t)/dt, calculated with Eq. 3 without and with the denominator) is shown in Fig. 6C. Initially, retardation is higher in the presence of Hsp104 but the effect decays rapidly and no longer occurs after ~20 h; in the absence of Hsp104, the initial elongation retardation factor is lower but its decay is slower, only disappearing at ~40 h. This phenomenon may reflect slight changes in the mechanism of elongation and its dependence on Aβ42 monomer concentration (Eq. 3) under conditions where the nuclei capable of elongating the chain are depleted by strong interactions with Hsp104. The effect of Hsp104 on the aggregation kinetics of Aβ42 is predicated on strong interactions of the chaperone with the Aβ42 nuclei P, which are produced in the course of aggregation in nanomolar quantities (typically, less than about 100 nM in Aβ42 by the end of the aggregation process). Tight binding of the nuclei P to Hsp104 ( ~ 14 nM; see Fig. 7A for the contour plot of the fraction of unliganded nuclei Pfree as a function of the concentrations of total nuclei P and Hsp104) takes the bulk of the nuclei out of the aggregation “loop,” as the Hsp104-bound nuclei, Pbound, are not capable of fibril elongation. At concentrations of Hsp104 below ~0.1 µM, fibrillization is progressively slowed down by increasing quantities of Hsp104 (Fig. 6 B, Left), as the fraction of Pfree progressively decreases (Fig. 7A). At Hsp104 concentrations higher than ~1 µM, fibrillization of Aβ42 is effectively abrogated as the concentration of Pfree becomes vanishingly small; at the same time, the process of off-pathway oligomer (O′) formation becomes somewhat faster with higher Hsp104 concentrations (Fig. 6 B, Left Inset). In effect, the action of Hsp104 is only apparently substoichiometric; that is, it may be considered to be such with respect to Aβ42 monomers that are present at micromolar concentrations for most of the aggregation profile, but not with respect to the nuclei P that never exceed ~100 nM in concentration. Fig. 7B shows the progressive changes in concentrations of mature fibrils M, off-pathway oligomers O′, free nuclei Pfree and bound nuclei Pbound during the course of Aβ42 aggregation. Formation of mature fibrils M is progressively slowed down as the concentration of Hsp104 is increased from 0.05 to 1 μM (in monomer units) and virtually completely shut down at higher Hsp104 concentrations (Fig. 7B, Top Left). Note the characteristic shallow maxima achieved for off-pathway oligomers O′ are a signature of a reversible process that competes with Aβ42 fibril formation proper in an Hsp104 concentration-dependent manner (Fig. 7B, Upper Right). The concentrations of Pfree and Pbound species along the aggregation time course follow opposing trends for Hsp104 concentrations up to 1 μM, progressively decreasing and increasing, respectively, with increasing Hsp104 concentrations (the curves for [Hsp104] up to 0.1 μM in Fig. 7B, Lower panels). For [Hsp104] ≥~1 μM, there is practically no Pfree left (Fig. 7B, Lower Left), while off-pathway oligomerization (that produces the species O′) is progressively accelerated at higher Hsp104 concentrations (Fig. 6B) and competes with residual production of nuclei P (which are all bound at these Hsp104 concentrations) further slowing the latter down (Fig. 7B, Lower Right).

