Steven E. Glynn

Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine.

ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.

Nucleotide Binding and Conformational Switching in the Hexameric Ring of a AAA+ Machine.

ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function.

Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine

In the Escherichia coli ClpXP protease, a hexameric ClpX ring couples ATP binding and hydrolysis to mechanical protein unfolding and translocation into the ClpP degradation chamber. Rigid-body packing between the small AAA+ domain of each ClpX subunit and the large AAA+ domain of its neighbor stabilizes the hexamer. By connecting the parts of each rigid-body unit with disulfide bonds or linkers, we created covalently closed rings that retained robust activity. A single-residue insertion in the hinge that connects the large and small AAA+ domains and forms part of the nucleotide-binding site uncoupled ATP hydrolysis from productive unfolding. We propose that ATP hydrolysis drives changes in the conformation of one hinge and its flanking domains and that the changes are propagated around the AAA+ ring through the topologically constrained set of rigid-body units and hinges to produce coupled ring motions that power substrate unfolding.

Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing

Feeding a protease step by step Proteins that degrade damaged or misfolded mitochondrial proteins are essential for mitochondrial function. A key player is the hexameric protease YME1, in which each subunit is anchored in the inner mitochondrial membrane by a helix and has an adenosine triphosphatase (ATPase) domain and a protease domain in the intermembrane space. Puchades et al. report a high-resolution structure that shows that the ATPase domains form an asymmetric spiral staircase that stacks above a planar protease ring. Conserved tyrosine residues in the central pore of the spiral staircase interact with a substrate peptide. The ATP hydrolysis cycle is sequential and coordinated with changes in the position of the tyrosine residues that result in stepwise translocation of the substrate into the protease chamber. Science, this issue p. eaao0464 Cryo–electron microscopy structure of a yeast mitochondrial inner membrane ATPase protease suggests a sequential mechanism for substrate translocation. INTRODUCTION Protein quality control is essential for mitochondrial function, and imbalances in this regulation are associated with numerous human diseases. YME1L is a hexameric AAA+ protease in the inner membrane (IM) that controls maintenance of the electron transport chain, protein import, lipid synthesis, and mitochondrial morphology. Every YME1L subunit contains an adenosine triphosphatase (ATPase) and a peptidase domain, which reside in the intermembrane space, tethered to the IM by a membrane helix. Protein substrates undergo adenosine triphosphate (ATP)–driven translocation through a central pore into a proteolytic chamber by a mechanism that is likely to be conserved in other AAA+ proteases. RATIONALE A compelling question is how YME1L couples ATP hydrolysis to processive substrate translocation. We sought to understand this mechanism by determining a near–atomic resolution cryo–electron microscopy (cryo-EM) structure of a solubilized form of the yeast homolog, YME1. By precisely visualizing how the nucleotide state of YME1 subunits allosterically controls their interaction with a translocating protein substrate, we can understand how cycles of nucleotide hydrolysis can drive stepwise translocation of protein substrates for degradation. RESULTS Our ~3.4 Å resolution structure shows that YME1 assembles into two stacked rings, with an asymmetric spiral staircase of ATPase domains atop a planar protease ring. Tyrosine residues in two conserved central ATPase pore loops grasp an unfolded 10–amino acid peptide and direct it toward a negatively charged proteolytic chamber. The four central subunits in the staircase bind ATP and intercalate their pore loop tyrosines with the substrate backbone in a configuration compatible with sequence-independent translocation. The lowest “posthydrolysis” subunit contains an adenosine diphosphate (ADP)–like EM density and only interacts modestly with the substrate, whereas the top apo-like step subunit does not contain well-resolved nucleotide density and is disengaged from both the substrate and the ATPase ring. Bound ATP is sensed by the adjacent protomer via two arginine fingers and an intersubunit signaling (ISS) motif that bridges the subunits across the nucleotide-binding pocket. Attachment of the ISS positions the pore loop tyrosines of the ATP-bound subunit to tightly grasp the substrate. Loss of the γ-phosphate releases the arginine fingers, retracting the ISS and repositioning the pore loops away from the substrate. The absence of nucleotide in the step subunit breaks coordination on both sides and sequesters the pore loops into helices away from the substrate. A glycine residue in the interdomain linker is required to accommodate large movements of the ATPase domains within the spiral staircase. CONCLUSION This structure of a substrate-bound single-polypeptide AAA+ protease allows us to define a tightly coordinated sequential ATP hydrolysis cycle. Hydrolysis in the lowest ATP-bound subunit abolishes coordination by the adjacent arginine fingers and ISS, repositioning the now posthydrolysis subunit to the lowest position of the staircase, which, in turn, triggers hydrolysis in the next-lowest ATP-bound subunit. Loss of coordination on both sides of the ADP-bound subunit breaks substrate interaction and displaces the subunit from the hexamer, where it can release ADP and rebind ATP at the top of the staircase. Iteration of this cycle drives stepwise translocation of the substrate into the proteolytic chamber. The high degree of structural conservation between YME1 and the 26S proteasome suggests that this mechanism may be conserved across ATP-driven proteases. Cryo-EM structure of AAA+ protease YME1 sheds light on the mechanism of substrate translocation. (1) A semitransparent surface representation shows how the asymmetric ATPase staircase is positioned above a planar C6-symmetric protease ring. Substrate (orange) surrounded by pore loop 1 tyrosines is shown in blue. Nucleotides are shown as gray densities. (2) A close-up view of the cryo-EM density reveals a spiral staircase organization with tyrosines intercalating into the substrate. (3) The nucleotide state could be identified for each subunit. (4) A cartoon representation of the YME1 ATPase hexamer depicts the asymmetric organization of the subunits surrounding the substrate. The ISS motif (represented as a Phe residue) protrudes into the nucleotide-binding pocket of the neighboring subunit only in the presence of ATP. We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo–electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.

Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate.

Engineered AAA+ proteases reveal principles of proteolysis at the mitochondrial inner membrane

The human YME1L protease is a membrane-anchored AAA+ enzyme that controls proteostasis at the inner membrane and intermembrane space of mitochondria. Understanding how YME1L recognizes substrates and catalyses ATP-dependent degradation has been hampered by the presence of an insoluble transmembrane anchor that drives hexamerization of the catalytic domains to form the ATPase active sites. Here, we overcome this limitation by replacing the transmembrane domain with a soluble hexameric coiled coil to produce active YME1L hexamers that can be studied in vitro. We use these engineered proteases to reveal principles of substrate processing by YME1L. Degradation by YME1L requires substrates to present an accessible signal sequence and is not initiated simply by substrate unfolding. The protease is also capable of processively unfolding substrate proteins with substantial thermodynamic stabilities. Lastly, we show that YME1L discriminates between degradation signals by amino acid composition, implying the use of sequence-specific signals in mitochondrial proteostasis.

Defects in fatty acid amide hydrolase 2 in a male with neurologic and psychiatric symptoms

BackgroundFatty acid amide hydrolase 2 (FAAH2) is a hydrolase that mediates the degradation of endocannabinoids in man. Alterations in the endocannabinoid system are associated with a wide variety of neurologic and psychiatric conditions, but the phenotype and biochemical characterization of patients with genetic defects of FAAH2 activity have not previously been described. We report a male with autistic features with an onset before the age of 2 years who subsequently developed additional features including anxiety, pseudoseizures, ataxia, supranuclear gaze palsy, and isolated learning disabilities but was otherwise cognitively intact as an adult.Methods and resultsWhole exome sequencing identified a rare missense mutation in FAAH2, hg19: g.57475100G > T (c.1372G > T) resulting in an amino acid change (p.Ala458Ser), which was Sanger confirmed as maternally inherited and absent in his healthy brother. Alterations in lipid metabolism with abnormalities of the whole blood acyl carnitine profile were found. Biochemical and molecular modeling studies confirmed that the p.Ala458Ser mutation results in partial inactivation of FAAH2. Studies in patient derived fibroblasts confirmed a defect in FAAH2 activity resulting in altered levels of endocannabinoid metabolites.ConclusionsWe propose that genetic alterations in FAAH2 activity contribute to neurologic and psychiatric disorders in humans.

Multifunctional Mitochondrial AAA Proteases

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

Cloning, purification, crystallization and preliminary crystallographic analysis of galactokinase from Pyrococcus furiosus.

Galactokinase catalyses the conversion of galactose to galactose-1-phosphate as the first step in the Leloir pathway, a metabolic route that eventually enables the degradation of galactose via the glycolytic pathway. Galactokinases have been isolated from a wide range of prokaryotic and eukaryotic organisms and the enzyme has been identified as a member of the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) superfamily. Pyrococcus furiosus galactokinase was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to the space group C222(1), with more than eight subunits in the asymmetric unit and with approximate unit-cell parameters a = 211.7, b = 355.4, c = 165.5 A, alpha = beta = gamma = 90 degrees. The crystals diffract X-rays to 2.9 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the molecular basis of substrate recognition and catalysis of this enzyme, for which no structures are currently available.

Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation.

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 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, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.