Biology subjects

Aricescu, A. R.

Publications and source records attributed to Aricescu, A. R..

8 recordsLinked to original sources

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↗

bioRxiv2021

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.

neuroscience↗

bioRxiv2021

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the worlds population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

immunology↗

bioRxiv2020

Single-particle cryo-EM at atomic resolution

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 [A] resolution cryo-EM reconstruction for a prototypical human membrane protein, the {beta}3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 [A] resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

biophysics↗

bioRxiv2020

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

Neuronal synapses undergo structural and functional changes throughout life, essential for nervous system physiology. However, these changes may also perturb the excitatory/inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we report the design and characterization of CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1 to interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors. CPTX induced the formation of excitatory synapses in vitro and in vivo and restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimers disease and spinal cord injury, respectively. Thus, CPTX represents a prototype for novel structure-guided biologics that can efficiently repair or remodel neuronal circuits. One Sentence SummaryStructural biology information was used to design CPTX, a synthetic protein that induces functional excitatory synapses and restores normal behaviors in mouse models of neurological diseases.

neuroscience↗

bioRxiv2019

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 [A] resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABAA {beta}3 homopentameric receptor bound to its small-molecule agonist histamine.

biochemistry↗

bioRxiv2017

Human PRDM9 Can Bind And Activate Promoters, And Other Zinc-Finger Proteins Associate With Reduced Recombination In cis

Across mammals, PRDM9 binding localizes almost all meiotic recombination hotspots. However, most PRDM9 motif sequence matches are not bound, and most PRDM9-bound loci do not become hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human and chimp PRDM9 binding sites in a human cell line, and measured PRDM9-induced H3K4me3 and gene expression changes. These data revealed varied DNA-binding modalities of PRDM9, and histone modifications that predict binding. At sites where PRDM9 binds, specific cis sequence motifs associated with TRIM28 recruitment, and histone modifications, predict whether recombination subsequently occurs. These results implicate the large family of KRAB-ZNF genes in consistent, localized meiotic recombination suppression. PRDM9 affects gene expression for a small number of genes including CTCFL and VCX, by binding nearby. Finally, we show that PRDM9s DNA-binding zinc finger domain strongly impacts the formation of multimers, with a pair of highly diverged alleles multimerizing less efficiently.

genetics↗