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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2011 Jan 25;108(6):2234–2239. doi: 10.1073/pnas.1014278108

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

Andrea Kravats 1, Manori Jayasinghe 1,1, George Stan 1,2
PMCID: PMC3038749  PMID: 21266546

Abstract

Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarse-grained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.

Keywords: AAA+ protease, molecular machine, protein translocation

Intracellular regulatory mechanisms for selective destruction of proteins and disassembly of protein aggregates are critical for the maintenance of vital cellular functions (1). Clp macromolecular machines, found in all domains of life from prokaryotes to multicellular eukaryotes (2), perform such protein quality control using powerful ATPase components (3–6) that effect protein unfolding and translocation through narrow pores. Clp (caseinolytic protease) ATPases, are members of the AAA+ (ATPases associated with diverse cellular activities) superfamily of proteins (7, 8) responsible for a variety of functions including intracellular transport, DNA replication and repair, and transcription regulation (9–11). The best understood Clp ATPases are ClpA; ClpX and ClpY (HslU) (12), which enable protein degradation by associating with peptidase subunits ClpP and ClpQ (HslV); and ClpB, which promotes disaggregation (13–15).

Functional forms of Clp ATPases are homohexameric assemblies that form in the presence of ATP (16). Monomers include one (ClpX, ClpY) or two (ClpA, ClpB) highly conserved nucleotide binding domains (17), referred to as AAA domains (7, 18). Crystal structures (19–21) and oligomeric models (22, 23) based on electron microscopy images (24, 25) reveal the presence of a central channel with a diameter of approximately 9–15 Å at the narrowest point. The channel is occupied by diaphragm-forming loops (one per AAA domain), which contain a conserved motif consisting of an aromatic-hydrophobic dipeptide flanked by glycine amino acids. These loops are implicated in the tightly regulated substrate selection mechanism that involves recognition of specific peptide tags, such as the Escherichia coli SsrA (sequence AANDENYALAA), fused to targeted proteins (26). Due to the narrow openings within the ATPase channel, SPs must be unfolded, a process that requires ATP hydrolysis for most proteins. Mechanical forces, that underlie the unfolding and translocation processes, arise from nucleotide-dependent displacements of channel loops coupled to strong loop-SP interactions (22, 27). The loop movements are suggested to be asymmetric around the ATPase ring in view of uneven ATP binding affinity in each monomer (28, 29).

Currently, the coupling between the allostery-driven motion of the loops and the unfolding and translocation of a fully flexible substrate is not completely understood. Recent computational studies (30), that describe conformational transitions of ClpY using a coarse-grained approach, support a proposed paddling action of central channel loops (27). These actions enable the unidirectional displacement of ClpY along an extended polypeptide chain restrained near the pore axis. Other computational studies (31–33), which examine mechanical pulling of SPs through a nonallosteric pore, have identified distinct unfolding mechanisms from those found when the protein ends are pulled apart, as in atomic force microscopy (AFM) studies. An alternative approach (34), focused on bulk unfolding by a constant pulling force modulated by a periodic force, determines shifts in AFM unfolding pathways. Overall, so far it has been a major challenge to perform computer simulations that capture the complex interplay of allostery, mechanical pulling, and translocation through nanopores that underlies the Clp ATPase function.

Here, we use a coarse-grained model to simultaneously probe these mechanisms over their associated long times cales. We identify the dynamic sequence of unfolding and translocation events of a model four-helix bundle protein (HBP) mediated by the ClpY ATPase (Fig. 1). An obligatory unfolding intermediate, which involves unraveling the C-terminal region, is minimally required to initiate substrate translocation. Further unfolding leads to low native-content SP conformations that are stabilized by the I domain of ClpY, which is located at the pore entrance, and are competent for translocation. Forces exerted by the loops onto the SP are found to be weaker relative to those required for SP unfolding in AFM experiments or for SP threading through a nonallosteric pore.

Fig. 1.

Fig. 1.

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Unfolding and translocation of the substrate protein mediated by the cyclical action of ATP-driven ClpY. (A) The substrate (HBP, purple; SsrA, yellow) approaches ClpY (green) from the proximal region. System size and orientation are as indicated. For clarity, two ClpY subunits are not shown. (B) Upon binding of the C terminus of HBP to the ClpY central channel loops (blue), the SP unfolds through an obligatory three-helix-bundle intermediate. (C) The SP is partially translocated by ClpY, in the absence of cofactors, through repetitive allosteric motions of ClpY channel loops. (D) The SP is completely translocated and partially refolds in the distal region (z > zloops) of ClpY through the combined action of ClpY and the ClpQ peptidase cofactor (not shown). Our simulations represent the SP–ClpQ interaction as harmonic restraints applied to the SP in the distal ClpY region. Protein images were created using VMD (56) and POV-Ray (www.povray.org).

Results and Discussion

AFM-Like Pulling of HBP Results in Multiple Unfolding Pathways.

