JBC Papers in Press. Published on February 21, 2017 as Manuscript M116.768978 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.768978 1

Covalently Linked HslU Hexamers Support a Probabilistic Mechanism that

Links ATP Hydrolysis to Protein Unfolding and Translocation

Vladimir Baytshtok1, Jiejin Chen1, Steven E. Glynn1, Andrew R. Nager1, Robert A. Grant1,

Tania A. Baker1,2, and Robert T. Sauer1* Downloaded from

1Department of Biology and 2Howard Hughes Medical Institute, Massachusetts Institute of

Technology, Cambridge, MA 02139 http://www.jbc.org/

by guest on March 12, 2017 current addresses: J.C., Takeda Pharmaceuticals, Cambridge, MA; S.E.G., Dept. of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794; A.R.N., Dept. of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305.

* corresponding author: [email protected] running head: Disulfide-linked HslU pseudohexamers keywords: AAA+ protease; mixed hexameric rings; protein unfolding; protein degradation;

HslUV

Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. 2

ABSTRACT

The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic-tethering and disulfide-bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring.

Genetic tethering impairs HslV binding and Downloaded from degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, http://www.jbc.org/ pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein

substrates and/or collaborate with HslV in by guest on March 12, 2017 their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism. 3

Enzymes of the AAA+ ATPase superfamily These results suggest that the six subunits of play roles in proteolysis, protein remodeling HslU assume non-equivalent functional and disaggregation, replication, roles within the hexamer and are more transcription, membrane fusion, vesicle consistent with sequential or probabilistic transport, and other cellular processes in all models. Here, we test different models by organisms (1-2). These enzymes share which ATP hydrolysis could power the conserved sequence and structural motifs mechanical functions of E. coli HslU by and typically function as homohexameric or introducing hydrolytically inactive subunits heterohexameric rings. Fueled by the energy at defined positions in its hexameric ring. of ATP binding and hydrolysis, AAA+ Using subunit crosslinking strategies enzymes act as molecular machines that involving genetic tethering or disulfide disassemble, remodel, or denature bonding, we find that HslU pseudohexamers macromolecule targets. There are three with mixtures of hydrolytically active and general models for how subunits in AAA+ inactive subunits retain protein unfolding hexamers hydrolyze ATP and generate the activity and support HslV degradation. mechanical power strokes required for These studies support a probabilistic Downloaded from function: (i) concerted ATP hydrolysis that mechanism in which ATP hydrolysis powers occurs simultaneously in all subunits (3); (ii) mechanical function in the HslU ring and sequential hydrolysis by individual subunits also reveal new information about that occurs in an invariant kinetic pattern interactions between HslU and HslV. http://www.jbc.org/ (4); and (iii) probabilistic hydrolysis in which following a power stroke, any ATP- bound subunit has some chance of RESULTS hydrolyzing ATP to drive the next power HslU dimers with covalent peptide stroke (5). by guest on March 12, 2017 tethers. HslU homohexamers containing the The HslUV protease consists of one or two Walker-B E257Q mutation are defective in AAA+ HslU hexamers and the dodecameric ATP hydrolysis and protein degradation but HslV peptidase (Fig. 1A; refs. 6-12). In retain the ability to bind HslV and protein ATP-dependent reactions, HslU hexamers substrates (13). We engineered genes to recognize protein substrates, unfold any encode tandem E. coli HslU subunits native structure that is present, and then connected by a 20-residue peptide tether translocate the unfolded polypeptide into the (Fig. 1B). One encoded dimer consisted of luminal chamber of HslV for degradation. In two wild-type subunits (W-W), another had all crystal structures of the Escherichia coli a wild-type subunit followed by a E257Q or Haemophilus influenza HslUV complexes subunit (W-E), and a third had a E257Q and some structures of HslU alone, the HslU subunit followed by a wild-type subunit (E- hexamer is highly symmetric and binds six W). These dimers behaved like wild-type ATP or ADP molecules, as might be HslU during purification, suggesting that expected for a concerted mechanism of they form W-W3, W-E3, and E-W3 hydrolysis (6-12). However, in other pseudohexamers. Indeed, the asymmetric structures of HslU alone, only three or four unit of a low-resolution W-E3 crystal nucleotides are bound to the hexameric structure contained four hexamers similar to HslU ring (6), and solution experiments wild-type HslU (Fig. 1C; Table 1), although show detectable binding of a maximum of 3 electron density for the C-terminal 8-10 to 4 nucleotides and the existence of at least residues was missing in alternating subunits two types of nucleotide-binding sites (13). of several hexamers or was generally poor 4 throughout a hexamer, as expected if the algorithm (18) to identify Glu47/Ala349 and tether disrupts normal C-terminal contacts. Gln39/Thr361 as sites for potential disulfide bonds across the rigid-body interfaces of an HslU hexamer. Fig. 3A shows a model of an W-W3 hydrolyzed ATP at about twice the otherwise Cys-free HslU pseudohexamer in rate of the W-E3 and E-W3 enzymes (Fig. which red subunits contain Cys47 and blue 2A), suggesting that ATP hydrolysis is subunits contain Cys349, potentially allowing largely restricted to the W subunits in these formation of three disulfide-linked dimers. enzymes. We constructed and expressed a SS 47 To make W W3 pseudohexamers, the Cys W-W-W trimer, but this protein was and Cys349 subunits both had wild-type insoluble. To assay protein unfolding, we SS I37A cp6 Walker-B ATPase motifs. To make W E3 constructed an Arc- GFP-st11-ssrA pseudohexamers, the Cys47 subunit had a fusion protein with a thrombin cleavage site wild-type Walker-B sequence and the Cys349 located between β-strands 5 and 6 (14). subunit contained the Walker-B E257Q I37A Arc is a denatured variant of Arc mutation to inactivate ATP hydrolysis. Fig. repressor that targets the substrate to HslU, 3B shows a pseudohexamer in which red Downloaded from and the C-terminal st11-ssrA sequence subunits contain Cys349, green subunits increases the substrate turnover rate ~2-fold contain Cys47 and Cys361, and blue subunits (15, 16). Following thrombin cleavage, contain Cys39. In this configuration, unfolding of the split substrate by wild-type formation of disulfide-linked trimers is http://www.jbc.org/ HslU results in an irreversible loss of GFP possible. We designed WSSWSSW trimers, fluorescence. In experiments performed at WSSESSW trimers, and WSSESSE trimers by different concentrations of the split changing which subunits had wild-type or substrate, unfolding by wild-type HslU and E257Q Walker-B sites.

