Cryo-EM structure of a delivery complex containing the SspB adaptor, an ssrA-tagged substrate, and the AAA+ ClpXP protease
Ontology highlight
ABSTRACT:
SUBMITTER: Robert Sauer
PROVIDER: EMPIAR-11349 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA. Reciprocally, N-end rule substrates increase ClpS affinity for ClpA(6). Enhanced binding requires the N-end residue and a peptide bond of the substrate, as well as multiple aspects of ClpS, including a side chain that contacts the substrate ?-amino group and the flexible N-terminal extension (NTE). Finally, enhancement also needs the N domain and AAA+ rings of ClpA, connected by a long linker. The NTE can be engaged by the ClpA translocation pore, but ClpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the protease.
| S-EPMC3168947 | biostudies-literature
Project description:Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-?7 insertions within both AAA domains important for Rix7 function.
| S-EPMC6355894 | biostudies-literature
Project description:The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960?nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82?Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the ??-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the ??-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of ?- and ?-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.
| S-EPMC7532537 | biostudies-literature
Project description:Setd2 methylate the nucleosome to form H3K36me3. Here we utilized the Cryo-EM to elucidate the structure of SETD2/Set2 bound with nucleosomes. Through this structure analysis, we found that histone H1 may interfere the enzymatic activity of SETD2/Set2 by inhibiting their binding affinity.
2021-05-20 | GSE148235 | GEO
Project description:According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP, as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.
| S-EPMC5424706 | biostudies-literature
Project description:The first structure of tetrameric mammalian acylaminoacyl peptidase, an enzyme that functions as an upstream regulator of the proteasome through the removal of terminal N-acetylated residues from its protein substrates, was determined by cryo-EM and further elucidated by MD simulations. Self-association results in a toroid-shaped quaternary structure, guided by an amyloidogenic β-edge and unique inserts. With a Pro introduced into its central β-sheet, sufficient conformational freedom is awarded to the segment containing the catalytic Ser587 that the serine protease catalytic triad alternates between active and latent states. Active site flexibility suggests that the dual function of catalysis and substrate selection are fulfilled by a novel mechanism: substrate entrance is regulated by flexible loops creating a double-gated channel system, while binding of the substrate to the active site is required for stabilization of the catalytic apparatus - as a second filter before hydrolysis. The structure not only underlines that within the family of S9 proteases homo-multimerization acts as a crucial tool for substrate selection, but it will also allow drug design targeting of the ubiquitin-proteasome system.
| S-EPMC9214879 | biostudies-literature
Project description:The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein-RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.
| S-EPMC6223129 | biostudies-literature
Project description:The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4?Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.
| S-EPMC5971111 | biostudies-literature
Project description:Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major high-affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+/Zn2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of 'ERAD' in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.
| S-SCDT-EMBOJ-2019-102935 | biostudies-other
Project description:Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major high-affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+ /Zn2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.
| S-EPMC7231995 | biostudies-literature