Biology subjects

Malinauskas, T.

Publications and source records attributed to Malinauskas, T..

11 recordsLinked to original sources

bioRxiv2026

Gag proteolysis drives HIV-1 envelope conformational closure through the gp41 cytoplasmic tail

HIV-1 must present a fusion-competent envelope glycoprotein (Env) to enter host cells while concealing the very conformation targeted by broadly neutralising antibodies (bnAbs). How the virus resolves this conflict has remained unclear. Using a minimally invasive nanobody-labelling strategy targeting Env's V4 loop, we combine single-virus FRET, fluorescence lifetime cross-correlation spectroscopy, and DNA-PAINT super-resolution microscopy to resolve Env conformation on individual, intact virions from the primary isolate JR-FL. We show that immature virions predominantly display an open Env conformation, while Gag maturation drives acquisition of the tightly closed, fusion-competent State 1 that harbours the principal bnAb epitope. Truncating the Env cytoplasmic tail abolishes this regulation, identifying the gp41 tail as the conduit linking Gag lattice maturation to Env's conformational fate. We further show that bnAb b12 preferentially binds open Env on immature virions, supporting a decoy mechanism that shields mature virions from immune recognition, with direct implications for HIV-1 vaccine design.

biophysics↗

bioRxiv2026

Molecular mechanism of action of a blood brain barrier shuttle antibody

The transferrin receptor has emerged as a prime target for transcytosis of antibody shuttles from the bloodstream into the brain parenchyma to deliver therapeutic payloads that treat neurological disorders. However, how the transferrin receptor-antibody binding mode impacts avidity, degradation, pH sensitivity and delivery remains underexplored. To address this, we determined the cryo-EM structure of mouse transferrin receptor 1 bound to the model brain shuttle antibody 8D3. In combination with cell binding and localisation studies we show that 8D3 can structurally support small-scale inter-receptor cross-linking, such as in self-contained pairs, that cause avidity but do not induce receptor redistribution or degradation. The structure can also explain pH-dependent binding modes, and we show how some of these antibody variants with graded sensitivities regulate brain penetration in vivo. Overall, our study illuminates how distinct molecular features of antibody binding impact transferrin receptor behaviour and brain delivery to inform on future shuttle design.

neuroscience↗

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

Site-specific steric control of SARS-CoV-2 spike glycosylation

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

biochemistry↗

bioRxiv2020

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

immunology↗

bioRxiv2020

Structure and Dynamics of HIV-1 Env Trimers on Native Virions Engaged in Living T Cells

The HIV-1 envelope glycoprotein (Env) mediates viral entry into the host cell. Although the highly dynamic nature of Env intramolecular conformations has been shown with single molecule spectroscopy in vitro, the bona fide Env intra- and intermolecular mechanics when engaged in live T cells remains unknown. We used both, two photon fast fluorescence lifetime imaging detection of single-molecule Forster Resonance Energy Transfer and single molecule photobleaching to reveal transitions between intramolecular and intermolecular conformations that mediate Env clustering. Furthermore, we show that three broad neutralizing anti-Env antibodies directed to different epitopes destabilise Env intramolecular dynamics and their clusters when engaged to living T cells. Importantly, our results show that Env clustering is a common conformation across different HIV-1 Env strains, which depends on efficient virus maturation, and that is disrupted upon binding of Env to CD4 or to neutralizing antibodies. Thus, this study illuminates how different intramolecular conformations and clusters of Env mediate HIV-1 Env-T cell interactions in real time and therefore might control immune evasion.

immunology↗

bioRxiv2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

microbiology↗

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

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 [A] of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. HighlightsO_LICR3022 neutralises SARS-CoV-2 C_LIO_LINeutralisation is by destroying the prefusion SPIKE conformation C_LIO_LIThis antibody may have therapeutic potential alone or with one blocking receptor attachment C_LI

microbiology↗

bioRxiv2018

Heteromeric GABAA receptor structures in positively-modulated active states

Type-A {gamma}-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of /{beta}/{gamma} subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human 1{beta}3{gamma}2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered 5 and 5{gamma}2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation.

biophysics↗