Forced unfolding simulations of the bulk SP are performed by pulling the C terminus at constant velocity, while keeping the N terminus fixed (see Methods and Table S1). We identify a major pathway (75% of simulations), in which the N-terminal helix unravels first, followed by the C-terminal helix and the contacts between the intermediate helices (Fig. S1 and Movie S1), and a minor pathway (25% of simulations), initiated by C-terminal unfolding. The force-extension curves obtained from our simulations indicate peak unfolding forces of 50–100 pN (Fig. S1), which are on the order of forces of 25–35 pN observed in AFM studies of the triple helix bundle protein spectrin (35). Our coarse-grained approach yields similar unfolding events to those obtained using implicit solvent simulations of the forced unfolding of spectrin and a four-helix bundle protein, the acyl-coenzyme A-binding domain (36). Although the latter simulations are performed at higher pulling speeds than in our approach and pulling forces are about an order of magnitude higher than in experiments, the major pathways of SP unfolding involve the same sequence of helix unraveling. Overall, comparison with AFM experiments and implicit solvent simulations show that our coarse-grained approach reproduces the range of experimental forces and the microscopic details of unfolding events obtained in atomistic simulations of proteins.

Unfolding of SP Occurs Upon Binding of its C Terminus to ATP-driven ClpY Channel Loops.

The initial step (Fig. 1A) in our ClpY-mediated unfolding simulations consists of presenting HBP to the nonallosteric open-state ClpY ring (see Methods and SI Text). In accord with experimental studies, which indicate that ATP hydrolysis is required for the proteolysis of substrates (6), we find that SsrA tag binding to ClpY central channel loops does not result in HBP translocation in the nonallosteric pore case. In our simulations, HBP retains its native structure upon tag binding to ClpY (Fig. 2).

Fig. 2.

Fig. 2.

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Unfoldase action of ClpY. The probability distribution P(QN) of the fractional number of native contacts QN of HBP is shown following substrate protein binding to the ClpY loops (blue) and after 50 cycles of ATP-driven, unassisted, ClpY action (black). The red line indicates P(QN) after 50 ClpY cycles during which HBP amino acids found in the distal ClpY region experience harmonic restraints due to the ClpQ peptidase component. P(QN) of the bulk HBP native contacts is represented by the green line.

To elucidate the factors involved in SP unfolding, we analyze the structural changes in HBP effected by the ATP-dependent conformational transitions of ClpY. In our simulations, sequential allosteric motions of ClpY, that result in cycling between open and closed pore states, are mimicked by conformational transitions of pairs of adjacent subunits (see Methods). We find that, during ATP-driven cycles, ClpY channel loops impart periodic mechanical forces on the bound SP. As a result, the folded part (HBP) of the HBP-SsrA fusion protein is brought in contact with the ClpY channel loops. The initial unfolding event, which consists of unraveling near the C terminus of HBP, takes place upon transient binding of the C-terminal amino acids to the ClpY channel loops. We quantify the strength of the interaction between the HBP C-terminal region and the ClpY channel loops through the interactions that involve two hydrophobic HBP amino acids, Leu70 and Leu71. As the attractive interaction between HBP amino acids Leu70 and Leu71 and ClpY channel loops strengthens from ≃-2 kcal/mol to ≃-8 kcal/mol, the fraction of HBP native contacts, QN, decreases rapidly from a native-like value of 0.85 to ≃0.45 (Fig. S2A). We conclude that binding of the HBP C terminus to the ClpY loops is nearly simultaneous with HBP unfolding (Fig. S2B and Movie S2). These events occur on a fast time scale for HBP owing to the weak mechanical resistance of its all-α fold. By examining the first passage time over a large number of short simulations (see SI Text and Table S1), we find that the characteristic time for unfolding is τu ≃ 0.68τ, where τ is the duration of one ClpY cycle. This mechanism of SP unfolding upon binding of the C-terminal region to ClpY channel loops, found in our simulations, is in accord with experimental studies (37), which indicate that protease-mediated unfolding depends on the local structural stability of the SP near the degradation tag.

SP Translocation Occurs Through Stepped Transitions Mediated by Repetitive Nucleotide-Dependent Motions of ClpY Channel Loops.

We distinguish two stages of ClpY translocase action. First, ClpY effects partial translocation of the unfolded chain without assistance from cofactors. Next, the ClpY action is complemented by the interaction of the translocated SP chain with the ClpQ peptidase, which is suggested to act through spring-like restraints for the polypeptide chain in the distal region (28). In our simulations (Table S1), these actions are described by strong hydrophobic–hydrophobic interactions (λ = 2.0, see Methods) between amino acids on the distal surface of ClpY and HBP–SsrA and, in the second stage, by harmonic restraints applied onto HBP–SsrA amino acids that are located within the distal region of ClpY (see Methods). During both stages, the power stroke of the ATPase cycle enables motions of ClpY channel loops that effect mechanical pulling of HBP. As a result, in the first stage, partial HBP translocation (shown as a transition between Rg ≃ 27–35 Å, after t ∼ 2τ, in Fig. 3A) takes place on a different time scale than unfolding (Rg ≃ 15–27 Å transition in Fig. 3A), with the characteristic time τt,1 ≃ 24.6τ. This unassisted action of ClpY (Movie S3) results in an average translocated chain length 〈L〉 ≃ 37.2 Å. A large dispersion of the translocated chain length results is observed, with Lmax ≃ 99 Å, which exceeds the propagation of the entire C-terminal helix (Lhelix = 60.8 Å) and its adjacent loop (Lloop = 11.4 Å). Our simulations of 50–100τ (Table S1) show that, in the unassisted stage, the translocated chain length is independent of the translocation time scale.

