Triggering protein folding within the GroEL-GroES complex

Abstract

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.

FIGURE 1.

EL43Py is a chemically modified GroEL variant that folds proteins more slowly than wtGroEL. A, the structure of the GroEL-GroES complex (14) is shown in cross-section with amino acid position 43 highlighted in yellow and the last crystallographically resolved amino acid at position 526 highlighted in red. The figure was prepared with PyMol (DeLano, W. L. (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos, CA). B, the structures of the reactive dyes N-(1-pyrene)maleimide (pyrene) and 5-(2-acetoamidotheyl)aminonaphthalene 1-sulfonate (AEDANS) are shown conjugated to the thiol side chain of Cys. C, the refolding of Rubisco by wild-type GroEL (wtGroEL), a single Cys mutant of GroEL (EL43C), pyrene-labeled EL43C (EL43Py), a second pyrene-labeled GroEL mutant (EL529Py), and an AEDANS-labeled GroEL mutant (EL43Ed) are shown. D, the refolding of rhodanese by the same GroEL variants is shown. Rubisco and rhodanese were denatured in acid-urea and mixed (100 nm) with the indicated GroEL variant (500 nm), GroES (1 μm), and ATP (2 mm). Error bars represent the standard deviation of n = 3 experimental replicates. Unless otherwise stated, all concentrations listed are post-mixing.

FIGURE 2.

The GroEL ATPase cycle in the presence of GroES and absence of substrate protein is shown. The hydrolytic reaction is divided into two phases, where the first phase (Phase I) illustrates the pre-steady-state behavior of the system when apo-GroEL is first mixed with ATP and GroES. An apo-GroEL ring first fills cooperatively with ATP, followed by rapid binding of GroES (2, 3). The resulting asymmetric GroEL-ATP-GroES complex (an “ATP bullet”) hydrolyzes the bound ATP with an intrinsic turnover rate of 0.12 s^-1, yielding an asymmetric GroEL-ADP-GroES complex (an “ADP bullet” (38)). When ATP is limiting, the reaction stops at the ADP bullet following a single hydrolytic turnover. In the presence of excess ATP, the binding of ATP to the open trans ring of the ADP bullet initiates the disassembly of the cis complex, leading to release of GroES and ADP (5, 9, 27). Nearly simultaneous binding of another GroES heptamer (indicated by the brackets) results in the formation of a new cis complex, regenerating the ADP bullet upon hydrolysis of the ATP within the cis complex (5, 9, 27). The steady-state cycling between ATP and ADP bullets constitutes the second phase of the reaction (Phase II), and disassembly of the ADP-bound cis complex is the rate-limiting step of this cycle in the absence of substrate protein (27, 38). Under in vivo conditions, the GroEL-GroES system persists almost exclusively in the steady-state cycle (Phase II).

FIGURE 3.

EL43Py completes a hydrolytic cycle more quickly than wtGroEL. A, the steady-state hydrolysis of ATP by wtGroEL, EL43Py, EL43C, EL529Py, and EL43Ed are shown in the presence of excess ATP. In each case, 250 nm GroEL tetradecamer was mixed with 500 nm GroES and 0.5 mm ATP, and the rate of ATP hydrolysis was monitored. B, in limiting ATP, the single-turnover kinetics of ATP hydrolysis by wtGroEL and EL43Py are identical. The rate of ATP hydrolysis from a single ring of wtGroEL and EL43Py was examined by rapidly mixing 3 μm chaperonin and 12 μm GroES with a limiting amount of radiolabeled ATP (21 μm) in a quench-flow apparatus at 25 °C. The single round hydrolysis data were fit to a single-exponential rate law with k_cat = 0.110 ± 0.006 s^-1 for wtGroEL and k_cat = 0.128 ± 0.008 s^-1 for EL43Py. Error bars represent the standard deviation of n = 3 experimental replicates. C, the pre-steady-state kinetics of ATP hydrolysis by EL43Py in the presence of GroES show a reduced burst amplitude. The ATPase activity of wtGroEL and EL43Py (3 μm) in the presence of GroES (6 μm) was examined by rapidly mixing each chaperonin with excess radiolabeled ATP (1 mm) in a quench-flow apparatus at 25 °C. Error bars represent the standard deviation of n = 5 experimental replicates. The pre-steady-state bursts observed with wtGroEL and EL43Py, obtained by extrapolation of the linear reaction phase to zero time, are illustrated with dashed lines. D, the release of fluorescently labeled GroES (ES98Fl, 200 nm) from wtGroEL and EL43Py (220 nm) during steady-state ATP turnover was monitored by gel filtration. In both cases, the dissociation of GroES was observed to follow a single-exponential decay with rate constants of k_obs = 0.030 ± 0.003 s^-1 for wtGroEL and k_obs = 0.079 ± 0.004 s^-1 for EL43Py. Error bars represent the standard deviation of n = 3 experimental replicates.