W-W3 occurred with steady-state Vmax rates by guest on March 12, 2017 that were similar, whereas Vmax for unfolding by the W-E3 and E-W3 Relatively efficient formation of disulfide- pseudohexamers was about half of the wild- linked HslU dimers or trimers was achieved type value (Fig. 2B). Thus, pseudohexamers by cytosolic coexpression of appropriate with alternating ATPase active and inactive variants in the oxidizing SHuffle strain of E. subunits retain substantial protein-unfolding coli (19). For example, following activity. However, compared to wild-type purification by Ni-NTA affinity HslU, W-W3 supported HslV degradation chromatography, non-reducing SDS-PAGE very poorly (Fig. 2C) and bound HslV ~20- showed ~50% formation of WSSW and fold more weakly (Table 2), probably WSSE and ~33% formation of WSSWSSW, because the peptide tether interferes with WSSESSW, and WSSESSE (not shown). To contacts between HslU and HslV (see further purify disulfide-linked Discussion). Thus, we explored a different pseudohexamers, we performed ion- method of constructing covalently linked exchange chromatography and gel-filtration HslU hexamers. chromatography once in the presence and once in the absence of urea, which destabilizes unlinked HslU hexamers more Construction of disulfide-crosslinked than linked hexamers. Following the final HslU pseudohexamers. HslU hexamers chromatography step, the WSSW, WSSE, consist of rigid-body units formed by the WSSWSSW, WSSESSW, and WSSESSE large and small domains of adjacent subunits variants had purities of >95% (Fig. 3C). The (17). We used the disulfide-by-design disulfide-linked pseudohexamers bound 5

SS SS HslV slightly more tightly than wild-type (Fig. 5A). Importantly, W E W2 and SS HslU (Table 2). W E3 also supported degradation at 35-45% of the wild-type rate, demonstrating that pseudohexamers with only three or four ATP hydrolysis by disulfide-linked hydrolytically active subunits also have pseudohexamers. Like the basal ATP- substantial degradation activity. Degradation hydrolysis activities of the genetically- SS SS supported by W E E2 proceeded very tethered pseudohexamers, those of the slowly, at ~3% of the wild-type rate. disulfide-linked enzymes were roughly proportional to the number of hydrolytically active W subunits in each pseudohexamer The degradation defects caused by E (Figs. 4A, 4B). Compared to wild-type subunits in pseudohexamers were more SS SS SS cp6 HslU, however, the W W3 and W W W2 severe for Arc- GFP-st11-ssrA, a stable pseudohexamers were ~3-fold more substrate that wild-type HslUV degrades ~5- SS hydrolytically active (Fig. 4A), possibly as a fold more slowly than Arc-cysA. W W3 SS SS consequence of small conformational and W W W2 supported HslV degradation Downloaded from changes stabilized by the disulfide bonds. In of a high concentration of Arc-cp6GFP-st11- SS SS the presence of HslV, the ATPase rate of ssrA at near wild-type rates, but W E W2 SS wild-type HslU was stimulated ~3-fold, as had only ~20% activity, W E3 had only SS SS previously observed (13, 20, 21), ATP ~10% activity, and W E E2 was inactive SS SS SS http://www.jbc.org/ hydrolysis by W W3 and W W W2 was (Fig. 5B). Degradation rates were stimulated ~2-fold, but ATP hydrolysis by normalized by dividing by the number of SS SS SS SS SS W E3, W E W2, or W E E2 was not wild-type subunits in HslU or different markedly stimulated (Fig. 4C). Thus, the variants and are plotted in Fig. 5C for the presence of E subunits suppressed normal Arc-cysA substrate and Fig. 5D for the Arc- by guest on March 12, 2017 HslV stimulation of ATP hydrolysis by cp6GFP-st11-ssrA substrate. For Arc-cysA, HslU pseudohexamers. there was a sharp discontinuity between 2 and 3 wild-type subunits. For Arc-cp6GFP- st11-ssrA, by contrast, the major Degradation supported by disulfide- discontinuity was between 4 and 6 subunits. linked pseudohexamers. Arc repressor, a good substrate for HslUV degradation, is a metastable dimer that unfolds/dissociates To determine the energetic efficiency of with a half-life of ~10 s but refolds in degradation of Arc-cp6GFP-st11-ssrA, we milliseconds to maintain a predominantly assayed the rate of ATP-hydrolysis for each native structure (15, 22). We assayed the disulfide-linked pseudohexamer in the ability of different disulfide-linked presence of HslV and Arc-cp6GFP-st11-ssrA pseudohexamers to support HslV (Fig. 5E). We then divided the ATPase rate degradation of Arc-cysA, where cysA by the degradation rate to determine the designates a unique cysteine labeled with an average number of ATPs hydrolyzed during Alexa-488 fluorophore (23), as auto- degradation of a single substrate (Fig. 5F). SS SS SS SS quenching of the fluorophores in the native Notably, W W3, W E3, W W W2, and protein is relieved upon degradation. The SS SS W E W2 all had similar energetic SS SS SS W W3 and W W W2 pseudohexamers efficiencies, hydrolyzing ~500 ± 100 ATPs supported HslV degradation of a near- for each substrate degraded. Assuming that saturating concentration of Arc-cysA at rates power strokes are tightly coupled to ATP comparable to the wild-type HslU hexamer hydrolysis, this result suggests that the 6

