Asymmetric Conformational Transitions in AAA+ Biological

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Asymmetric Conformational Transitions in AAA+ Biological Nanomachines Modulate Direction-Dependent Substrate Protein Unfolding Mechanisms Abdolreza Javidialesaadi, and George Stan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05963 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 9, 2017

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The Journal of Physical Chemistry

Asymmetric Conformational Transitions in AAA+ Biological Nanomachines Modulate Direction-Dependent Substrate Protein Unfolding Mechanisms Abdolreza Javidialesaadi and George Stan∗ Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, United States E-mail: [email protected] Phone: +1 (513) 556-3049. Fax: +1 (513) 556-9239

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Abstract Powerful AAA+ ATPases, such as ClpY, form hexameric ring structures which selectively process abnormal proteins targeted for degradation by unfolding and threading them through a narrow central channel. The molecular details of this process are not yet fully understood. We perform Langevin dynamics simulations using a coarse-grained model of Substrate Proteins (SPs), Titin I27 and its V13P variant, threading through the ClpY pore. We probe the effect of ClpY surface heterogeneity and changes in pore width on the SP orientation and the direction of applied force during SP unfolding. We contrast mechanisms of SP unfolding in a restrained geometry, as in single-molecule force spectroscopy experiments, and in an unrestrained geometry, as in the in vivo degradation process. In open pore configurations, unfolding of the unrestrained SPs occurs via an unzipping mechanism which involves force application along a weak mechanical direction. In the partially closed pore, unfolding occurs via a shearing mechanism with force application in a strong mechanical direction. By contrast, unfolding of the restrained I27 is limited to a shearing mechanism due to application of force along the strong mechanical direction. We propose that Clp nanomachine plasticity underlies direction-dependent pulling mechanisms that enable versatile SP remodeling actions.

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Introduction Protein degradation is an essential quality control mechanism that ensures cell viability by clearing defective proteins that result from errors in synthesis and by regulating cellular levels of stress-response proteins. 1–7 This action is performed by bacterial Caseinolytic protease (Clp) or eukaryotic proteasome machinery which thread substrate proteins (SPs) through stacked ring-shaped compartments specialized in unfolding and proteolysis. The requisite SP unfolding step is effected by the AAA+ (ATPases Associated with diverse cellular Activities) unfoldase to initiate the sequence of events leading to the ultimate polypeptide fragmentation within the peptidase chamber. 8–11 The homohexameric Clp ATPases, which comprise subunits with one (ClpX or ClpY) or two (ClpA or ClpB) catalytic sites, couple non-concerted ATP-driven motions of subunits with mechanical force applied onto the SP by a set of central channel pore loops. 12–18 Structural studies 19–23 reveal a non-uniform width of the pore channel with a diameter of ∼ 20-30 Å at the entrance and ∼ 9-15 Å at the narrowest point. Diverse substrates are targeted and degraded 24–27 by the Clp ATPase through short peptide tags, such as the E. coli SsrA, 26 fused at the SP terminal or through intrinsic sequence motifs. 28–30 The efficacy of the Clp machinery was shown to correlate with the local mechanical stability near the tagged terminus rather than the global stability of the SP. 31,32 This local stability model is a common feature in mechanical unfolding of proteins, as illustrated by single-molecule experiments in which pulling along the N-C direction probes the strength of terminal regions of the SP. In accord with this model, ClpXP, ClpAP, and the proteasome efficiently degrade the N-terminal tagged barnase, but not the dihydrofolate reductase (DHFR), which is at variance with the greater global stability of barnase. The contrasting behavior of these proteins is consistent with the presence of a mechanically stronger N-terminal structure of DHFR (β-sheet) compared with barnase (α-helical). From a kinetic perspective, topological features of SP regions presented to the Clp ATPase determine whether unfolding or translocation represent the rate-limiting step in the degradation reaction. As shown by ex3



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perimental and computational studies, small proteins with predominantly α-helical structure are unfolded on fast timescales compared with those involved in translocation, 33,34 whereas proteins with mostly β structure unfold on slow timescales. 35 Computer simulations indicate that the interplay between unfolding and translocation yields complex conformational pathways in the degradation process that include unfolding prior to or simultaneous with translocation. 33–35 In addition, competition between refolding and translocation observed for unfolding intermediates, such as GFP∆β11 of the Green Fluorescent Protein (GFP), results in stringent kinetic requirements for translocation. 16,36,37 An intriguing situation is illustrated by pulling knotted biopolymers through narrow pores, which can result in pore jamming and threading to take place through a reptation mechanism. 38–40 Simulations of mechanical pulling of proteins through model pores suggest diverse translocation scenarios as a function of knot topology. 41–44 For a given SP, anisotropic properties dictate that mechanical stability is strongly dependent on the direction of the pulling force applied. Single-molecule mechanical pulling experiments and computational studies identified distinct force requirements in directionaldependent mechanical unfolding pathways of diverse proteins, such as E2lip3, ubiquitin, GFP, SH3, and I27. 45–50 These studies highlighted, in particular, larger forces required for unraveling β-sheets by pulling along the parallel direction to the strand registry than along the perpendicular direction. The effect of force directionality can be a dramatic variation of the resistance to deformation, such as the wide range of critical unfolding forces, from 100 pN to 600 pN, required for GFP and its variants with altered N and C termini. 47 Anisotropic properties of SPs play a prominent role in the degradation process, in which the relevant direction of mechanical strength is determined by the SP orientation relative to the Clp ATPase pore axis. As the central channel loop-mediated pulling imposes restraints only on the tagged terminus of the SP, mechanical strength can be probed along multiple directions compatible with the two-dimensional motion of the SP on the unfoldase surface. 51,52 Thus, the direction of mechanical pulling probed by the Clp ATPase should be viewed as