FIGURE 4.

EL43Py correctly binds and encapsulates non-native substrate protein beneath GroES. A, binding of a non-native substrate protein to the trans ring of an EL43Py ADP bullet complex is comparable to binding to the trans ring of a wtGroEL ADP bullet complex. Each bullet complex (120 nm) was mixed with denatured, fluorescently labeled Rubisco (100 nm) for 5 min at 25 °C, and the samples were then loaded onto a gel-filtration column. The elution profile of each sample is shown. The elution position of the fluorescent, non-native Rubisco bound to the trans rings of each bullet complex is indicated. The total integrated area of each peak in arbitrary units (a), reflecting the amount of non-native Rubisco bound and retained by each complex over the course of the column run are indicated in parenthesis. B, the conformational state of the bound Rubisco monomer was examined by time-resolved, donor-side FRET using a previously described fluorescently labeled Rubisco variant (29, 31). In each case, fluorescently labeled Rubisco samples were denatured in acid-urea and mixed (100 nm) with either EL43Py or wtGroEL ADP bullets (120 nm) to allow trans ring binding (shown schematically in the inset). The donor intensity decay curves for non-native Rubisco carrying either donor-only (fluorescein; D) or donor and acceptor probes (fluorescein/rhodamine; D + A) are shown. The essentially identical decay curves demonstrate that the observed FRET efficiency of the labeled Rubisco monomer and, therefore, the average conformation of the non-native protein, on the two trans rings is the same. C, GroES encapsulates non-native Rubisco on an EL43Py ring. Denatured, fluorescein-labeled Rubisco (Rub454Fl, 100 nm) was bound to the trans ring of both GroEL and EL43Py ADP bullets (100 nm), formed in the presence of excess GroES. The protease sensitivity of the non-native Rubisco was examined either following (cis-3°) or without (trans) the subsequent addition of ATP, permitting GroES binding. To prevent cycling and allow a single round of GroES binding, added ATP was quenched after 5 s with hexokinase and glucose. Samples were supplemented with proteinase K for varying times and then subjected to SDS-PAGE. The location of the non-native Rubisco protein in the cis and trans complexes is shown schematically above the gel. The quantity of undigested Rubisco was examined using a laser-excited fluorescence gel scanner. The amount of intact Rubisco was quantified and plotted as a function of time in D. Error bars represent standard deviation of n = 4 experimental replicates.

FIGURE 5.

EL43Py fails to release non-native Rubisco upon GroES and ATP binding. A, the addition of excess wtGroEL to an EL43Py folding reaction does not rescue Rubisco folding. Denatured Rubisco (100 nm) was mixed with EL43Py (250 nm), and the sample was then supplemented with a 5-fold excess of wtGroEL (1.25 μm). Folding was initiated by the addition of ATP (2 mm) and excess GroES (3 μm, EL43Py rescue). The refolding curves of Rubisco (100 nm) in the presence of either wtGroEL or EL43Py (250 nm in both cases, with 500 nm GroES) are shown for reference. B, schematic of a FRET experiment designed to examine conformational changes in Rubisco following a single round of GroES binding to the trans ring of an ADP bullet. Fluorescently labeled, non-native Rubisco (29, 31) was first bound to the trans ring of an ADP bullet in the presence of excess GroES. Subsequent addition of ATP permits GroES binding and Rubisco encapsulation. To prevent cycling and permit long time observation of the complex, the excess ATP was quenched with hexokinase and glucose (Hex/Glc) ∼ 4 s after the addition of ATP. C, the change in FRET efficiency was monitored as a function of time following GroES binding to the Rubisco-occupied trans ring of wtGroEL and EL43Py ADP bullets. Rubisco-bound ADP bullet complexes in the presence of excess GroES were manually mixed (60 nm) with ATP (1 mm) in a standard fluorometer cuvette with magnetic stirring. Excess ATP was then quenched with hexokinase and glucose to prevent cycling. This mixing sequence precludes observation within the first ∼8 s of the experiment.

FIGURE 6.