Fig. 3.

Fig. 3.

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Substrate unfolding and translocation mediated by ATP-driven ClpY. (A) Radius of gyration of HBP (purple) as a function of time during initial unfolding and translocation mediated by ATP-driven ClpY (green). (B) Translocase action of ClpY. The length of the HBP chain translocated within the ClpY pore as a function of time during a single simulation trajectory. After 50 ClpY cycles, planar harmonic restraints are applied onto HBP amino acids in the distal region to mimic the action of the ClpQ peptidase.

The second stage of the ClpY action is initiated from configurations in which HBP is partially translocated through the ClpY pore (Table S1). The combined actions of allostery-mediated pulling by ClpY loops and the contribution of ClpQ facilitate the completion of the polypeptide translocation process within a characteristic time τt,2 ≃ 20.5τ. We find that SP translocation (Movie S4), which is monitored through the time evolution of the length of the HBP chain propagated in the distal region of ClpY, occurs through sharp stepped transitions (Fig. 3B). Abrupt jumps associated with these transitions indicate a high degree of cooperativity of the translocation events involving, at each step, partial or complete propagation of one helix and adjacent connecting loop(s) within HBP.

To examine the requirement of ATP-driven ClpY motions for the translocase activity during the second stage, we also performed a series of simulations of a nonallosteric ClpY pore with distal region restraints applied onto the SP (Table S1). Each of these simulations is initiated from a conformation of partially translocated HBP, obtained after the first stage action of ClpY. None of the simulations of the nonallosteric pore result in further HBP translocation within the time scale t = 30τ > τt,2 (Table S1), showing that nucleotide-dependent ClpY motions are required for translocation. Overall, our model supports a translocation mechanism by repetitive mechanical pulling, due to the SP interactions with the ATP-driven ClpY loops and SP unfolding prior to translocation.

The Unfolding Pathway Due to ATP-Driven Action of ClpY Involves an Obligatory Nonnative Intermediate of the HBP.

The interplay of unfolding and translocation, resulting from allostery-driven pulling by ClpY loops, leads to complex SP remodeling. As shown by the P(QN) distribution (Fig. 2), after 50 cycles of the first stage of the ClpY action the nonnative conformations of HBP partition into two clusters. One of these clusters comprises HBP conformations with QN ≃ 0.5–0.6, which contain an unfolded C-terminal region and a nearly native three-helix bundle formed by the remaining helices. The second cluster involves HBP states with low native content, QN ≃ 0.2–0.3. This partitioning is closely associated with the translocase action of ClpY, as significant unfolding of the substrate takes place during translocation. Consequently, the P(QN) of partially translocated SPs is dominated by the population of low native-content conformations (Fig. S3A). By contrast, in the absence of translocation, substrates are somewhat more likely to be found in a three-helix bundle than in a low native-content conformation (Fig. S3A). Importantly, initial SP translocation does not require extensive unfolding. As shown in Fig. S3B, immediately prior (Δt ≲ 0.001τ) to translocation, the fraction of substrate conformations with low native content (QN < 0.45) is approximately equal to that of the three-helix bundle state (0.45 < QN < 0.65). This behavior implies that the minimal unfolding requirement for partial translocation is the destabilization of the SP near the degradation tag.

During the second stage of ClpY action, continued HBP translocation results in accumulation and partial refolding of the protein chain within the distal region of ClpY. As a result, translocated HBP chains acquire a larger native content than the partially translocated chains resulting from the first stage action (Fig. S3A). The P(QN) distribution for the translocated HBP structures is broad, with values ranging between ≃0.2–0.6 (Fig. 2). These structures, which include a helical hairpin, are found among the nonnative structures of HBP obtained as a result of thermal denaturation (38).

Reaction pathways and maps of probability density of QN and Rg, shown in Fig. 4A–C, indicate the SP conformations that are accessed kinetically due to the ClpY action. The initial unfolding near the C terminus of HBP results in a three-helix bundle (QN ≃ 0.6) unfolding intermediate, termed U1. The N → U1 reaction is an obligatory step for unfolding, independent of the ultimate success of SP translocation (Fig. 4A and C). As shown in Fig. 4A, following the initial unfolding, translocation may take place either from the three-helix bundle conformation (the U1 state) or from a low native-content conformation (U2), leading to an extended conformation (U3) with large Rg (≃35 Å) and low native content QN (≃0.25). During the second stage of ClpY action, completion of translocation yields a state (U4) characterized by conformations with Rg > 40 Å (inset of Fig. 4A).