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a dynamic variable. Similar to bulk mechanical pulling, the emergence of single-molecule approaches for Clp ATPase-mediated threading of SPs enabled the selective investigation of directions of mechanical strength. 14–17,53 These studies revealed that Clp-mediated forces, with a lower bound of 20 pN, are used in unfolding proteins with distinct terminal topologies, such as filamin (β-sheet), I27 (β-sheet), GFP (β-barrel), and HaloTag (α-helical). Molecular dynamics simulations, using coarse-grained or atomistic representations of SPs pulled through narrow cylindrical pores or a circular aperture, highlight the role of force directionality and indicate unfolding pathways that are in accord with the local stability model. 48,54–56 In particular, translocation by pulling the C-terminal of ubiquitin or I27 yields unfolding mechanisms with lower barriers than in bulk pulling due to an SP orientation on the pore surface that favors force application perpendicular to the β-strand registry. By contrast, in the translocation setup that reproduces the N-terminal restraint imposed in single-molecule approaches, unfolding of I27 outside the pore entrance requires forces oriented parallel to the β-strand registry and larger than those in bulk pulling. 55 This restrained geometry probes the SP mechanical strength along the preselected N-C direction, as in bulk pulling approaches, in combination with constraints resulting from pinning the SP at the pore entrance. Pore widening in a hexameric model using spherical subunits results in faster translocation kinetics. 44 Overall, these observations suggest that the surface configuration of the Clp ATPase pore has a strong contribution to the unfolding mechanism. During allosteric cycles of the ATPase ring, the molecular shape of the surface is dynamically reconfigured. Non-concerted subunit motions that underlie the repetitive opening and closing of the ATPase pore yield asymmetric ring configurations consisting of heterogeneous nucleotide states of individual subunits. Currently, the combined effects of ATPase ring asymmetry, molecular surface heterogeneity and pore width on unfolding mechanisms are insufficiently understood. In this paper we perform coarse-grained simulations of SP threading through the ClpY∆I pore to probe the role of asymmetric ClpY ring plasticity on SP unfolding mechanisms. Prior simulations from our group 34,35 indicate that, for SPs with strong mechanical resistance, both

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SP threading through non-allosteric pores and SP remodeling by allosteric pores yield major pathways that involve simultaneous unfolding and translocation. For such substrates, important aspects are that unfolding represents the rate-limiting step and the unfolded SP fragment is located near the pore entrance. Both application of a continuous pulling force in a non-allosteric pore and the repetitive force mimicking the action of the allosteric pore reproduce these characteristics. To obtain information about the role of changes in the pore width during the ClpY allosteric cycle, we compare and contrast the unfolding of I27 upon mechanical pulling through open and partially closed pore configurations that result from non-concerted intra-ring allostery (Figure 1A-B). While these simulations assume that ClpY∆I configurations have infinite lifetimes and therefore the role of dynamic conformational transitions within ring subunits is omitted, they reveal distinct SP unfolding mechanisms favored in intermediate states in the ring cycle. We find that open pore configurations result in selecting I27 orientations that favor unfolding along a soft mechanical direction through an unzipping mechanism (Figure 1C). In this case mechanical forces are applied in the perpendicular direction to the β-sheet registry of I27 and inter-strand native contacts are removed sequentially. In closed pore configurations the SP is restricted to unfold along a strong mechanical direction through a shearing mechanism (Figure 1C). In this mechanism, the mechanical force is applied along the direction parallel to the β-sheet registry and inter-strand native contacts are removed simultaneously. Imposing an N-terminal restraint on the SP, as in single-molecule force spectroscopy experiments, limits the unfolding along the strong mechanical direction (Figure 1C). These results suggest strong effects of ring plasticity and force directionality on SP orientation and unfolding mechanisms.

Materials and Methods Coarse-grained model of ClpY∆I-SP system We describe the ClpY∆I-I27 system by using a coarse-grained representation that enables us to probe long time scales associated

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with substrate protein unfolding and translocation. In this model, each amino acid of ClpY∆I and the SP is represented as a virtual particle located at the Cα position. We consider three configurations of ClpY∆I, corresponding to the open pore and two partially closed pore states (Figure 1). The open pore configuration is obtained from the crystal structure with Protein Data Bank (PDB) ID 1DO2, 19 which comprises a trimer made of dimers of ATPbound and apo subunits. The structure of the ClpY∆I deletion variant was obtained by removing the auxiliary I-domain (residues 111-242) from the crystal structure. Partially closed pore states are modeled by replacing conformations of two (state I) or four (state II) subunits by their closed pore conformations. The closed pore state was described using the crystal structure with PDB ID 1DO0, 19 which comprises a dimer made of trimers of one apo and two ADP-bound subunits. The SP consists of the fusion peptide (SsrA)2 , with the SsrA sequence AANDENYALAA, attached covalently at the C-terminus or at both termini of the I27 domain of titin. The native conformation of I27 is obtained from the crystal structure with PDB ID 1TIT 58 and SsrA is modeled as a disordered chain. Coarse-grained Langevin dynamics simulations are performed using the CHARMM molecular modeling software. 59,60 In our simulations, intermediate allosteric states of ClpY∆I are assigned infinite lifetimes, therefore the positions of ATPase amino acids are fixed and interactions between ClpY∆I amino acids are not included in potential energy calculations. Interactions between I27 amino acids are described by the Karanicolas-Brooks (KB) Cα Go-model 61,62 in which all native contacts are attractive and non-native contacts are repulsive. Native contacts are assigned based on backbone-backbone hydrogen bonds and side chain-side chain interactions present in the atomistic structure. Hydrogen bonds are identified according to the method of Kabsch and Sander 63 by using partial charges on relevant atoms, C,O (+q1 ,−q1 ) and N,H (−q2 ,+q2 ):