Using a variety of biophysical techniques, including NMR, ThT fluorescence assays, negative stain EM and AFM, we demonstrate that Hsp104 inhibits fibril formation at substoichiometric ratios of Hsp104 to Aβ42 monomer. Time courses for Aβ42 aggregation in the presence of varying concentrations of Hsp104 were obtained by monitoring the disappearance of Aβ42 monomer using serially recorded 1H-15N SOFAST-HMQC experiments (Figs. 2, 4, and 6) and the kinetics analyzed quantitatively by global fitting. At high concentration of monomeric Aβ42 (50 μM), aggregation occurs via a branched mechanism (Fig. 4A): an irreversible “on-pathway” comprising primary and secondary nucleation to produce nuclei that subsequently undergo saturating elongation to generate mature fibrils (Fig. 3) which are ThT reactive (Fig. 1); and a reversible off-pathway resulting in the formation of “nonspecific,” nonfibrillar oligomeric species that do not react with ThT (Fig. 1) and therefore lack cross-β structure characteristic of amyloid fibrils. The off-pathway oligomers are too large to be observed directly by NMR (SI Appendix, Fig. S2) and too small to be imaged by negative stain EM and AFM (Fig. 3). It is unknown whether the off-pathway nonfibrillar oligomers detected in the present work have intrinsic pathogenic properties that have been ascribed previously to on-pathway fibrillar oligomers (60). The effect of Hsp104 on the Aβ42 aggregation profiles can be accounted quantitatively by the actions of Hsp104 on both the on- and off-pathways. In the on-pathway, Hsp104 binds tightly but reversibly to the Aβ42 nuclei (on a time scale that is fast relative to that of aggregation; Fig. 6A), thereby preventing elongation of nuclei into mature fibrils. Because the Aβ42 nuclei are sparsely populated and only generated in nanomolar quantities (Fig. 7), the initial concentration of Aβ42 monomers can be considerably higher than that of Hsp104, while still permitting Hsp104 to act in a kinetically “conventional” (i.e., noncatalytic) manner, with no need to invoke a special catalytic mechanism for the inhibition of on-pathway fibrillization by Hsp104. At concentrations of 0.05 and 0.1 μM Hsp104, corresponding to Aβ42:Hsp104 ratios of 1,000:1 and 500:1, respectively, formation of mature fibrils is slowed down (Fig. 6B, Left), but as the concentration of Hsp104 is raised to 1 μM and beyond corresponding to Aβ42:Hsp104 ratios of ≤ 50:1, fibril formation is virtually completely inhibited (Figs. 3, 6B, Right, and Fig. 7B, Top Left). Hsp104 also impacts the rate of formation of off-pathway oligomers, first slowing this process down at concentrations ≤ 0.1 μM, and thereafter speeding it up at concentrations ≥ 1 μM (Figs. 6B and 7B, Top). This work therefore shows that Hsp104 has the inherent ability, in the absence of ATP and/or co-chaperones, to block Aβ42 amyloid fibril formation by tightly binding to sparsely populated Aβ42 nuclei. Since ATP is not required for tight binding of Hsp104 to Aβ42 nuclei we can surmise that active translocation of substrate through the central channel of Hsp104 represents a separate activity that is required for disaggregation (38, 40, 41) but not for inhibition of fibril formation. It is, however, possible that, in the presence of ATP, translocation of Aβ42 nuclei through the central pore of Hsp104 may also occur, prying the nuclei apart to regenerate monomeric Aβ42. Aβ42 nuclei bind approximately five orders of magnitude more tightly to Hsp104 (Fig. 6) than Aβ42 monomers (Fig. 5). Since monomeric Aβ42 is an intrinsically disordered peptide, this observation implies that the Aβ42 nuclei are structurally ordered and present a well-defined surface for binding to Hsp104. Inhibition of fibril formation by tight binding and sequestration of nuclei likely represents a general mechanism employed by other chaperones, such as DNAJB6 (3–5), that also inhibit fibril formation in a so-called substoichiometric manner relative to the monomeric concentration of aggregating polypeptides. Knowledge of the kinetics and mechanism of Hsp104’s capabilities in preventing fibrilization, as shown in this work, will allow a better understanding of the activity of Hsp104 on aggregating clients.

