Biology subjects

Naydenova, K.

Publications and source records attributed to Naydenova, K..

7 recordsLinked to original sources

bioRxiv2025

ZNFX1, an immunoregulatory RNA helicase and E3 ubiquitin ligase, assembles into pleiomorphic polymers

ZNFX1 is an SF1-family RNA helicase essential for innate immunity. Patients with ZNFX1 mutations experience recurrent infections, yet the underlying mechanism remains unclear. We determined cryo-EM structures of ZNFX1 in apo and RNA-bound forms, revealing auto-inhibition of the helicase through a regulatory insertion occluding the RNA-binding groove. ZNFX1 also functions as a bi-catalytic E3 ubiquitin ligase, containing an RZ-finger homologous to RNF213 and a previously unidentified Miz-like domain that catalyze ubiquitylation independently or cooperatively, with activity enhanced by ubiquitin chains. Patient mutations demonstrate that the helicase, E3 ligase, and rigid zinc-finger stalk connecting them are required for function. ZNFX1 can assemble into structured, pleiomorphic polymers via multivalent protein-protein interactions, revealing a mechanism that may facilitate RNA sequestration in stress granules and antiviral activity. These findings establish ZNFX1 as a multifunctional enzyme in innate immunity that couples RNA sensing to ubiquitin signaling and assembles into higher-order structures for signal amplification.

biochemistry↗

bioRxiv2025

GABAA receptor gating imaged on the millisecond timescale

Type-A {gamma}-aminobutyric receptors (GABAARs) are fast pentameric ligand gated ion channels (pLGICs)1-5. Within a millisecond, saturating agonist concentrations trigger activity bursts consisting of high-frequency fluctuations between conductive and non-conductive states6,7. These can last for tens to hundreds of milliseconds until, stochastically, receptors adopt stable, long-lived, desensitised conformations2,8,9. This highly dynamic process, known as gating, controls transient passages of chloride ions across plasma membranes to enable neurotransmission and other fundamental processes in animal physiology10-12. The conformational transitions inside activity bursts, inferred from electrophysiology, have remained inaccessible to structural biology investigation. Here we describe the gating motions of three GABAA receptor variants imaged within the first 10 milliseconds of agonist application by cryogenic electron microscopy (cryo-EM)13. We illustrate how activation and desensitisation proceed through multiple asymmetric states, supported by major secondary, tertiary and quaternary structural rearrangements, and demonstrate that the same gating principles apply to both homomeric and heteromeric GABAARs. Furthermore, we show that cholesterol and phospholipids stabilise newly formed inter-subunit interfaces and obstruct channel pores in short-lived desensitised states, while phosphatidylinositol 4,5-bisphosphate (PIP2) precludes the opening of both 1{beta}3 and 1{beta}3{gamma}2 GABAAR channels. Our results provide a novel framework to interpret decades of electrophysiology observations and suggest a broadly applicable approach to investigate mechanistically the vast arrays of physiological and pharmacological modulators of GABAARs5,14 and other fast neurotransmitter receptors15,16. Moreover, the subunit interfaces and lipid-binding pockets that form and disappear during GABAAR gating provide new opportunities to discover modulators with improved specificity and therapeutic properties. One sentence summarySingle-particle cryo-EM was used to explore the dynamic conformational landscape of three human GABAA receptor variants within the first 10 milliseconds of interaction with their neurotransmitter agonists.

neuroscience↗

bioRxiv2024

Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology1-4. This diverse protein family mediates the ionotropic signals triggered by major neurotransmitters and includes{gamma} -aminobutyric acid receptors (GABAARs) and acetylcholine receptors (nAChRs). Receptor function is fine-tuned by a myriad of endogenous and pharmacological modulators3. A functional pLGIC is built from five homologous, sometimes identical, subunits, each containing a {beta}-scaffold extracellular domain (ECD), a four-helix transmembrane domain (TMD) and intracellular loops of variable length. Although considerable progress has been made in understanding pLGICs in structural and functional terms, the molecular mechanisms that enable their assembly at the endoplasmic reticulum (ER)5 in a vast range of potential subunit configurations6 remain unknown. Here, we identified candidate pLGICs assembly factors selectively associated with an unassembled GABAAR subunit. Focusing on one of the candidates, we determined the cryo-EM structure of an assembly intermediate containing two 1 subunits of GABAAR each bound to an ER-resident membrane protein NACHO. The structure showed how NACHO shields the principal (+) transmembrane interface of 1 subunits containing an immature extracellular conformation. Crosslinking and structure-prediction revealed an adjacent surface on NACHO for {beta}2 subunit interactions to guide stepwise oligimerisation. Mutations of either subunit-interacting surface on NACHO also impaired the formation of homopentameric 7 nAChRs, pointing to a generic framework for pLGIC assembly. Our work provides the foundation for understanding the regulatory principles underlying pLGIC structural diversity.

biochemistry↗

bioRxiv2024

Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213s dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.

molecular biology↗

bioRxiv2024

Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213

The evolutionary arms race between pathogens and hosts has resulted in pathogens acquiring diverse adaptive countermeasures that antagonize host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates eat-me signals for antibacterial autophagy but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here we show that Shigella flexneri, a professional cytosol-dwelling enterobacterium, actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to the LPS E3 ubiquitin ligase RNF213, ubiquitylates it, and targets it for degradation by the proteasome, thus preventing LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the structure of their complex using cryogenic electron microscopy. The specificity of the interaction is achieved via the leucine rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding of ubiquitin-charged E2 enzymes. Interestingly, IpaH1.4 also targets the E3 ligase LUBAC - required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213 - as well as multiple other E3 ligases involved in inflammation and immunity - through binding to the E2-interacting face of their RING domains. We conclude that IpaH1.4 has evolved to antagonize multiple anti-bacterial and pro-inflammatory host E3 ligases.

molecular biology↗

bioRxiv2020

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 [A]. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

molecular biology↗

bioRxiv2020

Structure of the light harvesting 2 complex reveals two carotenoid energy transfer pathways in a photosynthetic bacterium

We report the 2.4 [A] resolution structure of the light harvesting 2 complex (LH2) from Marichromatium (Mch.) purpuratum determined by electron cryo-microscopy. The structure contains a heptameric ring that is unique among all known LH2 structures, explaining the unusual spectroscopic properties of this bacterial antenna complex. Two sets of distinct carotenoids are identified in the structure, and a network of energy transfer pathways from the carotenoids to bacteriochlorophyll a molecules is shown. The geometry imposed by the heptameric ring controls the resonant coupling of the long wavelength energy absorption band. Together, these details reveal key aspects of the assembly and oligomeric form of purple bacterial LH2 complexes that were previously inaccessible by any technique. One Sentence SummaryThe structure of a heptameric LH2 antenna complex reveals new energy transfer pathways and the basis for assembling LH rings.

biochemistry↗