E = f q1 q2

1 rN O

+

1 rHC

−

1 rHO

−

8


1 rN C

(1)

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where f=332 Å kcal/e2 mol, e is the electron charge, q is the partial charge, and rXY is the distance, in Å between atoms X and Y. A hydrogen bond is present when E < -0.5 kcal/mol. Side chain-side chain interactions corresponding to native contacts involve distances between non-hydrogen side chain atoms of less than 4.5 Å. Native pairs interact through the 12-10-6 potential:

Vij

12 10 6 σij σij σij = ǫij 13 − 18 +4 rij rij rij

(2)

where rij is the distance between particles i and j and σij and −ǫij are the distance and energy corresponding to the minimum value of the non-bonded pairwise interaction potential. Interactions between ClpY∆I and the SP, as well as those between SsrA and I27 or SsrA are modeled as repulsive. To this end, we used the 12-10-6 potential with parameters rmin = 11.2 Å and ǫ = 1.32 × 10−4 kcal/mol. Omitting attractive interactions in this model is supported by our prior observations of ClpY-mediated unfolding of an α/β substrate which has strong mechanical resistance. 35 In that case, the major unfolding event primarily involves repulsive interactions of the native structure with the ClpY surface. The friction coefficient was set to 0.1 ps−1 . Covalent bonds between virtual particles of I27 and SsrA were constrained to their native values (≃ 3.8 Å) by using the SHAKE algorithm 64 with tolerance 1.0 × 10−6 Å. The timestep was set to 10 fs and frames were saved every 10 ps. Simulations were performed at T = 0.7 Tf , where the I27 folding temperature in the KB model, Tf , is 288 K. 65 Mechanical pulling of the SP To probe the mechanical resistance of the SP, we used the AFM module of CHARMM. 60,66 In the bulk setup, the N-terminal of I27 was fixed and the C-terminal was pulled at constant velocity of 105 µm/s by using a harmonic spring with force constant of 5 × 104 pN/µm (Table 1). In the setup corresponding to SP threading, ClpY∆I is oriented such that the z-axis coincides with the pore axis, the origin of the coordinate system corresponds to the center 9



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of mass of the ATPase and the proximal region of the pore is on the negative side of the z-axis (Figure 1). The center of mass of the ClpY∆I loops is located at zloops ≃ −4 Å. In the initial configuration, the largest principal axis of the SP is aligned with the ClpY∆I pore axis and the center of mass of the SP is located at z ≃ -40 Å, on the proximal side of ClpY∆I. Eight distinct initial orientations of the SP are obtained by successively rotating I27 by 45◦ around the z-axis. As in bulk simulations, the external mechanical force applied onto the C-terminal along the z-axis was implemented through a harmonic spring pulled at constant velocity (Table 1). We consider two geometries of ClpY∆I-mediated threading, one including a restraint applied onto the N-terminal and one with a free N-terminal. In the restrained case, the SP is (SsrA)2 -I27-(SsrA)2 and an opposing force of 100 pN was applied at its N-terminal (Figure 1). In addition, a harmonic cylindrical restraint, C(r) = kc r2 2

where kc = 100 kcal/(mol · Å ) is the force constant and r is the distance to the z-axis, was applied onto the N-terminal to maintain it along the pore axis. In the unrestrained geometry the SP is I27-(SsrA)2 . Orientation of I27 near the ClpY∆I pore surface To monitor the orientation of I27 with respect to the ClpY∆I pore axis, we use the angular degrees of freedom in the spherical coordinate system. The polar angle, θ, is determined by the angle between the first principal axis of the I27 and the z-axis. The azimuthal angle, φ, is determined by the angle between the projections of the principal axis of I27 and of the position vector of one subunit of ClpY∆I onto the plane perpendicular to the z-axis. In the case of unfolding intermediates, the principal axis of the folded fragment is used to determine the two angles. Fraction of Native Contacts in I27 We use the fraction of native contacts, QN = P (1/NC ) i6=j,j±1 Θ η − |rij (t) − rij0 | , to monitor unfolding of I27 substructures during threading. Here, NC is the number of native contacts generated by KB model, Θ(x) is the Heaviside

step function for which Θ(x) = 1 if x ≥ 0 and Θ(x) = 0 if x < 0, with tolerance η = 2 Å. In this model, I27 has 150 native contacts, of which 18 involve the A′ strand and 10 involve the 10


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A′ -G interface. The V13P variant was modeled by removing all six contacts involving V13 of which four were with G strand amino acids. We chose to represent the effect V13P mutation through removal of all of the native contacts as this approach results in the reduction of the average critical unfolding force by 78 pN, which is similar to the experimentally-determined force reduction by 68 pN. 67 The time scale is set relative to the major unfolding event, τ =0, which corresponds to QN ≃ 0.7 and (QN )A′ −G = 0. Data clustering algorithm The mean shift algorithm, 68 which does not require prior knowledge about clusters and their shapes, was used to cluster two dimensional (2D) QN − θ phase space and find the center of clusters. The algorithm uses the Euclidean metric in this 2D space and specifies separations (“bandwidths”) to determine clusters (“Kernels”). Convergence is assessed through the stability of the centroid of each Kernel. For restrained, unrestrained and bulk simulations, we used bandwidths of 0.11, 0.14 and 0.08, respectively. All probability density maps were calculated by using Gaussian Kernel Density Estimation.