The wild-type Saccharomyces cerevisiae Hsp104 plasmid containing a Tobacco Etch Virus (TEV)-cleavable N-terminal His6-tag was purchased from Addgene (Plasmid #1230) with a pROEX-HTb vector backbone. Expression and purification of Hsp104 was carried out essentially as described in ref. 61 with minor modifications. Hsp104 was expressed in Escherichia coli BL21-CodonPlus (DE3)-RP cells and induced overnight with 1 mM isopropyl β-D-thiogalactoside (IPTG) at 15 °C. Bacterial cells were pelleted, resuspended and lysed using a microfluidizer. After centrifugation at 22,000 rpm for 1 h at 4 °C, the soluble lysate was applied to a Ni-NTA Sepharose High Performance column, followed by a Q-Sepharose High Performance column. The protein was dialyzed overnight with TEV protease at 4 °C under high salt conditions (500 mM KCl) and then reapplied to the Ni-NTA column to remove the cleaved His-tag, TEV protease and any uncleaved His-tagged protein. Finally, the Hsp104 hexamer was isolated by size exclusion chromatography on a Superdex 200 26/60 column in 20 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.4, 140 mM KCl, 10 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT). After, the purified Hsp104 protein was concentrated using an Amicon Ultra centrifugal concentrator (MW 50,000 and volume 20 mL), buffer exchanged in 20 mM HEPES, pH 7.4, 140 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.05% (w/v) sodium azide and 10% (w/v) glycerol, and stored in aliquots at −80 °C. Concentrations of Hsp104 were determined from UV-visible absorbance at 280 nm using an extinction coefficient of ε = 32,500 M−1cm−1 and refer to the monomeric species. The molecular weight of the monomeric units was verified by electrospray mass spectrometry, and the hexameric species was characterized by size exclusion chromatography—multiangle light scattering (SEC-MALS) and analytical ultracentrifugation (AUC) (SI Appendix, Fig. S1).

To characterize the hexameric Hsp104, 250 μg of protein in 125 μL of buffer (20 mM HEPES, pH 7.4, 140 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10 % w/v glycerol) was injected onto an analytical size exclusion column (Superose 6; 10/300; GE Healthcare) at a flow rate of 0.5 mL/min. The molecular mass was determined with in-line MALS (DAWN Heleos-II; Wyatt Technology), refractive index (Optilab T-rEX; Wyatt Technology), and UV (Waters 2487; Waters Corporation) detectors. Molecular masses were calculated from the data using the software Astra, version 6.1.

Samples of Hsp104 in 20 mM HEPES, pH 7.4, 140 mM KCl, 10 mM MgCl2, 0.1 mM EDTA and 1 mM DTT were analyzed by sedimentation velocity at 20.0 °C and 30,000 rpm on a Beckman Optima XL-A or Beckman Coulter ProteomeLab XL-I analytical ultracentrifuge following standard protocols (62). Sedimentation scans were analyzed in SEDFIT 16.1c (63) using a continuous c(s) distribution of sedimenting species. The solution density, solvent viscosity, and partial specific volume for Hsp104 were calculated based on composition using SEDNTERP (64).

Aβ(1–42) (denoted as Aβ42) and 15N-labeled Aβ(1–42) peptides were purchased from rPeptide. The Aβ42 peptides were solubilized with Chelex-treated NaOH and lyophilized as described previously (65). The lyophilized Aβ42 peptides were dissolved in 10 mM NaOH (pH 12.0) and 40 mM sodium phosphate (pH 6.9) in a ratio of 1:3 to reach a final pH of 7.0. All Aβ42 sample preparations were conducted on ice.

Aβ42 aggregation kinetics were monitored by thioflavin T (ThT) fluorescence in the absence and presence of different concentrations of Hsp104. Each sample was analyzed in triplicate with a sample assay volume of 100 µL/well. Each sample had a final Aβ42 concentration of 8 µM and a ThT concentration of 5 µM. The samples were placed in a 96-well microplate (Microplate Greiner) and ThT fluorescence was measured every 5 min using a bottom read-out. Measurements were performed at 37 °C using an Infinite 200 PRO plate reader (Tecan Group Ltd.) with a 440 nm excitation filter and a 482 nm emission filter. The background signal from the buffer with 5 μM ThT was subtracted from all data.

All NMR samples were prepared in 95% (v/v) H2O/ 5% (v/v) D2O. A stock solution of Hsp104 was added in amounts corresponding to final concentrations between 0.05 and 75 µM. The Aβ42 peptide was then added to reach a final concentration of 50 μM in a volume of 500 μL. The final buffer conditions for the NMR samples were 9 mM HEPES, 63 mM KCl, 4.5 mM MgCl2, 45 µM EDTA, 0.45 mM DTT, 4.5% (w/v) glycerol, 1.2 mM NaOH, and 14.6 mM sodium phosphate with a final pH of 7.0.