SS SS SS W E W2 and W E3 pseudohexamers use bridge with a HslV lysine side chain (7, 11, approximately the same number of power 12). In E. coli HslUV structures, by contrast, strokes as hexamers with six wild-type the tails remain docked into HslU (8, 9). subunits to unfold and translocate Arc- Moreover, deletion of the five C-terminal cp6GFP-st11-ssrA. Thus, the slower residues of E. coli HslU does not prevent SS SS degradation activities of the W E W2 and stimulation of HslV peptidase activity or SS W E3 enzymes compared to degradation (20). Nevertheless, peptides pseudohexamers with only wild-type corresponding to the C-terminal residues of subunits are principally a consequence of HslU activate peptide cleavage by E. coli their slower rates of ATP hydrolysis. HslV, and mutations in HslV predicted to disrupt contacts with the C-terminal tails prevent HslU activation (20, 24). Our results DISCUSSION support the importance of the C-terminal tails in high-affinity HslV binding and Our studies show that E. coli HslU variants indicate that more than three tails of E. coli containing hydrolytically active and inactive HslU must interact optimally with HslV to Downloaded from subunits at specific positions in the allow tight binding and efficient proteolysis. hexameric AAA+ ring can hydrolyze ATP, unfold proteins, and degrade substrates in collaboration with HslV. As we discuss The pseudohexamers we studied have basal below, these results support a probabilistic ATP hydrolysis rates roughly proportional http://www.jbc.org/ model of ATP hydrolysis and provide to their total number of hydrolytically-active insights into the multivalent interactions W subunits. This result supports a model in between HslU and HslV that are required for which the W subunits in these

efficient protein degradation. pseudohexamers contribute independently to by guest on March 12, 2017 basal ATPase activity, making concerted or strictly sequential models unlikely. For Genetic tethering allowed us to express and example, if ATP hydrolysis in a specific purify HslU pseudohexamers consisting of a subunit required prior hydrolysis in a trimer of linked dimers. However, the W-W3 neighboring subunit, as expected in a strictly enzyme binds HslV poorly, suggesting that sequential model, then a non-linear the tether interferes with HslV binding. relationship between W subunits and ATP Consistently, disulfide-linked hydrolysis would be expected. HslV pseudohexamers bind HslV well. In crystal SS stimulates ATP hydrolysis by W W3 and structures of HslU hexamers alone, the C- SS SS W W W2 but caused little change in terminal tails dock into a pocket and the - SS SS SS α hydrolysis by W E W2, W E3, or SS SS carboxyl group forms a salt bridge with an W E E2. As the E subunits in these latter arginine in the sensor-2 motif of the same enzymes also increase pseudohexamer subunit (6, 10). These tail interactions were affinity for HslV, stronger interactions disrupted in several subunits in our low- between E subunits and HslV might restrict resolution structure of W-E3 HslV-induced conformational changes pseudohexamers, as expected if the attached required for higher ATPase activity in tether prevents proper packing of these neighboring W subunits and thus explain the residues. In the H. influenza HslUV lack of ATPase stimulation. Alternatively, complex, the C-terminal tails are detached HslV binding might slow ATP dissociation from HslU and pack into grooves on HslV, from inactive E subunits, which becomes with the HslU α-carboxylate forming a salt rate-limiting for hydrolysis in W subunits, 7