Results Anisotropy of the titin I27 domain results in strong mechanical resistance along parallel directions to the β-sheet registry and weak resistance along perpendicular directions. Previous atomic force microscopy (AFM) and computational studies of bulk mechanical pulling of I27 revealed that unfolding events along the N-C direction involve breaking contacts between A-B strands, followed by the rate-determining “mechanical clamp” A′ -G. 67,69–74 These unfolding events are consistent with the geometry of the native structure, in which the β-sheet registry is nearly parallel with the N-C direction (Figure 1) and, therefore, unfolding of the native structure requires cooperative removal of the A′ G contacts through shearing. AFM experiments found that the average force required for I27 unfolding is ≃ 200 pN 67,70 and several coarse-grained simulations, using distinct nativecentric models, yield average unfolding forces of 200-300 pN. 55,65,75 Unraveling I27 may 11



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bypass large force requirements by pulling along a softer mechanical direction perpendicular to the β-strand registry. Simulations of bulk pulling along such directions, using atomistic or coarse-grained descriptions, indicate sequential removal of inter-strand contacts through an unzipping mechanisms with lower force requirements, . 150 pN. 73,76 In the present study, we use the Karanicolas-Brooks (KB) coarse-grained model 61 (see Materials and Methods), which was used in the pulling studies of West et al. 65 As shown in Fig. S1, our bulk mechanical pulling simulations (Table 1), which are performed at lower temperature and loading rates than in the studies of West et al. 65 yield unfolding pathways in agreement with the previous studies. Larger critical forces ≃ 300 pN in our simulations are consistent with the higher energy barrier of unfolding at lower T. Table 1: Summary of coarse-grained simulations performed. System I27

I27 variant Restraint ClpY∆I v [µm/s] Ntraj a hFcritical i [pN]b WT N-fixedc 105 104 291±3 WT N-fixed 104 16 265±5 V13P N-fixed 105 104 196±3 4 V13P N-fixed 10 16 187±1 ClpY∆I − SP d WT Open 105 24 230±4 WT Open 5 × 104 24 226±5 WT State I 105 24 257±11 5 WT State II 10 24 390±18 5 V13P Open 10 24 197±1 V13P State I 105 24 219±2 5 V13P State II 10 24 351±17 5 WT N-pulled Open 10 24 368±5 WT N-pulled Open 5 × 104 24 372±0 WT N-pulled State I 105 24 402±6 WT N-pulled State II 105 24 549±33 5 V13P N-pulled Open 10 24 286±11 5 V13P N-pulled State I 10 24 321±11 V13P N-pulled State II 105 24 431±17 a Simulations are terminated when the end-to-end distance of bulk I27 reaches 260 Å or the C-terminal of the threaded SP has translocated, i.e. z = 160 Å. Duration of each trajectory is ≃ 100 − 200 ns if v = 105 µm/s and ≃ 2.5 µs if v = 104 µm/s. b Standard error in the mean is indicated. c C-terminal of SP was pulled. d SP consists of I27-(SsrA)2 and (SsrA)2 -I27-(SsrA)2 in unrestrained and restrained geometries, respectively. 12


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Rotational flexibility of the unrestrained SP enables application of Clp-mediated force along multiple directions to select soft mechanical direction. We probed I27 unfolding by mechanically pulling the C-terminal of the SP through the narrow pore of the rigid ClpY∆I structure in restrained and unrestrained geometries (see Materials and Methods and Table 1). In both setups, docking of the SP occurs within the approximately cylindrical vestibule (with radius R ≃ 18 Å) of ClpY∆I prior to initiation of unfolding (Figure 1 and Figure S2). For proteins that fit into this region (Rg /R . 1), such as I27 (Rg ≃ 12.5 Å), the Clp-mediated pulling force is applied close to the C-terminus of the SP, which renders the direction of force strongly affected even by small SP orientation changes. Using Flory’s scaling law for globular proteins, Rg ≃ aN 1/3 , where N is the number of amino acids and a = 3, 77 the cutoff value corresponds to proteins with N . 254 amino acids. By contrast, larger proteins with (Rg /R & 1) are docked on the surface of ClpY∆I and the pulling force is applied through a longer stretch (d ≃ 17 Å) of the peptide chain. In this case, the combination of two-dimensional constraints due to the ClpY∆I surface and the longer lever arm of the force yield a narrower solid angle sampled by the force direction than in the case of smaller substrates. As the direction of mechanical force applied onto the SP is dependent on the SP orientation relative to the ClpY∆I pore axis, we monitor the dynamic evolution of the polar angle θ (see Materials and Methods and Figure 2) during the pulling trajectories. To probe the effects of ring plasticity and pore width fluctuations during the allosteric cycle, we considered three ClpY∆I configurations (see Materials and Methods, Figure 1 and Table 1). These comprise the ATP-bound open pore configuration and two partially closed pore configurations obtained by switching the conformations of two (state I) or four subunits (state II) from the open to the closed state. As shown in Figure 2, the relative SP-ClpY∆I orientations sampled during mechanical pulling are strikingly different in the two geometries considered. In the unrestrained case, the native I27 samples a broad range of orientations corresponding to the angular interval 10◦ . θ . 35◦ . In the restrained geometry, the range of angles available

