Proteins are the workhorses of the body, fulfilling many roles essential for life processes. These molecules are made up of hundreds or thousands of small units called amino acids, which attach to each other to form a long chain. The exact sequence of amino acids determines how the protein will then fold to acquire its final, three-dimentional shape.

Enzymes called proteases can degrade unneeded or faulty proteins so that the amino acids can be recycled. For instance, in bacteria, the AAA+ protease ClpXP can recognize and ‘grab’ specific patterns of amino acids at the ends of a protein. This molecular machine then tugs on the segment and unfold the protein, the way a ball of yarn unwinds when pulled from one end. The unfurled protein is then fed into a different section of ClpXP, where it is chopped into short segments for recycling.

ClpXP is the best-characterized enzyme amongst AAA+ proteases. However, it is still unclear how it can grip target proteins tightly enough to allow unfolding. To investigate, Bell et al. attached different patterns of 12 amino acids to the end of a folded protein. How well ClpXP grasped each of these proteins was then measured in bacteria and in test tubes. This revealed that ClpXP attaches to six to eight amino acids at a time, suggesting that only part of the enzyme clasps on the protein. Large amino acids are better gripped than small amino acids, similar to how a knotted string is easier to hold than a smooth rope. Amino acids that are electrically charged also interfere with ClpXP attaching to the protein. Finally, ClpXP grasps multiple amino acids at the same time, which dramatically increases grip strength.

Many proteins, including some found in viruses, use ‘slippery’ patterns of amino acids to avoid being gripped and unfolded by proteases. By understanding how different patterns of amino acids are grasped, it may someday be possible to engineer enzymes able to target dangerous proteins.

Cells maintain homeostasis by balancing protein synthesis and degradation with growth. When nutrients are available, new proteins are constantly synthesized, whereas damaged, misfolded, or unneeded proteins are degraded. Regulated degradation typically requires a protein-unfolding motor of the AAA+ family (ATPases associated with various cellular activities) that associates with a self-compartmentalized protease (e.g., ClpX with ClpP, the 19S regulatory particle with the 20S proteasome) or is genetically tethered to a protease (e.g., Lon, FtsH, Yme1) (Sauer and Baker, 2011; Olivares et al., 2016; Glynn, 2017). When challenged with degrading a folded substrate, AAA+ motors couple ATP hydrolysis to mechanical motion that overcomes the resistance of the folded domain. Despite broad consensus on the overall mechanism of protein unfolding, it is largely unknown how interactions between a AAA+ motor and its substrate produce grip, the ability for the motor to maintain hold of the substrate while applying an unfolding force.

ClpX is a ring-shaped AAA+ homohexamer that functions autonomously in protein remodeling in bacteria and eukaryotic organelles and also associates with ClpP tetradecamers to form the ATP-dependent ClpXP protease (Baker and Sauer, 2012). Substrates are targeted to ClpX or ClpXP by N- or C-terminal peptide tails (also called degradation tags or degrons), which initially bind in the ClpX axial pore. Proteins marked with a sequence-defined degron or post-translational modification can be recruited to the AAA+ protease directly or with assistance from auxiliary adaptors (Sauer and Baker, 2011; Trentini et al., 2016). For example, during rescue of stalled ribosomes in Escherichia coli, the 11-residue ssrA tag is appended to the C-terminus of abortive protein products (Keiler et al., 1996), allowing ClpXP to recognize and degrade the attached protein (Gottesman et al., 1998; Farrell et al., 2005).

The ssrA tag and other degron tails interact with ClpX loops that line the axial pore. A Tyr-Val-Gly (YVG) sequence in the pore-1 loop is critical for substrate binding, unfolding, and translocation (Siddiqui et al., 2004; Martin et al., 2008a; Martin et al., 2008b; Iosefson et al., 2015). Other AAA+ unfolding motors contain related pore-1 loops and mutation of these loops typically abolishes function (Yamada-Inagawa et al., 2003; Schlieker et al., 2004; Hinnerwisch et al., 2005; Park et al., 2005). In several AAA+ unfolding motors, the pore-1 loops adopt a spiral staircase conformation within the pore, which facilitates multivalent interaction with the bound peptide tail (Monroe et al., 2017; Gates et al., 2017; Puchades et al., 2017; de la Peña et al., 2018; Majumder et al., 2019; Dong et al., 2019; White et al., 2018). ATP-dependent conformational changes are thought to draw the tail of a substrate into the pore until a folded domain too large to transit the pore impedes progress. For ClpXP, repeated cycles of ATP hydrolysis are then required to unfold the substrate and to translocate the polypeptide through the pore and into ClpP for degradation (Kenniston et al., 2003; Aubin-Tam et al., 2011; Maillard et al., 2011; Sen et al., 2013; Cordova et al., 2014).