Fig. 4.

Fig. 4.

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Unfolding and translocation reactions. (A) Probability distribution map of the fraction of native contacts QN and the radius of gyration Rg of the SP for the simulation trajectories that result in translocation without assistance from cofactors. (Inset) Map for complete translocation assisted by the ClpQ cofactor and Partial refolding in the distal ClpY region. (B) Reaction pathways of the unfolding and translocation reaction. An obligatory intermediate (U1) is identified for both unfolding and translocation. Partial translocation (U2) may also proceed from a low native-content conformation (U2) and complete translocation, with assistance from the ClpQ cofactor, results in extended conformations (U4). (C and D) Same as in A for simulations of (C) ClpY that do not result in translocation and (D) ClpYΔI.

Moderate Transient Interaction of the SP with the I Domain of ClpY Provides Unfolding and Translocation Assistance.

The extensive hydrophobic regions exposed by the I domain (residues 110–243) of ClpY are favorable for binding nonnative SP conformations. In most transient SP–ClpY complexes, this interaction contributes E ≃ -30 kcal/mol to the overall stabilization of the unfolded HBP conformation (Fig. S4 A and B). In simulations that do not result in translocation, the stability and strength of the SP–I domain interaction is greater, as illustrated by the higher density of states around E ≃ 30 kcal/mol and Rg ≃ 22 Å, and for E < -40 kcal/mol, respectively (Fig. S4 A and B). Successful translocation is characterized by multiple binding and unbinding events of the SP to the I domain, resulting in a high density of states around E ≃ 0 kcal/mol (Fig. S4A). Thus, a moderate interaction of the SP with the I domain stabilizes the nonnative substrate conformation and assists translocation for conformers that do not establish stable contacts with the I domain.

Deletion of the I domain results in nearly complete loss of the translocase function of ClpY with only 5% of ClpYΔI simulations resulting in SP translocation (Table S1). By contrast, the unfoldase function is preserved in the mutant system (Fig. 4D). Nevertheless, SP unfolding by ClpYΔI is mostly limited to unraveling the C-terminal region and the resulting SP conformations maintain significant native content particularly in the form of the three-helix bundle intermediate. Unfolding the substrate beyond the C-terminal region has a low probability due to a lack of favorable interactions between the nonnative conformation and the proximal surface of ClpYΔI. Overall, we surmise that the I domain is required to stabilize unfolded SP conformations to assist periodic mechanical pulling by the ClpY loops. These results are in accord with experimental studies (39–41), which suggest that the N domains of ClpA and ClpX stabilize unfolded conformations of substrate proteins and facilitate effective translocation.

Weak Mechanical Forces Exerted by the ClpY Central Loops onto the SP Effect Unfolding and Translocation.

Unfolding of the SP mediated by the allostery-driven conformational changes requires relatively small forces to be applied by the ClpY central loops (Fig. 5). We find that HBP is unfolded by maximal forces of 74 ± 9 pN exerted by the loops. The reduced force requirement for the allosteric pore compared to the AFM-type pulling is rationalized by the contribution of multiple interaction centers on the proximal surface of ClpY to destabilize the SP. Furthermore, the loop–HBP interaction need not be continuous (Fig. 5). After the initial unfolding event, further unraveling of the SP may be mediated exclusively by the I domain. Once the SsrA tag is propagated through the ClpY pore, HBP reenters the pore and experiences loop forces which promote productive translocation.

Fig. 5.

Fig. 5.

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Forces exerted by the ClpY channel loops onto the SP. The force in the direction parallel to the pore axis is shown as a function of time during a single trajectory that results in translocation (black). Also shown is the time evolution of the fraction of native contacts of the SP(red).

Mechanical Pulling of Substrate Protein Through Nonallosteric Pore Leads to Simultaneous Unfolding and Translocation.

In contrast to ATP-driven unfolding, pulling the HBP chain at constant force through the nonallosteric ClpY pore results in simultaneous unfolding and translocation (Movie S5) with little dispersion observed along the unfolding-translocation pathway (Fig. S5). We find that a minimum force of F = 105 pN is required to completely unfold and translocate HBP. This force threshold is consistent with the critical forces found in the AFM-type unfolding simulations (Fig. S1). However, in the nonallosteric pore case, unfolding is preceded by a slight enhancement of the native HBP structure, seen as a decrease in Rg (Fig. S6A) and an increase in QN (Fig. S6B). This effect is due to the combination between the pulling force and confinement near the proximal side of ClpY. Next, HBP unravels on a very fast time scale (∼0.065τ) (Fig. S6B) following a sequential pathway initiated from the C terminus. The unfolding is not strictly monotonic, as transient refolding involving helices 2 and 3 takes place. Overall, SP threading through a nonallosteric pore presents a distinct unfolding mechanism from both the ClpY ATPase and AFM-type experiments. We propose that the allosteric pore mechanism has been selected for its ability to effect unfolding and translocation without assistance from cofactors or environmental variations (for example, an electrostatic gradient).