especially if only a subset of subunits is The tethered W-E3 and E-W3 HslU nucleotide bound at any given time. pseudohexamers have ~50% of the protein unfolding activity of the parental W-W3 SS SS enzyme, and the disulfide-linked W E W2 Our strategies for engineering HslU rings SS and W E3 enzymes support HslV with defined mixtures of active and inactive degradation of an easily degraded Arc-CysA subunits were motivated by prior studies that substrate at 35-45% of the parental rates. applied these subunit-crosslinking methods Thus, protein unfolding and translocation by to different hexameric AAA+ unfoldases the AAA+ ring of HslU do not require ATP and remodeling machines (5, 25-27). Indeed, hydrolysis in adjacent subunits or in genetic-tethering experiments originally subunits immediately across the ring from showed that ClpX also operates by a each other. Again, these results support a probabilistic mechanism, as ClpX model in which probabilistic hydrolysis in pseudohexamers containing different the AAA+ ring powers unfolding and combinations of hydrolytically active and translocation. Slower rates of ATP inactive subunits unfold and degrade protein hydrolysis in rings with mixtures of active Downloaded from substrates in collaboration with the ClpP and inactive subunits correlate with their protease (5). Prior to the current study, reduced mechanical activities, as the ATP however, it was not obvious that HslU and cost of degradation of the more stable Arc- ClpX would operate using similar cp6GFP-st11-ssrA substrate is similar for the probabilistic mechanisms of ATP- SS SS SS http://www.jbc.org/ W E W2 and W E3 pseudohexamers and hydrolysis. First, HslU and ClpX contain their disulfide-linked parental enzymes unique family specific auxiliary domains. containing six W subunits. Although optimal The I-domain of HslU emerges from the top rates of ATP hydrolysis, unfolding, and of the AAA+ ring, between the Walker-A degradation require six hydrolytically active by guest on March 12, 2017 and Walker-B ATPase motifs, and regulates HslU subunits, rings with only three or four ATP hydrolysis, degradation, and active subunits use approximately the same autoinhibition (9, 20, 23). The N-domain of number of power strokes to degrade this ClpX, by contrast, serves as a docking site substrate. for some adaptors/substrates but its deletion has little effect on ATP hydrolysis or SS SS degradation of many ClpXP substrates (28). We note that the W E E2 pseudohexamer SS Second, in crystal structures, the large and hydrolyzes ATP at ~40-60% of the W E3 small AAA+ domains of each HslU subunit rate but supports no degradation of Arc- assume orientations that create a potential cp6GFP-st11-ssrA. These results suggest that nucleotide binding site, whereas the some subunit-subunit communication is corresponding domains of ClpX adopt required for efficient unfolding and SS SS structures that allow nucleotide binding in degradation by W E E2 and/or that a some subunits but prevent binding in other minimum rate of ATP hydrolysis is required subunits (6-12, 29). Third, HslU hexamers to unfold Arc-cp6GFP-st11-ssrA. make symmetric interactions with hexameric Probabilistic models of ATP hydrolysis do rings of HslV, whereas ClpX hexamers not exclude coordination between ring make asymmetric interactions with subunits for efficient ATP hydrolysis, heptameric rings of ClpP (30). substrate binding, or mechanical function. In fact, the rate of ATP hydrolysis and the degree of substrate binding by HslU change in a positively cooperative fashion with ATP 8 concentration, as expected for functional the E257Q mutation. Cysteine-free HslU linkage between different subunits (13). In supports robust ATP hydrolysis and the AAA+ ring of ClpX, communication substrate degradation (38-40). To make between subunits is necessary to allow disulfide crosslinked dimers, a gene staged ATP binding to drive conformational encoding untagged E47CHslU was cloned into changes needed for function (31-33). the first multiple cloning site (MCS1) of the Moreover, optical-trapping experiments pCOLADuet-1 (Novagen) vector between reveal that kinetic bursts of power strokes in the NcoI and BamHI sites and a gene A349C the ClpX ring result in random patterns of encoding His6-ENLYFQS- HslU was shorter and longer translocation steps, cloned into MCS2 between the NdeI and supporting a probabilistic but coordinated XhoI sites, where His6 is the hexahistidine mechanism (34-35). Similar themes of tag and ENLYFQS is the sequence probabilistic hydrolysis but coordinated recognized and cleaved by the TEV function are seen in AAA+ unfolding rings protease. E47CHslU had a glycine residue assembled from non-identical subunits. In inserted after the initiator methionine as a the Yta10/Yta12 m-AAA protease, for result of cloning. This pCOLADuet-1 vector Downloaded from example, mutating the Walker-A motif of was transformed into the of E. coli SHuffle either the Yta10 or the Yta12 subunits does T7 Express strain (New England Biolabs) for not affect hydrolysis in the remaining wild- expression. To make disulfide crosslinked type subunits, but Walker-B mutations in trimers, a gene encoding untagged http://www.jbc.org/ Yta12 trap ATP and prevent robust A349CHslU was cloned into MCS1 of hydrolysis in adjacent Yta10 subunits (36). pCOLADuet-1 between the NcoI and Finally, in the six distinct subunits of the BamHI sites and a gene encoding His6- E47CT361C Rpt1-6 AAA+ ring of the 26S proteasome, ENLYFQS- HslU was cloned into

Walker-B mutations in individual subunits MCS2 between NdeI and XhoI sites. This by guest on March 12, 2017 have a wide range of effects on ATP vector was co-transformed with a pet12b hydrolysis and mechanical activity, (Novagen) vector encoding the untagged Q39C requiring a model with some subunit-subunit HslU variant into the SHuffle T7 coordination within the context of an Express strain. Both A349CHslU and inherently probabilistic mechanism of ATP Q39CHslU contained an additional glycine hydrolysis (37). after the initiator methionine as a result of cloning. A gene encoding the Arc repressor from phage P22 followed by a cysteine EXPERIMENTAL PROCEDURES residue and a hexahistidine tag (Arc-cys- His6) was cloned and expressed in a pet21b Cloning, expression, and protein vector (Novagen). Wild-type HslU and HslV purification. Mutants were constructed and were expressed from pet12b vectors. Arc- cloned by standard PCR techniques unless cp6GFP-st11-ssrA, and I37AArc-cp6GFP- noted. To construct genetically linked HslU β5/β6-st11-ssrA (a dimers, a gene encoding two HslU subunits GGTEGSLVPRGSGESGGS sequence separated by the 20-residue between β-stands 5 and 6 allows thrombin ASGAGGSEGGGSEGGTSGAT linker was cleavage and generation of a split substrate), cloned into the pet11a vector (Novagen). and Arc-st11-ssrA were expressed from HslU mutants used to make disulfide- pet21b vectors as described (14, 15, 23). crosslinked pseudohexamers were constructed in the cysteine-free C262A/C288SHslU background with or without 9