EL43Py only slowly completes the ATP-driven transition that causes forced substrate unfolding. A, schematic of a stopped-flow FRET experiment designed to examine conformational changes in non-native Rubisco upon encapsulation beneath GroES on the trans ring of an ADP bullet (31). B, ATP and GroES binding results in the forced unfolding and compaction of the Rubisco monomer. Rubisco-bound ADP bullet complexes in the presence of excess GroES were rapidly mixed in a stopped flow (60 nm) with ATP (1 mm). Each FRET trace is the average of n = 10 replicates of matched experimental pairs, calculated from donor-only (Rubisco 454-D) and donor-acceptor (Rubisco 454-D/58-A) samples. The inset shows the FRET change over the first second of data. The wtGroEL data shown are from Ref. and are re-plotted here for reference. C, schematic of a stopped-flow FRET experiment designed to follow conformational changes in a substrate protein upon the binding of ATP alone to the trans ring (31). For this experiment, an excess of the single ring GroEL variant SR1 is used as a GroES trap (8). D, the allosteric transition triggered by ATP binding that causes forced unfolding proceeds slowly on an EL43Py trans ring. Stopped-flow FRET experiments were conducted in essentially the same manner as in B, except that SR1 was present in a 20-fold excess (1.2 μm). The inset shows the FRET change over the first second of data. The decrease in FRET efficiency, reflecting an increase in the intra-probe distance, was fit to a double exponential rate law for EL43Py with rate constants of k_fast = 2.9 ± 0.4 s^-1 and k_slow = 0.05 ± 0.02 s^-1 for EL43Py. The wtGroEL data shown are from Ref. and are re-plotted here for reference.

FIGURE 7.

ATP hydrolysis by a newly formed GroEL-GroES complex on a trans ring is delayed by the slow release of ADP from the opposite ring. The steps involved in the pre-steady-state hydrolysis of ATP by apo-GroEL (A) and the trans ring of an ADP bullet (B) are illustrated. In both cases, the pre-steady-state steps are followed by the same steady-state cycle. C, the pre-steady-state kinetics of ATP hydrolysis by apo-GroEL and the GroEL ADP bullet trans ring are shown. In both cases, GroEL samples (1 μm) were mixed with 1 μm acidurea-denatured Rubisco and incubated for 10 min at room temperature prior to the addition of radiolabeled ATP (250 μm). GroES was present at a total concentration of 3.5 μm for both reactions. Error bars show the standard deviation of n = 3 experimental replicates. The rates of the linear, steady-state phase of the hydrolysis reactions are very similar (∼0.02 s^-1 per active subunit). However, in comparison to the apo-GroEL sample, the pre-steady-state burst for the ADP bullet sample is dramatically reduced and only just detectable. D, the rate of GroES release from an ADP bullet cis complex was monitored with a previously described FRET assay employing fluorescently labeled GroEL and GroES (27). Non-native Rubisco was first bound to the trans ring of ADP bullets formed from donor-labeled GroEL and acceptor-labeled GroES. The Rubisco-bound ADP bullets (50 nm) were then mixed with a 20-fold excess of unlabeled GroES (4 μm) in a stopped-flow in the absence (+dRub and -ATP) or presence (+dRub and +ATP) of 500 μm ATP. Dissociation of the labeled GroES was monitored as a loss in FRET efficiency. The FRET traces shown represent the average of n = 14 experimental replicates of matched donor-only and donor-acceptor experiments. The rate of ADP release from the cis complex was monitored using radiolabeled ADP ([α-³²P]ADP). The trans ring of wtGroEL ADP bullets containing ³²P-labeled ADP in the cis cavity was saturated with non-native Rubisco and then briefly supplemented with either buffer (+dRub and -ATP) or 1 mm ATP (+dRub and +ATP). The sample was then applied to a rapid spin column that separated free from bound ADP in 4 s. Scintillation counting was employed to monitor the amount of GroEL-bound, radiolabeled ADP at each time point. Error bars show the standard deviation of n = 3 experimental replicates.

FIGURE 8.

The stages of the protein folding trigger of GroEL. A simplified reaction cycle is shown (left), illustrating the sequential progression of allosteric states (R₁, R₂, and R₃) of the ATP-saturated GroEL cis ring required to trigger protein folding. Initial binding of ATP leads to population of the R₁ state. The R₁ state was not directly observed in the current work but has been previously described (25). The transition from the R₁ to the R₂ state, involving the directed movement of the GroEL apical domains, is responsible for forced substrate unfolding (1). The movement of the apical domains also permits GroES binding and the subsequent compaction of the non-native protein (2). The R₂ state simultaneously binds the substrate protein and GroES (2 and 3). Following GroES binding to the R₂ state, the GroEL-GroES complex undergoes an additional transition to the R₃ state, which releases the substrate protein into the stable GroEL-GroES cavity and initiates folding. ATP hydrolysis in the cis ring is delayed by an event on the opposite GroEL ring, most likely ADP release, allowing GroES binding to reach completion. The stages of substrate, ATP, and GroES binding for EL43Py are similar to wtGroEL. However, the EL43Py ring only rarely populates the R₃ state, usually stalling in R₂ (3, left arrow). Because the bound ATP is committed to hydrolysis, and these EL43Py rings usually fail to populate the folding-competent allosteric state of the GroEL ring (R₃), the slowest step of the GroEL steady-state ATPase cycle (GroES and ADP release from the folding competent GroEL-GroES cis complex) is usually bypassed by EL43Py, resulting in faster steady-state ATP hydrolysis and much less efficient folding.