SsrA Tag Mutations Impair Substrate Protein Recognition and Remodeling Actions.

Mutagenesis experiments (42, 43) show that substitutions A10D and A11D within the SsrA peptide inhibit degradation of the GFP-SsrA by ClpX. Mutations at positions 1–8 of the tag show reduced substrate degradation compared with the wild-type SsrA (43). To obtain insight into the mechanistic implications of these sequence variations of SsrA, we performed simulations of the fusion protein with three tag variants (Table S1). Within the coarse-grained level representation adopted here, we find that the A10A11–DD variant results in 50% lower binding probability and reduced translocation yield compared to the wild type (Table S1). In accord with experimental studies (43), the glycine-rich variant G8LAA maintains the wild-type binding affinity, but it is not unfolded efficiently (Table S1). The third SsrA variant, D2AD5AD2, is unable to bind to ClpY. Allosteric-driven remodeling of fusion proteins that involve SsrA variants results in nonnative SP states similar to those of the wild-type HBP–SsrA (Fig. S3C). After 10 ClpY cycles, the A10A11–DD variant results in SP unfolding to the three-helix bundle intermediate (QN ≃ 0.5–0.6) and HBP conformations with low native content (QN ≃ 0.2–0.4). The G8LAA variant results in limited unfolding to the three-helix bundle intermediate.

Concluding Remarks

Our coarse-grained simulations reveal the unfolding and translocation mechanisms of a four-helix bundle substrate protein as a result of cyclical ClpY action. The crucial unfolding event, required for SP translocation, is the unraveling of the SP near the tagged C-terminal region. While the resulting obligatory three-helix bundle intermediate is competent for translocation, extensive unfolding may also take place at the pore entrance. The unfolded conformations must be stabilized through interactions with the I domain. If these interactions are transient and fall within a moderate range, the nonnative SP is translocated across the pore.

It is interesting to compare unfolding actions of nanomachines that are involved in distinct quality control mechanisms—namely, protein degradation (Clp ATPases) and protein folding (the GroEL chaperonin). GroEL binds misfolded conformations at the hydrophobic sites and encapsulates them in a predominantly hydrophilic cavity (44). Distinct substrate remodeling actions result from allosteric-driven cycles of GroEL and Clp ATPases. GroEL effects iterative annealing of the substrate by alternating random disruptions of misfolded SP conformations with substrate confinement (45, 46). Clp ATPases act indiscriminately on both native and nonnative contacts, because their ultimate function is to destroy the SP tertiary structure.

An important functional aspect of the ClpY ATPase mechanism concerns the efficacy of the allosteric mechanism for protein remodeling and translocation. Experimental studies point toward nonconcerted allostery, but the precise ordering of subunit motions is not clear (47). Computer simulations of chaperonins mediated protein folding (48) also suggest that sequential and concerted motions are optimized for different substrate types. In accord with experiments, concerted simulations using our coarse-grained model (Table S1) do not result in SP translocation and unfolding of the substrate is limited to the three-helix bundle conformation (Fig. S3D). Nevertheless, our current model, which describes sequential allostery through motion of pairs of subunits, does not provide the resolution to discriminate ordered and random motions. A more elaborate model will be developed to address the details of the allosteric mechanism.

Methods

Coarse-Grained Model of the ClpY ATPase-SP Interaction.

Large time scales and length scales associated with Clp actions are prohibitively large for solvated, all-atom, simulations. To effectively address these challenges we use a residue-level model that incorporates the fundamental factors that are expected to contribute to SP unfolding and translocation. These are periodic conformational changes in Clp subunits that result in cycling between “open” and “closed” pore states and favorable interactions between central loops and SP. We use a “united atom” description of the protein chain that represents amino acids, classified as hydrophobic (B), hydrophilic (L), or neutral (N) (38), as spherical particles located at Cα atom positions (49). The CHARMM program (50) is used to perform Langevin dynamics simulations at T = 300 K of the ClpY-mediated unfolding of HBP-SsrA (Table S1) using a friction coefficient of 1 ps-1 and a time step of 25 ps. Minimalist model studies of mechanical pulling of a bulk GFP monomer (51) are found to be in good agreement with experiments (52).

Protein Model and Protein–Protein Interactions.

We choose the designed four-helix bundle protein (HBP) (53) for probing ClpY-mediated translocation and unfolding. The interaction energy, in terms of the cartesian coordinates ri (Inline graphic), is Vtot = VBL + VBA + VDA + VNB where VBL is the bond length, VBA and VDA are the bond angle and the dihedral angle potentials, respectively (38). In this BLN model the nonbonded potential is Inline graphic and rij is the distance between residues. The nonbonded potential includes attractive B-B interactions, and repulsive interactions for any other pairs (see SI Text for interaction parameters). Local (dihedral angle) interactions favor either α-helical (for B and L residues) or loop conformations (N). The SsrA tag is covalently fused at the C terminus of HBP and all of its bonded interactions are modeled to favor loop conformations. Nonbonded ClpY·HBP–SsrA interactions are given by VGi,Hj = λGi,HjVHBPi,HBPj where G = {ClpY,SsrA}, H = {SsrA,HBP} and ij = {B,L,N} (see SI Text for interaction parameters).