Genetically linked HslU dimers were 200 16/60 column equilibrated in buffer F expressed and purified as previously (50 mM Tris, pH 7.5, 300 mM NaCl, 10% described for wild-type HslU (20). Disulfide (v/v) glycerol, 1 mM EDTA). Fractions with crosslinked HslU dimers were expressed and the highest purity of crosslinked dimer were purified as follows. E. coli SHuffle T7 pooled and concentrated. The concentration Express cells carrying the pCOLA-Duet1 of crosslinked dimer was determined in vector coding for the appropriate HslU hexameric equivalents by measuring the ° -1 - mutants were grown at 30 C until OD 0.6- absorbance at 280 nm using 148,545 M cm 0.8, the temperature was shifted to 18 °C, 1 as the molar extinction coefficient. and protein expression was induced with 0.5 Concentrated protein was divided into small mM IPTG for 20 h. Cells were pelleted and aliquots and was flash frozen at –80 °C. resuspended in buffer A (50 mM Tris, pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.5 mM EDTA), and 0.5 tablets of Complete Disulfide-crosslinked HslU trimers were Ultra EDTA-free protease-inhibitor cocktail expressed and purified largely as described

(Roche) and 1.5 µL benzonase (250 for crosslinked dimers. His6 tags were Downloaded from 349 units/µL, Sigma) per liter of the original present on Cys subunits for dimers and 47 361 culture were added. Cells were sonicated, Cys /Cys subunits for trimers. After the lysate was cleared by centrifugation, and precipitation with PEI, the sample was 0.1% v/v polyethyleneimine (PEI) was cleared by centrifugation and the http://www.jbc.org/ added to the supernatant. This precipitate supernatant was loaded onto a 5 mL HisTrap was cleared by centrifugation and the HP column (GE Healthcare) equilibrated in supernatant was loaded onto Ni++-NTA buffer B. The column was washed with 75 beads equilibrated in buffer B (50 mM Tris, mL of buffer B, the sample was eluted with pH 7.5, 300 mM NaCl, 20 mM imidazole). a gradient of 20 to 500 mM imidazole in by guest on March 12, 2017 The Ni++-NTA beads were washed buffer B (100 mL total), and 2 mL fractions extensively with buffer B and protein was were collected. Fractions were pooled and eluted with buffer C (50 mM Tris, pH 7.5, dialyzed overnight against buffer G (50 mM 300 mM NaCl, 250 mM imidazole). The Tris, pH 7.5, 150 mM NaCl, 10% glycerol eluate was diluted 3-fold with buffer D (50 (v/v), 1 mM EDTA) at 4 °C. The dialyzed mM Tris, pH 7.5, 10% (v/v) glycerol, 1 mM material was loaded onto a Mono Q 10/100 EDTA), loaded onto a Mono Q 10/100 GL GL column and purification then proceeded column (GE Healthcare), and eluted with a by the method described for crosslinked linear gradient from 150 to 500 mM NaCl in dimers. The concentration of the crosslinked buffer D (120 mL total). Appropriate Mono trimer was determined in hexameric Q fractions were pooled, concentrated using equivalents by measuring absorbance at 280 -1 -1 an Amicon Ultra-15 centrifugal filter unit, nm using 147,180 M cm as the molar chromatographed on a Superdex 200 16/60 extinction coefficient. Concentrated protein column (GE Healthcare) equilibrated in was aliquoted and flash frozen at –80 °C. buffer E (50 mM Tris, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, 1.5 M urea, 1 mM A pet21b vector carrying Arc with a C- EDTA) and 1 mL fractions were collected. terminal CHHHHHH tail (Arc-cys-His ) Fractions with the highest purity of the 6 was transformed into the E. coli X90 (λDE3) crosslinked dimer as judged by non- slyD::kan hslUV::tet strain and cells were reducing SDS-PAGE were pooled, grown to an OD of 0.6-0.8 at 37 °C. concentrated, and run on another Superdex 600 Protein expression was induced by the 10 addition of 1 mM IPTG and continued for 4 (monomer equivalents) was measured by h at room temperature. Cells were monitoring the decrease in GFP resuspended, lysed, and Arc-cys-His6 was fluorescence on a Spectramax M5 plate purified by Ni++-NTA affinity reader (excitation 467 nm; emission 511 chromatography as described for nm). Degradation reactions contained 5 mM purification of crosslinked HslU dimers. ATP and a regeneration system consisting of ++ After Ni -NTA purification, protein was 16 mM creatine phosphate and 10 µg/mL concentrated and injected onto a Superdex creatine kinase. HslV activation assays were 75 16/60 column (GE Healthcare) performed at 25 °C in the presence of ATP equilibrated in buffer H (50 mM Tris, pH as described (13, 23), and K1/2 values were 7.5, 300 mM NaCl, 10% glycerol (v/v), 1 determined by fitting to a hyperbolic mM EDTA, 2 mM DTT). Fractions equation. containing the Arc protein were concentrated and flash frozen at –80 °C. Arc-cys-His6 was labeled with the maleimide-derivative of Crystallography. The W-E3

