Biology subjects

Baker, T. A.

Publications and source records attributed to Baker, T. A..

10 recordsLinked to original sources

bioRxiv2024

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH{middle dot}HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsHs function in the degradation of membrane-embedded proteins.

biochemistry↗

bioRxiv2023

The membrane-cytoplasmic linker defines activity of FtsH proteases

Pandemic Pseudomonas aeruginosa clone C strains encode a xenolog of FtsH (PaFtsH2), an inner-membrane associated ATP-dependent protease. FtsH1 supports growth and intrinsic antibiotic resistance but cannot be replaced by ftsH2. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Swapping residues of a short MC peptide that links transmembrane helix-2 with the cytosolic AAA+ ATPase module from PaFtsH1 into PaFtsH2 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. FtsH1 MC peptides are glycine rich. Introducing three glycines into the membrane-proximal end of PaFtsH2s MC linker is sufficient to elevate activity in vitro and in vivo. Electron microscopy including PaFtsH2 indicates that MC linker identity influences FtsH flexibility. Our findings establish that the efficiency of substrate processing by two PaFtsH isoforms depends on how they are attached to the membrane and suggest that greater linker flexibility/length allows FtsH to degrade a wider spectrum of substrates. As FtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is not a latent enzyme, rather recognizes specific substrates or is activated in specific contexts or biological niches. We hypothesize that such linkers might play a more determinative role in functionality and physiological impact of FtsH proteases than previously thought.

biochemistry↗

bioRxiv2022

The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery

Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in organisms ranging from simple bacteria to humans. In E. coli and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structures reveal that binding of the substrate*adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer. SIGNIFICANCEIntercellular proteases, including ClpXP, degrade damaged or unneeded proteins. Peptide tags allow specific protein substrates to be recognized by the ClpX unfoldase/translocase component of ClpXP and by an adaptor, SspB, which tethers itself to ClpX and enhances ClpXP degradation of the tagged protein. Our cryo-EM structure of ClpXP bound to SspB and a tagged substrate shows that SspB and ClpX simultaneously contact the degradation tag and reveal changes in the structure of ClpX and its interaction with ClpP. These structural changes appear to be a prelude to an initial ClpX translocation step that pulls the substrate away from SspB and initiates degradation by allowing substrate unfolding and further translocation of the unfolded substrate into the proteolytic chamber of ClpP.

biochemistry↗

bioRxiv2022

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

Intracellular proteases must be specific to avoid degrading the wrong proteins. Here, we present cryo-EM structures of E. coli ClpXP, a AAA+ protease, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate. An open-channel ClpX mutation stimulates degradation of casein, a non-specific substrate, indicating that channel closure contributes to increased degradation specificity. We demonstrate that ClpX activates ClpP cleavage of a degron-free decapeptide by a channel-independent mechanism, in which the peptide substrate appears to pass through a symmetry mismatched gap in the interface between ClpX and ClpP before entering the degradation chamber via the axial portal of ClpP. The peptide products of ClpXP protein degradation are likely to exit the chamber by the reverse route.

biochemistry↗

bioRxiv2022

AAA+ protease-adaptor structures reveal altered conformations and ring specialization

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here, we present high-resolution cryo-EM structures of ClpAPS complexes showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two structural classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated and tucked away from the channel, and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies demonstrate new structures and functions for key AAA+ elements. In addition to ClpAPS delivery, pore-loop tucking may be used broadly by AAA+ unfoldases, for example during enzyme pausing/unloading.

biophysics↗

bioRxiv2020

ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

AAA+ proteases, which perform regulated protein degradation in all kingdoms of life, consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX must rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP omplexes unfold, translocate, and degrade protein substrates, albeit modestly slower han uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP therefore is ot required for ClpAP protease activity, although some flexibility in how the AAA+ ring ocks on ClpP may be necessary for optimal function.

biochemistry↗

bioRxiv2020

Structural basis of ClpXP recognition and unfolding of ssrA-tagged C-degron substrates

When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In E. coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer-degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

biophysics↗

bioRxiv2020

Multistep substrate binding and engagement by the AAA+ ClpXP protease

E. coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the ATP-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound ClpXP complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial binding complex, followed by an intermediate complex, and then an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrateengagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate. SignificanceAAA+ proteases play key regulatory and quality-control roles in all domains of life. These destructive enzymes recognize damaged, unneeded, or regulatory proteins via specific degrons and unfold them prior to processive degradation. Here, we show that E. coli ClpXP, a model AAA+ protease, recognizes ssrA-tagged substrates in a multistep binding and engagement reaction. Together with recent cryo-EM structures, our experiments reveal how ClpXP transitions from a machine that checks potential substrates for appropriate degrons to one that can unfold and translocate almost any protein. Other AAA+ proteases in organelles and bacteria are likely to use similar mechanisms to specifically identify and then destroy their target proteins.

biophysics↗

bioRxiv2019

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

biophysics↗

bioRxiv2018

Mitochondrial ClpX activates an essential metabolic enzyme through partial unfolding

Mitochondria can control the activity, quality, and lifetime of their proteins with their autonomous system of chaperones, but the signals that direct substrate-chaperone interaction and outcome are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting incorporation of cofactor. Here, we ask how unfolding by mtClpX directs activation. We identified sequence and structural features in ALAS that position mtClpX and provide a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding was limited to a subdomain extending from the mtClpX-binding site to the active site. Unfolding along this path was required for mtClpX to gate cofactor access to the ALAS active site. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and suggests how substrate-chaperone interactions can direct the outcome of remodeling.

biochemistry↗