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to the native SP is limited to the 0◦ . θ . 20◦ and the principal axis of the SP is nearly parallel to the direction of the ClpY∆I axis. For I27, these distinct SP orientations have important implications for the unfolding mechanism. As the A′ and G strands are nearly parallel to the principal axis of I27, the angle θ characterizes the approximate orientation of these strands with respect to the ClpY∆I pore axis. Thus, in the unrestrained geometry, the broad range of polar angles sampled implies that the pulling forces include significant parallel components, i.e. applied along the direction of inter-strand contacts. In the open and state I configurations, which include a larger ClpY∆I pore width, the effect of these parallel forces is to enable unfolding of I27 by sequential removal of the A′ -G contacts (Figure 2A and Figures S3-S4). The first unfolding event results in the transition from the native cluster N to the I1 intermediate (0.80 . QN . 0.92) and in tilting the SP to a nearly perpendicular direction to the ClpY∆I axis (50◦ . θ . 70◦ ). Complete removal of the G and F strands from the SP core, which corresponds to transition to the I2 cluster (hQN i ≃ 0.44) shown in Figure 2, restores the I27 orientation to the direction parallel to the ClpY∆I axis. Interestingly, in state II, the major unfolding pathway involves simultaneous removal of A′ -G contacts of I27, which corresponds to the direct transition N → I2 (Figure 2A). In this state, the narrow width of the pore results in stabilization of tilted SP conformation near the pore surface, which hinders the sequential loss of inter-strand contacts and prevents access to the I1 intermediate. In the restrained geometry, mechanical force is applied perpendicular to inter-strand contacts and, as unfolding requires cooperative breaking of A′ -G contacts, the native structure is largely preserved (0.88 . QN . 1.0) prior to the major unfolding event N → I2 (similar to bulk simulations that are shown in Figure S5). Overall, we surmise that rotational flexibility of I27 in the unrestrained geometry allows ClpY∆I to probe SP mechanical resistance along multiple directions. In ClpY∆I configurations with an “open” pore, this enables selection of an unfolding pathway corresponding to a soft direction associated with sequential removal of inter-strand contacts. In “closed” pore configurations, soft mechanical directions of the bulk I27 are stabilized by the pore surface and are no longer

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associated with low mechanical force requirements. In such pore configurations, the major unfolding pathway consists in concomitant removal of A′ -G contacts, similar to the case of SP threading in the restricted geometry. Broad regions of the unrestrained SP interact with the open-pore ClpY∆I to facilitate SP unfolding via unzipping. To understand, at the microscopic level, the distinct unfolding mechanisms in each geometry, we examined the force exerted between interacting regions of I27 and open-pore ClpY∆I and the time evolution of the native content, QN , of the total structure and of single strands (Figure 3). Interactions between ClpY∆I and the SP are highlighted by mapping the sites of application of mutual repulsion forces onto protein surfaces prior to major SP unfolding events. As shown in Figure 3A, in both restrained and unrestrained cases, only the vestibule region of the ClpY∆I surface interacts with the SP and the largest forces are applied onto the ClpY∆I central loops and onto I27 loops near the C-terminus that are in contact with them. This strong interaction is in agreement with the major role played by the central channel loops in promoting substrate unfolding and translocation. The pattern of forces applied to the rest of the SP is highly specific for each geometry. In the unrestrained case, the force is applied to a broad region that includes A′ and G strands, in agreement with sequential removal of the corresponding inter-strand contacts. By contrast, in the restrained case, ClpY∆I exerts force on a narrow C-terminal region of I27 around A′ and G strands (Figure 3A). In this geometry, the concentrated ClpY∆I-mediated force applied near the C-terminal of the SP is complemented by the N-terminal opposing force to yield mechanical pulling of the SP near its two termini, which results in probing the mechanical strength of similar interfaces to the ones in the bulk pulling setup. The evolution of the native content of I27 reflects the distinct unfolding mechanisms in the two geometries. As shown in Figure 3B, in the unrestrained case, the hallmark of the major unfolding pathway (N → I1 → I2) is the gradual loss of native contacts involving A′ and G

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strands primarily through unzipping of inter-strand contacts. This loss of native contact is responsible in large part for the corresponding gradual reduction of the total QN . Strands C and F, which are coordinated with the terminal strands, maintain a large fraction of their native contacts until the transition, while the core structure comprising strands A, B, D, and E, is only weakly perturbed by this unfolding event. Consistent with these observations, the N → I1 → I2 transitions involve large values of h∆QN iG(F ) ≃ −0.8 only for strands G and F which are completely removed from the I27 structure and translocated. The transition involves a significant shift in force application by ClpY∆I regions onto I27 (Figure 3B). The force exerted by the loop region is reduced from hF iloops ≃ 150 pN to ≃ 125 pN primarily due to the loss of interactions that involve Val89 following the N → I1 transition, while the contribution of vestibule amino acids increases from hF ivestibule ≃ 10 pN to ≃ 75 pN. Due to these compensating contributions, the total force applied by ClpY∆I onto the SP increases slightly from hF iClpY ∆I ≃ 150 pN to ≃ 225 pN. As shown in Figure 3C, a drastically different unfolding mechanism is identified in the restrained geometry. Very few native contacts are lost before the major unfolding event and the N → I2 transition is marked by the abrupt drop in native content, h∆QN i ≃ −1.0, in all regions except the core strands B, D, and E. Thus, shearing of A′ -G contacts is simultaneous with a nearly complete destruction of the I27 structure (total h∆QN i ≃ −0.6 ) as observed in the bulk mechanical unfolding. Forces required for this dramatic unraveling of the SP build up from hF iClpY ∆I ≃ 175 pN to ≃ 250 pN in the interval leading to the N → I2 transition (Figure 3B). The contribution of central channel loop amino acids increases from hF iloops ≃ 150 pN to hF iloops ≃ 225 pN primarily due to the Tyr91 site which constricts the pore entrance. These results support the concerted removal of the I27 contacts through the interaction with central channel loops. Overall, the geometry-specific mechanisms of removal of strand contacts are consistent with the distinct directionality of mechanical forces applied onto the SP.