ClpY Model.

To model the open (closed) ClpY state we use the Protein Data Bank (PDB) structure 1DO2 (1DO0) (19). These two asymmetric structures (28) were highlighted due to the observation that at least two subunits in the ClpX ring remain nucleotide-free (29). The 1DO0 structure has a narrower pore than 1DO2 (diameter d ≃ 8 Å vs. 19 Å), and its conserved channel loops (GYVG motif) are displaced by ≃8 Å toward ClpQ. ClpY residues are either constrained to fixed positions corresponding to the crystal structures or move along straight paths as the allosteric transitions take place, a procedure in the spirit of targeted molecular dynamics (54, 55). Allosteric transitions, induced by ATP, are mimicked as follows. The open and closed structures of ClpY are aligned to minimize the rmsd between the two structures. During the transition, ClpY residues of subunits that undergo allosteric changes are constrained to move at a constant velocity along a path connecting their open and closed positions (46). The motion of the ClpY residues is a reasonable approximation of the rigid-body motion of the domains of ClpY subunits. A complete ClpY cycle is realized by six moves of equal duration involving two adjacent subunits. Within each subunit pair, a single subunit has ATPase activity. Each cycle is simulated over 6,000,000 steps (0.15 μs); therefore ClpY amino acids move at speeds on the order of (105–104 μm/s). While these speeds are large, they are unlikely to affect the qualitative results of SP unfolding due to ClpY allostery.

Supplementary Material

Supporting Information
supp_108_6_2234__index.html (1.2KB, html)

Acknowledgments.

The authors gratefully acknowledge stimulating discussions with Sue Wickner, Mike Maurizi, D. Thirumalai, Bernard Brooks, and Rich Pastor. This work has been supported by a grant from the American Heart Association and the National Science Foundation CAREER Grant MCB-0952082 to G.S. and a University Research Council fellowship at the University of Cincinnati to M.J.

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1014278108/-/DCSupplemental.