Alexa-488 (Thermo Fisher Scientific) to pseudohexamer was crystallized by the Downloaded from hanging-drop method using 100 mM Bis- generate Arc-cysA (23). His6-tagged wild I37A Tris (pH 5.8), 26% (w/v) PEG 3350, and type HslU, His6-tagged HslV, Arc- cp6GFP-β5/β6-st11-ssrA, Arc-cp6GFP-st11- 260 mM ammonium sulfate as the well solution. Molecular replacement using ssrA, and Arc-st11-ssrA were expressed and http://www.jbc.org/ purified as described (23). Phaser (42) was initially used to solve the structure using a 1HQY hexamer (17) as the search model. We then replaced each 1HQY Biochemical assays. Unless noted, assays subunit with a 5JI3 subunit (23) to improve were performed at 37 °C in PD buffer (25 geometry and used rigid-body refinement of by guest on March 12, 2017 mM HEPES, pH 7.5, 5 mM KCl, 10% individual domains, refinement of one B- glycerol (v/v), 20 mM MgCl2, 0.032% factor and TLS group per subunit, and very Igepal CA-630). Hydrolysis of 5 mM ATP tightly constrained positional refinement was measured using an NADH-coupled with torsional NCS constraints in Phenix assay (41) by monitoring the loss of (43). Coot (44) was used for model building, absorbance at 340 nm on a Spectramax M5 and MolProbity (45) was used to assess the plate reader (Molecular Devices). Cleavage geometry of the model. of I37AArc-cp6GFP-β5/β6-st11-ssrA with thrombin was performed as described (14). Acknowledgements. Supported by NIH Rates of unfolding of different grant AI-16892. T.A.B. is an employee of concentrations of thrombin-split I37AArc- cp6 the Howard Hughes Medical Institute. GFP-β5/β6-st11-ssrA were determined by changes in cp6GFP fluorescence (excitation 467 nm; emission 511 nm) in assays that Conflict of interest. The authors declare no contained 0.5 µM HslU or tethered variants conflict of interest. and 5 mM ATP. Degradation of 30 µM Arc- cysA (monomer equivalents) was measured on a Spectramax M5 plate reader by Author contributions. J.C. performed monitoring the increase in fluorescence experiments with HslU subunits linked by (excitation 480 nm; emission 520 nm). genetically encoded tethers. A.R.N. Degradation of 20 µM Arc-cp6GFP-st11-ssrA designed the split substrate for unfolding 11 experiments. S.E.G., R.A.G., and R.T.S. performed crystallographic experiments. V.B. performed all remaining experiments. V.B. and R.T.S. wrote the manuscript. All authors contributed to the design and interpretation of experiments and approved the final manuscript.

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20. Seong, I.S., Kang, M.S., Choi, M.K., Lee, J.W., Koh, O.J., Wang, J., Eom, S.H., 27. Yamasaki, T., Oohata, Y., Nakamura, T., and Chung, C.H. (2002) The C-terminal tails and Watanabe, Y.H. (2015) Analysis of the of HslU ATPase act as a molecular switch cooperative ATPase cycle of the AAA+ for activation of HslV peptidase. J. Biol. chaperone ClpB from Thermus thermophilus Chem. 277, 25976–25982 by using ordered heterohexamers with an 14 alternating subunit arrangement. J. Biol. 34. Sen, M., Maillard, R.A., Nyquist, K., Chem. 290, 9789–9800 Rodriguez-Aliaga, P., Pressé, S., Martin, A., and Bustamante C. (2013) The ClpXP protease unfolds substrates using a constant 28. Baker, T.A., and Sauer, R.T. (2012) rate of pulling but different gears. Cell 155, ClpXP, an ATP-powered unfolding and 636–646 protein-degradation machine. Biochim. Biophys. Acta 1823, 15–28 35. Cordova, J.C, Olivares, A.O., Shin, Y., Stinson, B.M., Calmat, S., Schmitz, K.R., 29. Glynn, S.E., Martin, A., Nager, A.R., Aubin-Tam, M-E. Baker, T.A., Lang, M.J., Baker, T.A., and Sauer, R.T. (2009) and Sauer R.T. (2014) Stochastic but highly Structures of asymmetric ClpX hexamers coordinated protein unfolding and reveal nucleotide-dependent motions in a translocation by the ClpXP proteolytic AAA+ protein-unfolding machine. Cell 139, machine. Cell 158, 647–658 744–756 Downloaded from

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40. Yakamavich, J.A. (2008) Control of Richardson, D.C., Richardson, J.S., HslUV protease function by nucleotide Terwilliger, T.C., and Zwart, P.H. (2010) binding and hydrolysis (Massachusetts PHENIX: a comprehensive Python-based Institute of Technology Ph.D. Thesis) system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 41. Nørby, J.G. (1988) Coupled assay of Na+,K+-ATPase activity. Methods Enzymol. 156, 116–119 44. Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. D 42. McCoy, A.J., Grosse-Kunstleve, R.W., Biol. Crystallogr. 66, 486–501 Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007) Phaser crystallographic software. J. Appl. 45. Chen, V.B., Arendall, W.B., III, Headd, Crystallogr. 40, 658–674 J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, Downloaded from J.S., and Richardson, D.C. (2010) 43. Adams, P.D., Afonine, P.V., Bunkoczi, MolProbity: all-atom structure validation for G., Chen, V.B., Davis, I.W., Echols, N., macromolecular crystallography. Acta

Headd, J.J., Hung, L.W., Kapral, G.J., Crystallogr. D Biol. Crystallogr. 66, 12–21 http://www.jbc.org/ Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J.,

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Figure legends contained 5 mM ATP and a regeneration system.