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Asymmetric conformational transitions yield ClpY ATPase configurations with distinct propensity for SP unfolding mechanisms via unzipping or shearing. Non-concerted allosteric conformational changes of Clp ATPases (Figure 1B) yield large, asymmetric, perturbations of the pore surface. To probe the effect of the heterogeneous shape of the Clp surface on SP unfolding, we monitored the azimuthal angle φ (see Materials and Methods) that quantifies the rotation of the I27 substrate on the ClpY∆I surface. In the unrestrained geometry, we find that, prior to the major unfolding event, I27 orientation preferentially populates directions that reflect the three-fold symmetry of the open-pore configuration of ClpY∆I (Figure 4 and Figure S6). Based on these results, we propose that the effect of ClpY∆I surface heterogeneity is to select I27 orientations that favor interaction with subunits which are likely to undergo ATP-driven conformational transitions, thereby promoting SP unfolding. In state I, in which subunits A and B are in their ADP-bound state, the three-fold symmetry of ClpY∆I is broken and the distribution of the I27 orientation is bimodal and strongly biased towards regions near the ATP-bound subunits C and E (Figure 4A). Comparison with the open-state angular distribution indicates that conformational changes in the adjacent subunits A and B result in replacement of favorable orientations in the open state, centered on ATP-bound subunits A and C, by a single favorable orientation centered on subunit C. This behavior is consistent with the local increase of the vestibule depth as a result of the conformational change in subunits A and B (Figure 1B and Figure S2). By contrast, the favorable SP orientation centered around subunit E is largely unaffected as the local vestibule depth is unchanged. This suggests that preferred SP orientations enable interactions with the two ATP-bound subunits E and C to promote unfolding and translocation. Nevertheless, the larger probability density associated with the orientation towards the C subunit suggests that initiation of the allosteric cycle biases ClpY∆I conformations towards clockwise intra-ring processing of the SP by ClpY∆I subunits. In state II, ClpY∆I ring plasticity yields a nearly unimodal φ distribution of the preferred SP orientation disfavoring the interaction with the sole ATP-bound subunit E (Figure 4). Overall, these

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results suggest that the ClpY∆I surface heterogeneity plays an active role in SP remodeling and the alteration of the ClpY∆I vestibule during the allosteric cycle modulates clockwise SP processing. Reduction in the ClpY∆I pore width during the active part of the allosteric cycle results in an increased entropic barrier for the SP. We probe this effect by comparing and contrasting force requirements for unfolding of the restrained or unrestrained I27 SP in the three ClpY pore configurations considered in this study with the bulk unfolding case. As shown in Figure 4B, mechanical unfolding of the bulk I27 yields a multi-modal distribution which corresponds to critical force requirements that average 250 pN, 275 pN or 300 pN. These force requirements can be attributed to distinct mechanisms of simultaneous removal of A′ G and neighboring contacts. The lowest critical forces are required when the neighboring contacts involve F-C strands, intermediate forces involve G-F strands and the largest forces involve A-B contacts. I27 unfolding by ClpY∆I requires the least amount of force in the (open)

unrestrained geometry, hFcritical i ≃ 230 pN, compared to all the other cases. In state I, in (I)

the unrestrained geometry, the force requirement is only slightly larger, hFcritical i ≃ 240 pN, and smaller than the bulk requirement and the unzipping mechanism is not hindered by (II)

this perturbation. By contrast, in state II, the average critical force required is hFcritical i ≃ 390 pN and the unfolding mechanism involves shearing of A′ -G contacts (Figures 5A-B). We surmise that the progressive closing of the ClpY pore during each active hemicycle enforces degradation selectivity by promoting unfolding through unzipping A′ -G contacts in the initial stages (open pore) and hindering it by favoring a shearing mechanism in the final stages (closed pore). In contrast to these mechanisms, in the restrained geometry, I27 unfolding requires larger critical forces in all ClpY pore configurations than in bulk (open)

mechanical pulling. The average critical force requirement increases from hFcritical i ≃ 360 (I)

(II)

pN in the open state to hFcritical i ≃ 400 pN and hFcritical i ≃ 550 pN in partially closed pore configurations, which reflects increasingly unfavorable distribution of interaction forces between the ClpY∆I surface and I27. We note that, in the restrained geometry, the shape of

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the force distribution has a multimodal aspect as do bulk simulations. In particular, the I27 unfolding in the open-pore configuration yields force peak separations with approximately the same value as in the bulk, which indicates similar unfolding mechanisms. In state I and state II configurations, the shape of the force distribution increasingly diverges from the bulk one. In the extreme case of state II, the arrangement of ClpY∆I subunits deviates significantly from the three-fold symmetry found in the open state and the pore has a spiral structure (Figures 1B and S2). The change in pore structure coupled with the narrow diameter and the restraint applied onto the N-terminal of the SP, result in large unfolding force requirements and distinct unfolding mechanisms compared with those identified for wider pore configurations (see Figure 5C and Figure S7). Lower unfolding barrier of the V13P variant reduces force requirements but preserves geometry-specific mechanisms. Mutations that weaken the mechanical resistance of A′ -G contacts are able to significantly reduce the unfolding force requirement. I27 variants that include such mutations are of interest in single-molecule experiments as their lower unfolding barrier facilitates more frequent observation of unfolding events than in the wild-type case. 17 We consider the V13P variant, which involves disruption of inter-strand hydrogen bonds and requires an average unfolding force of 132 pN. 67 In the coarse-grained KB model used in our study, the effect of the mutation is included through removal of native contacts at the corresponding site (see Materials and Methods). As a result of this pertur(bulk)