References

  • 1.Inobe T, Matouschek A. Protein targeting to ATP-dependent proteases. Curr Opin Struct Biol. 2008;18:43–51. doi: 10.1016/j.sbi.2007.12.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Gottesman S. Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol. 2003;19:565–587. doi: 10.1146/annurev.cellbio.19.110701.153228. [DOI] [PubMed] [Google Scholar]
  • 3.Weber-Ban EU, Reid BG, Miranker AD, Horwich AL. Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature. 1999;401:90–93. doi: 10.1038/43481. [DOI] [PubMed] [Google Scholar]
  • 4.Hoskins JR, Singh SK, Maurizi MR, Wickner S. Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP. Proc Natl Acad Sci USA. 2000;97:8892–8897. doi: 10.1073/pnas.97.16.8892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kim Y-I, Burton RE, Burton BM, Sauer RT, Baker TA. Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. Mol Cell. 2000;5:639–648. doi: 10.1016/s1097-2765(00)80243-9. [DOI] [PubMed] [Google Scholar]
  • 6.Singh S, Grimaud R, Hoskins J, Wickner S, Maurizi M. Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP. Proc Natl Acad Sci USA. 2000;97:8898–8903. doi: 10.1073/pnas.97.16.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Neuwald AF, Aravind L, Spouge JL, Koonin EV. AAA+: A class of chaperone-like atpases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 1999;9:27–43. [PubMed] [Google Scholar]
  • 8.Lupas AN, Martin J. AAA proteins. Curr Opin Struct Biol. 2002;12:746–753. doi: 10.1016/s0959-440x(02)00388-3. [DOI] [PubMed] [Google Scholar]
  • 9.Vale R. AAA proteins: Lords of the ring. J Cell Biol. 2000;150:F13–F19. doi: 10.1083/jcb.150.1.f13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ogura T, Wilkinson A. AAA+ superfamily ATPases: Common structure-diverse function. Genes Cells. 2001;6:575–597. doi: 10.1046/j.1365-2443.2001.00447.x. [DOI] [PubMed] [Google Scholar]
  • 11.Hanson PI, Whiteheart SW. AAA+ proteins: Have engine, will work. Nat Rev Mol Cell Biol. 2005;6:519–529. doi: 10.1038/nrm1684. [DOI] [PubMed] [Google Scholar]
  • 12.Katayama-Fujimura Y, Gottesman S, Maurizi M. A multiple-component, ATP-dependent protease from Escherichia coli. J Biol Chem. 1987;262:4477–4485. [PubMed] [Google Scholar]
  • 13.Goloubinoff P, Mogk A, Zvi APB, Tomoyasu T, Bukau B. Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc Natl Acad Sci USA. 1999;96:13732–13737. doi: 10.1073/pnas.96.24.13732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Motohashi K, Watanabe Y, Yohda M, Yoshida M. Heat-inactivated proteins are rescued by the DnaK. J-GrpE set and ClpB chaperones. Proc Natl Acad Sci USA. 1999;96:7184–7189. doi: 10.1073/pnas.96.13.7184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zolkiewski M. ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli. J Biol Chem. 1999;274:28083–28086. doi: 10.1074/jbc.274.40.28083. [DOI] [PubMed] [Google Scholar]
  • 16.Baker T, Sauer R. ATP-dependent proteases of bacteria: Recognition logic and operating principles. Trends Biochem Sci. 2006;31:647–653. doi: 10.1016/j.tibs.2006.10.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Schirmer EC, Glover JR, Singer MA, Lindquist S. HSP100/Clp proteins: A common mechanism explains diverse functions. Trends Biochem Sci. 1996;21:289–296. [PubMed] [Google Scholar]
  • 18.Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1:945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bochtler M, et al. The structures of HslU and the ATP-dependent protease HslU-HslV. Nature. 2000;403:800–805. doi: 10.1038/35001629. [DOI] [PubMed] [Google Scholar]
  • 20.Guo F, Maurizi M, Esser L, Xia D. Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J Biol Chem. 2002;277:46743–46752. doi: 10.1074/jbc.M207796200. [DOI] [PubMed] [Google Scholar]
  • 21.Kim DY, Kim KK. Crystal structure of ClpX molecular chaperone from Helicobacter pylori. J Biol Chem. 2003;278:50664–50670. doi: 10.1074/jbc.M305882200. [DOI] [PubMed] [Google Scholar]
  • 22.Hinnerwisch J, Fenton WA, Furtak KJ, Farr GW, Horwich AL. Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell. 2005;121:1029–1041. doi: 10.1016/j.cell.2005.04.012. [DOI] [PubMed] [Google Scholar]
  • 23.Diemand A, Lupas A. Modeling AAA+ ring complexes from monomeric structures. J Struct Biol. 2006;156:230–243. doi: 10.1016/j.jsb.2006.04.011. [DOI] [PubMed] [Google Scholar]
  • 24.Rohrwild M, et al. The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome. Nat Struct Mol Biol. 1997;4:133–139. doi: 10.1038/nsb0297-133. [DOI] [PubMed] [Google Scholar]
  • 25.Ishikawa T, et al. Translocation pathway of protein substrates in ClpAP protease. Proc Natl Acad Sci USA. 2001;98:4328–4333. doi: 10.1073/pnas.081543698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Flynn JM, Neher SB, Kim Y, Sauer RT, Baker TA. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol Cell. 2003;11:671–683. doi: 10.1016/s1097-2765(03)00060-1. [DOI] [PubMed] [Google Scholar]
  • 27.Martin A, Baker TA, Sauer RT. Protein unfolding by a AAA+ protease is dependent on ATP-hydrolysis rates and substrate energy landscapes. Nat Struct Mol Biol. 2008;15:139–145. doi: 10.1038/nsmb.1380. [DOI] [PubMed] [Google Scholar]
  • 28.Wang J, et al. Nucleotide-dependent conformational changes in a protease-associated ATPase HslU. Structure. 2001;9:1107–1116. doi: 10.1016/s0969-2126(01)00670-0. [DOI] [PubMed] [Google Scholar]
  • 29.Hersch GL, Burton RE, Bolon DN, Baker TA, Sauer RT. Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: Allosteric control of a protein machine. Cell. 2005;121:1017–1027. doi: 10.1016/j.cell.2005.05.024. [DOI] [PubMed] [Google Scholar]