How the amino acids in the bound substrate tail contribute to grip during unfolding remains poorly understood. When degrading unfolded substrates, the rate of substrate translocation through ClpXP is largely insensitive to amino-acid charge, size, or peptide-bond spacing (Barkow et al., 2009). In contrast, when directly abutting a folded domain, sequences rich in glycine can result in failed unfolding by ClpXP or the 26S proteasome, leading to release of partially processed intermediates (Lin and Ghosh, 1996; Levitskaya et al., 1997; Sharipo et al., 2001; Hoyt et al., 2006; Daskalogianni et al., 2008; Too et al., 2013; Kraut, 2013; Vass and Chien, 2013). Abortive unfolding caused by Gly-rich motifs occurs as a result of slower domain unfolding rather than rapid substrate dissociation, suggesting that these motifs bind normally but are gripped poorly during unfolding (Kraut, 2013; Kraut et al., 2012). These results suggest that AAA+ motors struggle to efficiently grip sequences with very small side chains. Alternatively, sequence complexity rather than composition may dictate grip strength (Tian et al., 2005).

Here, we use green fluorescent protein (GFP) reporter substrates to interrogate the contributions of individual amino acids in the peptide tail to grip strength by ClpXP. In degradation assays performed in vivo and in vitro, we observe that substrate grip by ClpX is primarily mediated by interactions with a block of five amino acids, located two to six residues from the native GFP domain. Through systematic mutation, we characterize the ability of each amino acid to promote ClpX grip, and find that aromatic and large hydrophobic residues are gripped well, whereas charged and polar residues impair grip. Finally, we analyze synergistic contributions of multiple residues to unfolding, and show that contacts with more than one side chain lead to stronger grip and faster substrate unfolding. Our results provide unprecedented detail into the mechanism by which AAA+ motors grip terminal substrate tails during protein unfolding.

To unfold target proteins, ClpX and other AAA +protein remodeling machines use cycles of ATP binding and hydrolysis to pull on the degron tail of a substrate, thereby transmitting force to the native domain, but how these machines interact with individual tail residues during unfolding was poorly understood. Here, we identify and quantify the abilities of different tail residues to promote substrate grip during unfolding by ClpXP. Our experiments are enabled by the observation that placing 12 Gly residues between native GFP and a degron eliminates ClpXP degradation in vitro and markedly slows degradation in vivo. GFP unfolding/degradation was not inhibited by 12-residue sequences containing mixtures of Gly and Ala (GA cassette); Gly, Lys, and Arg (basic cassette); or Gly, Asp, and Glu (acidic cassette). Compared with these sequences, ClpXP probably grips Gly₁₂ poorly because of the absence of β-carbons and distal side-chain atoms or increased backbone flexibility. We use ‘grip’ in a functional rather than strictly physical sense, although the two concepts are undoubtedly related.

Ensemble and single-molecule experiments show that ClpXP can translocate an enormous number of different amino-acid sequences, including long Gly tracts, with only minor velocity differences (Aubin-Tam et al., 2011; Maillard et al., 2011; Sen et al., 2013; Cordova et al., 2014; Barkow et al., 2009). For example, in assays requiring ATP-dependent translocation, ClpXP degraded peptide substrates containing Gly₁₀, [Val-Gly]₅, or [Phe-Gly]₅ sequences at similar rates (Barkow et al., 2009). However, if ClpX can translocate poly-Gly sequences, then why does Gly₁₂ inhibit or slow unfolding/degradation? When ClpX pulls on a native protein, Newtonian mechanics dictate that the folded domain resists with an opposing force, which would be absent during translocation of an unstructured polypeptide. Hence, when ATP hydrolysis is coupled to molecular motion during an unfolding power stroke, we imagine that ClpX's grip on the Gly₁₂ sequence is insufficient to resist the opposition of the folded domain, causing an unproductive power stroke in which the pore-1 loops slip and fail to advance the substrate tail. In support of this model, we find that poor grip correlates with substantial increases in the ATP cost of degradation for the position-4 and Tyr-scan variants, an indication of slipping and futile power strokes. For example, degradation of one molecule of the Tyr-3, Tyr-4, and Tyr-5 substrates required hydrolysis of an average of ~180–240 ATPs, whereas degradation of the Tyr-2 or Tyr-6 substrates required hydrolysis of ~1900 and~3700 ATPs, respectively. These findings corroborate a previous report of futile power strokes during unsuccessful unfolding of a difficult substrate by ClpXP (Kraut, 2013). Furthermore, substrate-tail contacts with the axial pore that stimulate ATP hydrolysis by ClpX do not fully overlap with the contacts that determine grip.