bation, the critical unfolding force requirement is reduced from hFcritical iW T ≃ 300 pN in (bulk)

wild-type I27 to hFcritical iV 13P ≃ 200 pN in the V13P variant and removal of A′ -G contacts is effected in two successive shearing steps (Figure S1). The lower unfolding barrier yields faster unfolding of V13P which is illustrated by increased sampling of non-native states (Figure S5). We also note that the mutation significantly reduces the unfolding cooperativity as indicated by sampling of short-lived intermediates with 0.5 ≤ QN ≤ 0.9. These results are consistent with the weaker mechanical resistance of V13P compared with wild-type I27

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indicated by AFM studies. 67 Consistent with the weaker A′ -G interactions of V13P, unfolding forces required for threading of the unrestrained V13P through the open and state I of the partially closed pore of ClpY∆I are lower than for the wild-type (Figure 4B). Similar to WT (Figure 5A) an unzipping mechanism is employed in both cases (Figure S8). The average critical force requirement for V13P in these pore configurations are similar or slightly larger than the (bulk)

corresponding bulk unfolding force hFcritical iV 13P ≃ 200 pN (Figure 4B and Table 1), which is consistent with the weak cooperativity of bulk V13P shearing. In state II, V13P unfolds through a shearing mechanism (Figure S8), which imposes stronger force requirements than in the bulk as also noted in the wild-type case. In the restrained case, the critical force of unfolding in all pore configurations is higher than in the bulk (Figure 4C), which is similar to the wild-type case. Overall, the similarities between SP unfolding mechanisms of the wildtype and V13P variant of I27 suggest that the entropic barrier associated with the ClpY∆I pore configurations is the dominant factor in the unfolding mechanism. As the two SPs considered here have similar shapes, the force directionality favored in each pore configuration is not changed therefore supporting qualitatively similar SP remodeling actions.

Discussion and Conclusions Simulations presented in the current study indicate that the SP orientation relative to the AAA+ surface represents a complementary variable to the axial mechanical force in determining the unfolding pathways. Prior experimental and computational studies revealed that the central mechanism employed by AAA+ ATPases to effect unfolding and translocation in the degradation pathway involves mechanical pulling by central channel loops. 14–17,34,35,53,78 The basic step consists of stochastic SP gripping by an “active” loop, i.e. undergoing allosteric conformational changes, to impart mechanical force over its axial excursion of ≃ 1 nm. Coordinated transfer of the SP between 2-4 loops in a single AAA+ cycle allows translocation

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bursts of up to 4 nm. Simulations suggest that loop collaboration in the single-ring ClpY and double-ring p97 machines is mediated by a structural clockwise (CW) bias in the loop motion, which results in torque application onto the SP. 34,35 In combination with transient binding and release of the SP, 79 this action yields an efficient collaboration between multiple loops. Mechanical forces exerted by the central channel loops using this coordinated action yields unfolding pathways determined by the underlying energy landscape of the SP. 78 In the current study, we found that heterogeneous nucleotide states of the AAA+ subunits result in preferred SP configurations on the AAA+ surface, therefore introducing a bias in the mechanical pulling direction. This evolution of the AAA+ molecular surface during the functional cycle is particularly important for SPs, such as I27, that may be unfolded through multiple direction-dependent mechanisms. As the position of the folded SP domain is a function of the dynamic configuration of the AAA+ molecular surface and the constraint imposed by the translocated polypeptide segment, the pulling direction is continuously modified and the mechanical strength of distinct interfaces can be probed. Interestingly, reconfiguration of the Clp pore as a result of allosteric conformational transitions of subunits results in switching the application of force from a weak mechanical direction of the SP in the open pore to a strong mechanical direction in the closed pore. This suggests that the Clp ATPase is able to turn unfolding on and off during the allosteric cycle to reset the machine and to prevent unregulated unfolding in the closed pore state. Following each unfolding event, the directional bias is altered to reflect the interactions between the AAA+ machine with unfolding intermediates. Distinct shape and mechanical anisotropy of intermediates identified in experimental and computational studies 14,16,33,35 should yield diverse SP remodeling actions by the Clp ring. In the present simulations, we find that orientations probed by unfolded intermediates of I27 are significantly different compared with the native structure. Overall, we surmise that the plasticity of the AAA+ surface plays an active role in SP remodeling by altering the direction of mechanical pulling. Orientational bias may also result from the presence of extrinsic factors, such as co-