Figure 1. HslUV structure. (A) An HslU hexamer (cartoon representation) bound to Fig. 3. Design and purification of an HslV dodecamer (surface representation; disulfide-crosslinked HslU variants. (A) pdb code 1g3i). The large and small AAA+ Spheres show the positions of Cys47 domains of HslU and its C-terminal tails are (normally Glu) in red subunits and Cys349 colored blue, cyan, and red, respectively. (B) (normally Ala) in blue subunits of an HslU Tandem HslU subunits connected by a hexamer, suggesting that Cys47-Cys349 genetically encoded peptide tether. (C) disulfides would stabilize a pseudohexamer Three W-E3 hexamers in the asymmetric consisting of three linked dimers. (B) unit of structure 5TXV are shown in cartoon Spheres show the positions of Cys349 in red representation; the fourth hexamer is shown subunits, Cys47 and Cys361 (normally Thr) in Downloaded from in ribbon representation with electron green subunits, and Cys39 (normally Gln) in density from a composite omit map blue subunits. Disulfide-bond formation in contoured at 1 σ. this configuration would stabilize a

pseudohexamer consisting of two linked http://www.jbc.org/ trimers. (C) Non-reducing SDS-PAGE of Fig. 2. Activity of genetically tethered purified wild-type HslU, purified WSSW, pseudohexamers. (A) Rates of hydrolysis purified WSSE, purified WSSWSSW, purified of 5 mM ATP by the genetically tethered W- WSSESSW, and purified WSSESSE. In each by guest on March 12, 2017 W3, W-E3, and E-W3 pseudohexamers. case, 0.5 µM of the purified protein (in Values are averages (N ≥ 5) ± SD. (B) Rates hexamer equivalents) was loaded on the gel. of unfolding of different concentrations of The first lane contains molecular weight I37A cp6 thrombin-split Arc- GFP-β5/β6-st11- standards. ssrA by wild-type HslU or genetically- tethered variants. Lines are non-linear least- squares fits to the Michaelis-Menten Fig. 4. ATP hydrolysis by disulfide- equation. KM’s values for all enzymes were crosslinked variants. (A) Basal rates of 1-3 µM but were not well determined ATP hydrolysis by disulfide-crosslinked because of the small number of low- variants and wild-type HslU. Assays concentration data points. Average Vmax contained 0.3 µM HslU or pseudohexamers values ± SD calculated from the highest four and 5 mM ATP. Values are averages of at substrate concentrations were 0.155 ± 0.003 least three replicates ± 1 SD. (B) Basal ATP min-1 enz-1 (HslU), 0.143 ± 0.008 min-1 enz-1 hydrolysis rates for disulfide-crosslinked -1 -1 (W-W3), 0.0802 ± 0.007 min enz (E-W3), pseudohexamers plotted as a function of the -1 -1 and 0.0716 ± 0.006 min enz (W-E3). number of W subunits. (C) Rates of ATP Fitted Vmax values were 10-15% higher. (C) hydrolysis determined in the presence of 0.9 The kinetics of degradation of Arc-st11-ssrA µM HslV dodecamer (other conditions as in (10 µM) by HslV (10 µM) and HslU (0.3 panel A). The numbers above each bar µM) or W-W3 (0.3 µM) at 50 °C was represent the rate in the presence of HslV monitored by SDS-PAGE. Reactions divided by the rate in the absence of ATP. 17

Fig. 5. Degradation rates and energetic and 1 µM HslV. (F) Energetic efficiency efficiencies of disulfide-crosslinked determined by dividing the ATPase rates by variants. (A) Rates of degradation of Arc- the degradation rates. For panels A-D, cysA (30 µM) assayed by increased experiments were performed using 0.3 µM fluorescence. (B) Rates of degradation of HslU or variants and 0.9 µM HslV. For Arc-cp6GFP-st11-ssrA (20 µM) assayed by panels, E and F, experiments were decreased fluorescence. (C) Degradation performed using 0.5 µM HslU or variants rates for Arc-cysA from panel A were and 1 µM HslV. All assays contained 5 mM divided by the number of wild-type subunits ATP and were performed at 37 °C. Values in the HslU variant and plotted against this in panels A-E are averages (N ≥ 3) ± 1 SD. number. (D) Degradation rates for Arc- The error bars in panel F represent cp6GFP-st11-ssrA from panel B were divided propagated errors. In panels C and D, values by the number of wild-type subunits in the for the three variants with six wild-type HslU variant and plotted against this subunits were offset slightly on the x-axis to number. (E) Rates of ATP hydrolysis in the allow visualization of error bars. cp6 presence of 20 µM Arc- GFP-st11-ssrA Downloaded from http://www.jbc.org/ by guest on March 12, 2017 18