A Tyr-scan experiment shows that tail-position 4 is most important for grip, with flanking positions showing diminishing effects. We expect that amino-acid substitutions at positions 3 and 5 would show side-chain grip trends similar to those observed at position 4. This is not true at tail-position 1, where Tyr did not improve grip but Ala did, perhaps because this part of the tail interacts with different residues in ClpX or the folded GFP domain than downstream positions. A single Ala at tail-position four is gripped poorly but a second Ala at certain positions can improve unfolding/degradation. Contacts between the second Ala and the ClpX pore may contribute to stronger grip. Alternatively, the second Ala might affect ClpX contacts made by the first Ala by altering the substrate conformation.

In our GFP substrate with a single Tyr at tail-position 4, this side chain is likely to contact a pore-1 loop close to the folded GFP domain. In an extended chain, four residues would span ~12 Å, a distance that modeling suggests would allow interaction with either the highest or second highest pore-1 loop of ClpX (Figure 5A; Puchades et al., 2017; X. Fei, personal communication). This is also an area where the axial channel is most tightly constricted around substrate. The distribution of Tyr-effects at positions 2–6 could reflect interactions with different pore-1 loops or the probability that Tyr side chains at different positions contact one specific pore-1 loop. Optical trapping studies indicate that 5–8 residues are moved by a single ClpXP power stroke (Aubin-Tam et al., 2011; Maillard et al., 2011). Thus, once the Tyr side chain at position 4 is engaged by a pore-1 loop, one successful translocation event probably unfolds GFP. Indeed, although many unsuccessful power strokes and ATP hydrolysis events occur while ClpXP is attempting to unfold a stable domain, hydrolysis of a single ATP ultimately results in unfolding. We find it notable that the Tyr-scan distribution is only two residues wide at half height. Hence, one tyrosine at position 4 mediates robust unfolding, whereas one tyrosine at position 7 has no effect. If a position-4 side chain can contact a pore-1 loop high in the ClpX pore, then a position-7 side chain should be able to contact another pore-1 loop lower in the pore. If this model is correct, then it implies that physical contacts between substrate tail residues and the upper pore-1 loops of ClpX are far more important for grip than interaction with the lower loops.

Figure 5

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We can imagine several different mechanisms for the asymmetry in grip between pore-1 loops in the upper and lower sections of the ClpX pore. In one model, the stronger grip of upper pore-1 loops occurs because these loops maintain relatively static interactions with substrate throughout a power stroke (Figure 5B, left). Several translocation models have been recently proposed for AAA+ unfoldases in which ATP-bound subunits with pore-1 loops oriented near the top of the pore move together as a rigid unit in response to ATP hydrolysis in a lower subunit (Monroe et al., 2017; Puchades et al., 2017; de la Peña et al., 2018; Dong et al., 2019). Furthermore, a previous study demonstrated that pore-1 loop mutations disrupt substrate unfolding most dramatically when they are in neighboring ClpX subunits, consistent with grip mediated by a clustered subset of pore-1 loops (Iosefson et al., 2015). Alternatively, the substrate tail in the pore could absorb some unfolding force through elastic expansion, diverting part of the energy of each power stroke away from unfolding (Figure 5B, right). Substrate interactions with the uppermost pore-1 loops would minimize the expansion length of the substrate tail, whereas interactions with lower pore-1 loops would allow the tail to absorb more force.

In an otherwise all-Gly cassette context, we find that Val, Ile, Leu, Met, Phe, and Tyr at tail-position 4 all promote reasonable levels of grip. If we think of poly-Gly as a smooth and relatively featureless rope, then these larger and generally non-polar side chains can be viewed as knots in the rope that afford better grip. However, grip is not a simple function of side-chain size. For example, Trp supports slower GFP unfolding than the better-gripped residues, suggesting that there may be an upper limit on the size of a side chain that can be efficiently gripped. Polar atoms, especially those close to the peptide backbone of the substrate, weaken grip. For example, Val is one of the best residues in terms of grip, whereas Thr, which differs only by substituting a hydroxyl for a methyl group, barely supports unfolding. Similarly, Ser alone does not support GFP unfolding, but removing the hydroxyl group (Ala) or substituting a less-polar thiol group (Cys) restores low-level unfolding activity. ClpXP may grip polar side chains less tightly because oxygen or nitrogen atoms bearing partial or full charges are not fully solvated when they are in productive contact with a pore-1 loop and thus incur an energetic penalty. Our finding that large hydrophobic and aromatic side chains are gripped well by ClpX is consistent with a model in which van der Waal’s or hydrophobic interactions between the pore-1 loops and specific side chains in the tail are largely responsible for grip.