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chaperones and adaptors involved in the degradation process, and intrinsic structural components, such as auxiliary domains of Clp ATPases. 6 Adaptors, such as SspB 80,81 and RssB, 82 facilitate delivery of specific SPs to the ClpXP system and assist in the threading mechanism, while co-chaperones, such as the DnaK/DnaJ/GrpE system collaborating with the ClpB disaggregase, 83,84 form complexes with the Clp ATPases and actively participate in SP remodeling. Restraints on the SP orientation due to transient complexes formed by the Clp ATPase ring with adaptors or co-chaperones are likely to limit the directions of mechanical pulling probed. Increased degradation rates of SPs that use adaptor-mediated delivery mechanisms 80,81 may involve orientational bias along soft mechanical directions. Auxiliary domains, such as the I-domain of ClpY, and N-terminal domains of ClpX or ClpB, interact with specific SPs to either increase or decrease the degradation/disaggregation rate. 33,85–89 These complex effects on the SP degradation may result from the interplay between intrinsic structural flexibility of auxiliary domains and restraints on SP motions. The strong functional contribution of auxiliary domains is supported by lower disaggregation activity of ClpB variants that involve mutations which impair flexibility and alter the orientation of the N-terminal domain. 90 From a biological standpoint, these studies highlight the versatile mechanisms used by Clp ATPases in remodeling substrates with diverse structure. The hallmark of protein unfolding mediated by Clp nanomachines is to bypass stringent requirements of thermal unfolding of stable protein structures by applying mechanical force at one terminal of the polypeptide. This mechanism presents the advantage of replacing the need to collectively disrupt a large set of native contacts with local destabilization of the protein structure, which may succeed by removing few native contacts. Nevertheless, the presence of local structure with strong mechanical resistance, such as a β-sheet, can diminish or cancel altogether this advantage if the mechanical force is applied along the direction of large mechanical strength. Clp ATPases efficiently process these substrates by orienting the protein structure such that mechanical force is applied along soft directions. As illustrated in our simulations, this

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mechanism is enabled by the Clp ATPase ring plasticity which dynamically modulates the relative orientation of the substrate and the pore surface and favors interaction with active pore loops. Mechanisms of orientation of the substrate along specific directions should play an important role for the proteasome, the eukaryotic counterpart of bacterial Clp proteases. Structural studies of the proteasome, which reveal a functional spiral-staircase organization of AAA+ subunits, 91 suggest that the action of central channel loops is specifically tuned to guide substrate orientation. Non-concerted allostery, which underlies asymmetric conformational changes in the ClpY ring, is also used by group II chaperonins such as the eukaryotic CCT. 92 These nanomachines assist protein folding of SPs which are unable to fold spontaneously through repeated SP binding, encapsulation into a dynamic cavity and release into the cellular environment. Multi-valent binding of substrates to specific CCT chaperonin subunits 93–95 is suggested to enable domain-by-domain folding of the SP. Due to non-concerted conformational changes in group II chaperonins, SP remodeling is effected through stepwise release from the apical domains of individual subunits.

Acknowledgement The authors thank Sue Wickner, Michael Maurizi and Matthew Lang for extensive discussions. This work has been supported by the National Science Foundation grant MCB-1516918 to G.S.

Supporting Information Available Figures S1-S8: Bulk simulations of I27, details of ClpY∆I surface, and simulations of I27-WT and I27-V13P in ClpY∆I-SP system. This material is available free of charge via the Internet at http://pubs.acs.org/.

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Page 39 of 45



A ClpYΔI and Titin I27

The Journal of Physical Chemistry Unrestrained N-terminal

ClpYΔI 1 91 91 2 V13P 3 89 89 4 5 Top View 6 7 I27 Titin Z=0 8 9 ClpYΔI Z 10 11 (SsrA)2 Pulled 12 (Constant Velocity) C-Terminal 13Side View 14 15 ClpYΔI Plasticity 16 17 Open Vestibule State I State II 18 19 20 21 22 23 24 126 Å 90 ° 25 26Unfolding Mechanism 27 B 28 A 29 E 30 31 I27 C Titin F 32 33 D ACS Paragon Plus Environment 34 ClpY Force A' 35 G Unzipping Mechanism 36

B

Page 40 of 45 Restrained N-terminal Pulled (Constant Force)

N-terminal (SsrA)2

(SsrA)2

36 Å 16 Å

111 Å

17 Å

C

48 Å

20 Å

ClpY Force Shearing Mechanism

APage Unfolding of unrestrained I27The upon threading through the ClpYΔI pore 41 of 45 Journal of Physical Chemistry θ Z 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Unfolding 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

B

Open

State I

State II

of restrained I27 upon threading through the ClpYΔI pore Open

State I


State II

A ClpYΔI-I27 interaction forcesThe prior to major unfolding event Journal of Physical Chemistry

Page 42 of 45

Restrained

Unrestrained N I27 Side View

ClpYΔI Top View

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Major 16 17 18 19 20 21 22 23 24 25 26Major 27 28 29 30 31 32 33 34 35

N

C

B

unfolding event of unrestrained I27

C

unfolding event of restrained I27


C

I2743 orientation prior to major The unfolding APage of 45 Journalevent of Physical Chemistry Open

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Critical 24 25 26 27 28 29 30 31 32 33 34 35 Critical 36 37 38 39 40 41 42 43 44 45

State I

B

force required to break A'-G contacts in unrestrained I27

C

force required to break A'-G contacts in restrained I27


State II

I27 major unfolding event A Unfolding mechanism of unrestrained The Journal ofduring Physical Chemistry Open

1 2 3 4 5 6 7 8 B9 Orientation 10 11 12 13 14 15 16 17 18 19 20 21 Orientation C22 23 24 25 26 27 28 29 30 31 32 33 34 35

State I

of unrestrained I27 prior to major unfolding event

of restrained I27 prior to major unfolding event


Page 44 of 45 State II

Open Pore

Partially Closed Pore

Page 45 TheofJournal 45 of Physical Chemistry in

I27

Tit ClpYΔI 1 2 3 4 ACS Paragon Plus Environment 5 Low 6 Surface Heterogeneity High Surface Heterogeneity Unfolding via Shearing 7Unfolding via Unzipping

Asymmetric Conformational Transitions in AAA+ Biological

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