Table 1. Crystallographic statistics. PDB code 5TXV wavelength (Å) 0.979

space group P 1 21 1 unit-cell dimensions (Å) a = 86.5; b = 420.9; c = 176.5 unit-cell angles (°) α = γ = 90; β = 98.6 resolution range (Å) 49.2–7.1 (7.3–7.1) unique reflections 18630 (1784) completeness (%) 98.3 (94.6) redundancy 4.5 (4.5)

Rmerge 0.105 (0.694)

Rmeas 0.119 (0.782)

Rpim 0.055 (0.315)

Rwork 0.274 (0.356)

Rfree 0.298 (0.347) MolProbity score (percentile) 100 RMS bonds (Å) 0.003 RMS angles (°) 0.61 Downloaded from clash score 5.8 favored rotamers (%) 99.0 poor rotamers (%) 0.39 Ramachandran favored 98.0 Ramachandran outliers (%) 0 http://www.jbc.org/ bad bonds / angles 0 / 1 Cβ deviations 0

Values in parenthesis represent the highest resolution shell. by guest on March 12, 2017

19

Table 2. HslV binding.

protein K1/2 (nM) wild-type HslU 78 ± 11

W-W3 1603 ± 278 SS W W3 59 ± 9 SS SS W W W2 56 ± 5 SS SS W E W2 21 ± 7 SS W E3 23 ± 11 SS SS W E E3 28 ± 19

Downloaded from Titration assays, monitored by changes in HslV peptidase activity, were performed at 25 °C in the presence of 5 mM ATP. http://www.jbc.org/

by guest on March 12, 2017

A large AAA+ domains

small AAA+ HslU domains B peptide tethered HslU dimer

first HslU subunit C-tail

small C-tails large AAA+ largeB AAA+ peptide HslV second HslU subunit tether small large AAA+ AAA+

C-tail Downloaded from http://www.jbc.org/ C

W-E3 by guest on March 12, 2017 hexamers

Figure 1 Figure 2 Downloaded from A B 0.2 ) ) -1

-1 HslU 250 http://www.jbc.org/

enz W-W3 enz -1

-1 200

150 0.1 by guest on March 12, 2017 E-W3 W-E3 100

50 unfolding rate (min 0 0

ATP hydrolyzed (min ATP 0 10 20 30 40 W-W3 W-E3 E-W3 [protein substrate] (µM) C degradation time (min) 0 5 10 30 60 90 0 5 10 30 60 90

–Arc-st11-ssrA

HslU/HslV W-W3/HslV A C349-C47 C 3 SS-linked W trimerW trimerE trimer dimers form SS SS SS pseudohexamer W dimerE dimerW E E MW SS SS SS SS SS

standardswild-typeW HslUWDownloaded from W W W kDa 250 150 http://www.jbc.org/ 100 75

C349-C47 C349-C47 by guest on March 12, 2017 50

B 37 C349-C47 2 SS-linked trimers form 25 pseudohexamer 20 C361-C39

C361-C39

Figure 3 C349-C47 ) 300 A -1

enz 250 -1 200

150

100

50

ATP hydrolyzed (min ATP 0 3 2 2 3 2 W E SS E SS SS W SS W SS E HslU W E W SS SS W SS W W W ) -1 B Downloaded from 250 enz

-1 200 http://www.jbc.org/ 150

100 by guest on March 12, 2017 50

0 ATP hydrolyzed (min ATP 0 1 2 3 4 5 6 number W subunits

C fold-stimutation

) 2.2 1.9 1.1 1.2 0.87 3.4 600 -1

500 enz HslV present -1 400

300

200

100

ATP hydrolyzed (min ATP 0 3 2 2 3 2 W E SS E SS SS W SS W SS E E SS HslU W W SS W Figure 4 SS W W W A 6 B 1.2

5 ) 1.0 -1

) 4 enz 0.8 -1 -1 3

enz 0.6 -1

2 GFP-st11-ssrA 0.4 (min cp6

Arc-cysA degraded Arc-cysA 1 0.2 Arc- degraded (min 0 0 3 2 2 2 3 E 3 2 2 3 2 W W W SS E SS W E SS E SS SS SS SS SS W SS W SS HslU SS E HslU E W W SS E W W E W SS SS SS W SS W W W W W W Downloaded from

C D cp6 1.0 Arc-cysA 0.2 Arc- GFP-st11-ssrA ) ) -1

-1 0.8

0.6 http://www.jbc.org/ enz

enz 0.1

0.4 -1 -1 0.2 degradation (min degradation (min per W subunit per W subunit 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 by guest on March 12, 2017 number W subunits number W subunits E F

) 500 700 -1 600 enz 400 -1 500 300 400

200 300 200

100 substrate degraded ATP hydrolyzed per ATP 100

ATP hydrolyzed (min ATP 0 0 3 2 2 3 3 2 2 3 W E W E SS SS W SS W SS SS SS W SS W SS HslU W E W HslU W E W SS W SS SS W SS W W W W Figure 5 Covalently Linked HslU Hexamers Support a Probabilistic Mechanism that Links ATP Hydrolysis to Protein Unfolding and Translocation Vladimir Baytshtok, Jiejen Chen, Steven E. Glynn, Andrew R. Nager, Robert A. Grant, Tania A. Baker and Robert T. Sauer J. Biol. Chem. published online February 21, 2017

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