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Covalently Linked Hslu Hexamers Support a Probabilistic Mechanism That 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
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