  • 30.Koga N, Kameda T, Okazaki K, Takada S. Paddling mechanism for the substrate translocation by AAA+ motor revealed by multiscale molecular simulations. Proc Natl Acad Sci USA. 2009;106:18237–18242. doi: 10.1073/pnas.0904756106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Huang L, Kirmizialtin S, Makarov DE. Computer simulations of the translocation and unfolding of a protein pulled mechanically through a pore. J Chem Phys. 2005;123:124903. doi: 10.1063/1.2008231. [DOI] [PubMed] [Google Scholar]
  • 32.West DK, Brockwell DJ, Paci E. Prediction of the translocation kinetics of a protein from its mechanical properties. Biophys J. 2006;91:L51–53. doi: 10.1529/biophysj.106.089490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Feng G, Lu H. Computer simulation of I27 translocation through ClpY reveals a critical role of protein mechanical strength and local stability; Proceedings of the 29th Annual International Conference of the IEEE EMBS; Lyon, France: Cité Internationale; 2007. pp. 1213–1216. [DOI] [PubMed] [Google Scholar]
  • 34.Szymczak P, Janovjak H. Periodic forces trigger a complex mechanical response in ubiquitin. J Mol Biol. 2009;390:443–456. doi: 10.1016/j.jmb.2009.04.071. [DOI] [PubMed] [Google Scholar]
  • 35.Rief M, Pascual J, Saraste M, Gaub HE. Single molecule force spectroscopy of spectrin repeats: Low unfolding forces in helix bundles. J Mol Biol. 1999;286:553–561. doi: 10.1006/jmbi.1998.2466. [DOI] [PubMed] [Google Scholar]
  • 36.Paci E, Karplus M. Unfolding proteins by external forces and temperature: The importance of topology and energetics. Proc Natl Acad Sci USA. 2000;97:6521–6526. doi: 10.1073/pnas.100124597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lee C, Schwartz MP, Prakash S, Iwakura M, Matouschek A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol Cell. 2001;7:627–637. doi: 10.1016/s1097-2765(01)00209-x. [DOI] [PubMed] [Google Scholar]
  • 38.Guo Z, Thirumalai D. Kinetics and thermodynamics of folding of a de novo designed four—helix bundle protein. J Mol Biol. 1996;263:323–343. doi: 10.1006/jmbi.1996.0578. [DOI] [PubMed] [Google Scholar]
  • 39.Singh SK, et al. Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis. J Biol Chem. 2001;276:29420–29429. doi: 10.1074/jbc.M103489200. [DOI] [PubMed] [Google Scholar]
  • 40.Lo JH, Baker TA, Sauer RT. Characterization of the N-terminal repeat domain of Escherichia coli ClpA-A class I Clp/HSP100 ATPase. Protein Sci. 2001;10:551–559. doi: 10.1110/ps.41401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Hinnerwisch J, Reid BG, Fenton WA, Horwich AL. Roles of the n-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease. J Biol Chem. 2005;280:40838–40844. doi: 10.1074/jbc.M507879200. [DOI] [PubMed] [Google Scholar]
  • 42.Gottesman S, Roche E, Zhou Y, Sauer R. The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 1998;12:1338–1347. doi: 10.1101/gad.12.9.1338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Flynn JM, et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc Natl Acad Sci USA. 2001;98:10584–10589. doi: 10.1073/pnas.191375298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Viitanen PV, Gatenby AA, Lorimer GH. Purified chaperonin 60 (GroEL) interacts with the non-native states of a multitude of Escherichia coli proteins. Protein Sci. 1992;1:363–369. doi: 10.1002/pro.5560010308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Todd MJ, Lorimer GH, Thirumalai D. Chaperonin—facilitated protein folding: Optimization of rate and yield by an iterative annealing mechanism. Proc Natl Acad Sci USA. 1996;93:4030–4035. doi: 10.1073/pnas.93.9.4030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Stan G, Lorimer GH, Thirumalai D, Brooks BR. Coupling between allosteric transitions in GroEL and assisted folding of a substrate protein. Proc Natl Acad Sci USA. 2007;104:8803–8808. doi: 10.1073/pnas.0700607104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Martin A, Baker TA, Sauer RT. Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines. Nature. 2005;437:1115–1120. doi: 10.1038/nature04031. [DOI] [PubMed] [Google Scholar]
  • 48.Jacob E, Horovitz A, Unger R. Different mechanistic requirements for prokaryotic and eukaryotic chaperonins: A lattice study. Bioinformatics. 2007;23:i240–i248. doi: 10.1093/bioinformatics/btm180. [DOI] [PubMed] [Google Scholar]
  • 49.Honeycutt JD, Thirumalai D. The nature of folded states of globular—proteins. Biopolymers. 1992;32:695–709. doi: 10.1002/bip.360320610. [DOI] [PubMed] [Google Scholar]
  • 50.Brooks BR, et al. CHARMM: The biomolecular simulation program. J Comput Chem. 2009;30:1545–1614. doi: 10.1002/jcc.21287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Mickler M, Dima RI, Hyeon HDC, Thirumalai D, Rief M. Revealing the bifurcation in the unfolding pathways of GFP using single molecule experiments and simulations. Proc Natl Acad Sci USA. 2007;104:20268–20273. doi: 10.1073/pnas.0705458104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Fernandez JM, Li H. Force-clamp spectroscopy monitors the folding trajectory of a single protein. Science. 2004;303:1674–1678. doi: 10.1126/science.1092497. [DOI] [PubMed] [Google Scholar]
  • 53.Ho SP, DeGrado WF. Design of a 4-helix bundle protein: Synthesis of peptides which self-associate into a helical protein. J Am Chem Soc. 1987;109:6751–6758. [Google Scholar]
  • 54.Ma J, Karplus M. The allosteric mechanism of the chaperonin GroEL: A dynamic analysis. Proc Natl Acad Sci USA. 1998;95:8502–8507. doi: 10.1073/pnas.95.15.8502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.van der Vaart A, Ma J, Karplus M. The unfolding action of GroEL on a protein substrate. Biophys J. 2004;87:562–573. doi: 10.1529/biophysj.103.037333. [DOI] [PMC free article] [PubMed] [Google Scholar]

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