Several recent cryo-EM structures of AAA+ proteases and protein-remodeling motors reveal a spiral arrangement of subunits in which aromatic residues in the pore-1 loops interact with substrate side chains spaced two residues apart (Monroe et al., 2017; Gates et al., 2017; Puchades et al., 2017; de la Peña et al., 2018; Majumder et al., 2019; Dong et al., 2019; White et al., 2018). Our observation that substrate tails with two tyrosines can in some cases specifically enhance grip when spaced by a multiple of two residues is consistent with these structures. However, substrates with two Val residues do not exhibit the same periodic grip enhancement as Tyr, and multiple Ala residues together promote strong grip regardless of their relative spacing. It is clear that nonspecific interactions between the axial pore and substrate side chains are sufficient to promote strong grip independent of precise pore-1 loop intercalation. In specific cases, periodic side chain intercalation could enhance grip for aromatic side chains through the establishment of π-stacking networks with the pore-1 loop Tyr residues, possibly by optimizing the bound substrate conformation.

A recent cryo-EM structure of the AAA+ motor NSF, which disassembles SNARE complexes following vesicle fusion, contains well-resolved density for substrate side chains, revealing interactions with the pore-1 loop tyrosine (White et al., 2018). Although NSF disassembles SNARE complexes in a single round of ATP turnover in a mechanism distinct from ClpX (Ryu et al., 2015), the structural similarities between NSF and many AAA+ unfoldase proteases suggests a common mode of substrate interaction and grip. The assembled SNARE complex is remarkably stable, and NSF likely requires strong grip to disassemble the complex. Consistent with our biochemical observations for ClpX, this structure indicates that the strongest substrate contacts are formed with pore-1 loops high in the upper ring of NSF, and that two substrate residues (Met and Leu) that are gripped well by ClpX participate in these interactions.

A previous study found that a Gly₁₅ sequence placed between GFP and an ssrA tag did not slow ClpXP degradation (Barkow et al., 2009). However, their substrate contained six additional residues (Thr¹-His²-Gly³-Met⁴-Asp⁵-Glu⁶) between folded GFP and the Gly₁₅ sequence. As we find that Met at position four supports robust unfolding, it is likely that interactions with the extra sequence mediate unfolding of the Gly₁₅ substrate. Our findings suggest that evolutionary placement of tail residues that are gripped well by ClpX may tune degradation of substrates that unfold non-cooperatively or that have multiple-folded domains. Complete, processive unfolding of multi-domain substrates depends critically on interactions between ClpX and the peptide-tail remnants from unfolding and degradation of the previous domain. For example, ClpXP degradation of Domain III of C. crescentus DnaX is inhibited by a Gly-rich sequence between Domains III and IV, which acts as a partial processing mechanism essential for DNA replication (Vass and Chien, 2013). Gly-rich tracts also inhibit unfolding/degradation of E. coli DHFR, although multiple alanines in the tail do not improve degradation of this substrate (Too et al., 2013). Thus, ClpX unfolding of different native substrates probably requires different degrees of grip strength, which could be mediated by more and/or better-gripped amino acids adjacent to the folded domain.

ClpXP contains just two types of subunits, whereas the 26S proteasome consists of more than 30 subunit types (Budenholzer et al., 2017). Nevertheless, our work is reminiscent of and reinforces studies of proteasomal degradation by Matouschek and colleagues. For example, they find that low-complexity sequences primarily composed of Gly, Ser, or Thr residues can inhibit proteasomal degradation (Tian et al., 2005); these residues individually are also insufficient to promote ClpXP degradation of GFP. Similarly, sequences that include Phe and Tyr residues can improve or rescue degradation by both the proteasome and ClpXP. These similarities may arise because the Rpt_1-6 unfolding ring of the proteasome, despite containing six distinct subunits, has pore-1 loops very similar to those of ClpX. Given the structural similarities between many AAA+ protein remodeling machines, we expect that the principles underlying grip in ClpX reflect those of the broader family.

All data generated during this study are included in the manuscript and supporting files as Tables 1 and 2 and Figure 2 - source data 1.

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Author details

1. Tristan A Bell

   Department of Biology, Massachusetts Institute of Technology, Cambridge, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   tribell@mit.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3668-8412

2. Tania A Baker

   1. Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
   2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States

   Contribution

   Supervision, Funding acquisition

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0737-3411

3. Robert T Sauer

   Department of Biology, Massachusetts Institute of Technology, Cambridge, United States

   Contribution

   Supervision, Funding acquisition, Project administration, Writing—review and editing

   For correspondence

   bobsauer@MIT.EDU

   Competing interests

   No competing interests declared

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