Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/105866

Structure of a AAA+ unfoldase in the process of unfolding substrate

Ripstein, Z. A.·Huang, R.·Augustyniak, R.·Kay, L. E.·Rubinstein, J. L.
View original source ↗Export metadata ↓

Abstract

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 [A] stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Ripstein, Z. A., Huang, R., Augustyniak, R., Kay, L. E., Rubinstein, J. L.. 2017-02-03. Structure of a AAA+ unfoldase in the process of unfolding substrate. https://doi.org/10.1101/105866

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Direct Bioconjugations of Diverse Payloads using an Extraordinarily Donor-Promiscuous Flavin Transferase

Site-specific protein conjugation is a widely used tool in chemical biology research and is the foundation of many biotechnological and biomedical applications. A key enabling technology are repurposed post-translational protein modifying enzymes, where variants with broad donor and acceptor substrate promiscuity were developed into general-purpose bioconjugation platforms. However, direct enzymatic attachment of bulky or structurally diverse payloads is often limited by low reaction efficiencies and difficulties in preparing suitable donors. Consequently, conjugation typically relies on two-step strategies in which an enzyme first installs a bioorthogonal handle on the target protein, followed by chemical attachment of the payload. These enzyme-first, chemistry-second approaches limit chemistry to bioorthogonal reactions that leave a molecular footprint. Here, we introduce direct ADDing, an enzymatic bioconjugation platform based on our discovery of the exceptional donor promiscuity of the flavin transferase ApbE. We show that ApbE accepts a remarkably broad range of donor substrates provided they carry an adenosine diphosphate (ADP) anchor. The prevalence of ADP-containing compounds in nature provides access to diverse building blocks and an extensive biochemical toolbox for donor generation. Exploiting these resources, we demonstrate multiple strategies for preparing compatible donors and achieve direct, site-specific enzymatic conjugation of diverse small molecules, peptides, proteins, and synthetic polymers.

biochemistry↗
bioRxiv2026

A respiratory microdomain links ubiquinone biosynthesis to succinate dehydrogenase in an apicomplexan mitochondrion lacking Complex I

Mitochondrial complex I (CI) is a major entry point for NADH-derived electrons and an important site of redox regulation in most eukaryotes. However, apicomplexan parasites, including Toxoplasma gondii, lack CI, raising a fundamental question of how mitochondrial respiration is organized in these parasites. Here, we identify a parasite-specific mitochondrial microdomain that physically and functionally couples ubiquinone (UQ) biosynthesis to succinate dehydrogenase (SDH). Using proximity labeling, membrane-based yeast two-hybrid analyses, reciprocal co-immunoprecipitations, and blue native PAGE, we show that UQ biosynthesis proteins associate with parasite-specific SDH subunits and co-migrate as a high-molecular-weight assembly. Knockdown of TgCoq4, a component of the UQ biosynthesis pathway, reduced SDH activity, and adding exogenous UQ to the assay did not restore it, indicating that UQ availability is not sufficient for efficient succinate oxidation. FCCP stimulates malate- and TMPD/ascorbate-supported respiration to maximal capacity but fails to do so with succinate as the substrate. This result, combined with reactive oxygen species (ROS) data, suggests that succinate causes over-reduction of the UQ pool, limiting further electron flow. We also find that CIII associates with the UQ synthesis machinery, suggesting a larger protein assembly. Together, these findings reveal that UQ biosynthesis and electron transport are physically and functionally integrated in T. gondii, defining a previously unrecognized organization of mitochondrial respiration in an organism lacking CI.

biochemistry↗
bioRxiv2026

Human senataxin uses distinct peripheral elements to unwind R-loops and act on RNA polymerase II

Persistent R-loops and paused or stalled RNA polymerase II (RNAPII) elongation complexes can threaten genome stability. Senataxin (SETX) contributes to R-loop resolution and RNAPII regulation, but their intimate connection during transcription makes the individual contributions of these activities difficult to distinguish in cells. To determine whether these activities rely on the same molecular features, we investigated the structure and biochemical properties of the SETX helicase domain (SETX-HD). Cryo-EM defines the conserved helicase core and RNA-binding channel of SETX-HD, while biochemical analyses identify distinct roles for two peripheral elements: a previously uncharacterised C-terminal RNA-binding domain (CT-RBD) and the prong subdomain. In reconstituted mammalian transcription assays, SETX-HD promotes passage beyond a defined RNAPII pause and elongation-complex dissociation on the same template. The CT-RBD stabilises RNA engagement and is required for efficient R-loop unwinding, whereas its deletion preserves both RNAPII-associated activities. Conversely, replacing the native SETX prong with its fungal Sen1 counterpart preserves R-loop unwinding but impairs both RNAPII outcomes. These reciprocal separation-of-function effects establish that a common SETX-HD RNA-translocation core depends on distinct peripheral elements for R-loop unwinding and action on mammalian RNAPII. This separation provides a direct route to determine how these activities contribute to SETX function in cells.

biochemistry↗