All NMR experiments were recorded at 20 °C using a Bruker 600 MHz spectrometer, equipped with a TCI triple resonance inverse cryogenic probe equipped with x, y, z gradients. All NMR data were processed with the NMRPipe/NMRDraw software suite (66). Aggregation of 15N-labeled Aβ42 was monitored by serially acquired two dimensional (2D) 1H-15N SOFAST-HMQC experiments using the pulse sequence of Schanda and Brutscher (42, 48). Each spectrum was acquired using eight dummy scans, a recycle delay of 0.2 s, and a total of 100* × 2,048* complex data points in the indirect (15N) and direct (1H) dimensions, with respective acquisition times of 37 and 136 ms. The total measurement time for each 2D 1H-15N SOFAST HMQC spectrum was 20 min. 1H-15N cross-peak intensities (peak height) and peak volumes from each 2D 1H-15N SOFAST HMQC spectrum were plotted to monitor the aggregation kinetics. 15N-R1 and R1ρ experiments were recorded at 5 °C using Bruker 600 and 800 MHz spectrometers, equipped with a cryogenic probe and z-gradients, using previously described pulse sequences (67) on samples containing 50 μM 15N-labeled Aβ42, in the absence and presence of 65 μM hexameric Hsp104 (monomer concentration). For the 15N-R1ρ measurements, the spectra were recorded in an interleaved fashion and variable spin lock periods were applied for 3, 23, 43, 73, 113, 153, and 203 ms at radiofrequency (RF) field strengths of 1.3 and 1.8 kHz at 600 and 800 MHz, respectively. For the 15N-R1 measurements, the relaxation delays used were 40, 120, 240, 360, 480, and 640 ms at both 600 and 800 MHz. A single exponential function was used to fit the R1 and R1ρ decay curves. 15N-R2 values were determined from the relationship R2 = (R1ρ – R1cos2θ)/sin2θ, where θ is the angle between the effective spin-lock field and the z axis of the laboratory frame given by tan−1(ω1/Ω), where ω1 is the spin-lock RF field strength and Ω the offset of a given resonance from the 15N carrier frequency. Lifetime line-broadening 15N-ΔR2 values are given by the difference in 15N-R2 values obtained in the presence and absence of Hsp104.

After completion of the SOFAST-HMQC experiments, aliquots from each sample were taken for AFM and negative stain EM measurements. For EM, 5 μL samples were blotted onto carbon-coated copper grids (3 to 4 nm carbon, Electron Microscopy Sciences) for 1 min, washed with water and then stained with 2% uranyl acetate for 1 min. Images were recorded with a FEI Tecnai T12 electron microscope at 120 kV using a Gatan US1000 CCD camera. For AFM, 10 μL samples were added to freshly cleaved mica, incubated for 2 min, washed with water, and then air dried. A Nanosensors PPP-NCSTAuD probe, oscillating at 160 kHz, was used with a nominal tip radius curvature of 10 nm and a nominal force constant of 7.4 N/m. AFM images were taken at a scan rate of 1.07 Hz, 256 sampling points per line, and 256 lines in the tapping mode using a Veeco MultiMode instrument with a Nanoscope IV controller. AFM images were analyzed using the Gwyddion software (68).

The decay profiles of averaged normalized NMR signal intensities, I, measured at an Aβ42 concentration mtot of 50 µM, were analyzed by minimization of the target function, where Iexp and Icalc are the experimental and calculated normalized intensities, respectively; σ is the SE (assumed to be equal to 1/4 of the SD of the distributions of peak intensities for 21 residues of Aβ42); the index j in the inner sum of Eq. 5 runs over all time-points obtained, and the index i runs over all concentrations of added Hsp104 (0, 0.05, 0.10, 1, 5, 25, 50, and 75 µM). Eqs. 1.1–1.3 and 3 were integrated using an explicit pair of Runge-Kutta formulas of orders 4 and 5 (69), implemented in the Matlab “ode45” solver for nonstiff ordinary differential equations (70) with integration steps corresponding to the time-points used in NMR experiments with minimization carried out using the least-squares trust-region-reflective algorithm. Calculations were also carried out using the program FACSIMILE which employs a modified Gear method with a variable timestep integrator for numerical integration of systems of stiff ordinary differential equations in conjunction with modified Powell minimization (involving optimization of the natural logarithm of the various optimized parameters) to produce rapid convergence of the residual sum of squares for nonlinear systems (71, 72).

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