Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.
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ABSTRACT:
SUBMITTER: Rizo AN
PROVIDER: S-EPMC6546751 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
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Project description:Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a β-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.
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Project description:The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.
| S-EPMC9934407 | biostudies-literature
Project description:Hsp100 polypeptide translocases are conserved members of the AAA+ family (adenosine triphosphatases associated with diverse cellular activities) that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from Saccharomyces cerevisiae solubilizes stress-induced amorphous aggregates and amyloids. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-electron microscopy structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80-angstrom-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore loop-substrate interactions by two amino acids. These changes are coupled to activation of specific nucleotide hydrolysis sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and substrate-responsive flexibility during Hsp104-catalyzed disaggregation.
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Project description:Hexameric ATP-dependent proteases and protein remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. Whether loops from multiple subunits act independently or coordinately in these processes is a critical aspect of the mechanism but is currently unknown for any AAA+ machine. By studying covalently linked hexamers of the Escherichia coli ClpX unfoldase bearing different numbers and configurations of wild-type and mutant pore loops, we show that loops function synergistically, and the number of wild-type loops required for efficient degradation is dependent on the stability of the protein substrate. Our results support a mechanism in which a power stroke initiated in one subunit of the ClpX hexamer results in the concurrent movement of all six pore loops, which coordinately grip and apply force to the substrate.
| S-EPMC4333055 | biostudies-literature
Project description:The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore-loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.
| S-EPMC8503904 | biostudies-literature
Project description:Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.
| S-EPMC5413351 | biostudies-literature
Project description:The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings.
| S-EPMC5319980 | biostudies-literature
Project description:Human ATP-binding cassette (ABC) subfamily A (ABCA) transporters mediate the transport of various lipid compounds across the membrane. Mutations in human ABCA transporters have been described to cause severe hereditary disorders associated with impaired lipid transport. However, little is known about the mechanistic details of substrate recognition and translocation by ABCA transporters. Here, we present three cryo-EM structures of human ABCA4, a retina-specific ABCA transporter, in distinct functional states at resolutions of 3.3-3.4 Å. In the nucleotide-free state, the two transmembrane domains (TMDs) exhibit a lateral-opening conformation, allowing the lateral entry of substrate from the lipid bilayer. The N-retinylidene-phosphatidylethanolamine (NRPE), the physiological lipid substrate of ABCA4, is sandwiched between the two TMDs in the luminal leaflet and is further stabilized by an extended loop from extracellular domain 1. In the ATP-bound state, the two TMDs display a closed conformation, which precludes the substrate binding. Our study provides a molecular basis to understand the mechanism of ABCA4-mediated NRPE recognition and translocation, and suggests a common 'lateral access and extrusion' mechanism for ABCA-mediated lipid transport.
| S-EPMC8219669 | biostudies-literature
Project description:AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.
| S-EPMC6593972 | biostudies-literature
Project description:ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.
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