Biology subjects

Hardwick, S. W.

Publications and source records attributed to Hardwick, S. W..

10 recordsLinked to original sources

bioRxiv2026

Rapid Production and Cryo-EM Structure Determination of GPCR-Nanodisc Complexes via Cell-Free Synthesis

G protein-coupled receptors (GPCRs) represent major therapeutic targets, yet cryo-EM structure determination of unliganded or antagonist-bound GPCRs remains severely bottlenecked by small receptor mass, detergent-induced instability, and multi-week cell-based production pipelines. Here, we combine intracellular loop 3 (ICL3) rigid fiducial engineering with a high-throughput, cell-free protein synthesis workflow to rapidly generate cryo-EM-ready GPCR-nanodisc assemblies. Using the eProtein Discovery(TM) digital microfluidics system, we performed automated nanoliter-scale screening of construct fusions and nanodisc lipid compositions, establishing that co-translational insertion into nanodiscs is strictly required for receptor solubilization and affinity purification. Direct scale-up of screening conditions yielded structural-grade protein within four days from gene assembly to grid vitrification. We demonstrate this workflow with a 3.38 Angstrom cryo-EM structure of antagonist-bound human adenosine A2A receptor (A2AR)-beta-lactamase fusion in complex with beta-lactamase inhibitory protein II (BLIP-II). To demonstrate generalizability of protein quality, we extended this detergent-free co-translational pipeline to an expanded panel of 12 wild-type GPCRs spanning Classes A, B, and C, as well as six orphan receptors. All targets were purified with biophysical quality comparable to the A2AR benchmark. By removing the reliance on cellular expression or agonist/G-protein stabilization and compressing construct assembly, nanodisc screening, biophysical quality control, and grid vitrification into a four-day turnaround, this detergent-free cell-free pipeline provides a rapid Design-Build-Test-Learn (DBTL) engine to accelerate structure-based drug discovery for challenging and unliganded GPCR targets.

biochemistry↗

bioRxiv2026

Affinity purification contaminants identified by cryo-EM and mass spectrometry

Affinity chromatography is a powerful and therefore popular method for the purification of proteins for structural studies. The success of the technique relies on the specificity of the interaction between the target protein and the affinity resin. Here, we present the identification of two protein contaminants isolated from HEK293 cell lysate following affinity purification of twin Strep-tagged or FLAG-tagged proteins. The contaminants were identified as human propionyl-coenzyme A carboxylase (hPCC) and protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) via a combination of cryo-EM data processing and proteomic analyses. This report serves to illustrate how these contaminants may appear in cryo-EM datasets and to highlight the paramount importance of affinity chromatography resin specificity for efficient protein purification.

biochemistry↗

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

DNA polymerase Lambda is anchored within the NHEJ synaptic complex via Ku70/80

Non-homologous end joining (NHEJ) is the predominant pathway by which double-strand DNA breaks (DSBs) are repaired in mammals. To enable final break closure, various NHEJ end-processing factors respond to the chemistry of the damaged DNA ends. Amongst these factors is DNA polymerase lambda (Pol {lambda}), a member of the Pol X family. How members of the Pol X family engage with the NHEJ complex is unknown. Here, we present cryo-EM structures of Pol {lambda} in complex with the Ku70/80 DSB sensor whilst engaged with the DNA-PK holoenzyme in a long-range synaptic complex. These structures reveal a specific interaction site between Ku70/80 and the Pol {lambda} BRCT domain. The functionality of this interaction is assessed by generating point mutations on either side of the Pol {lambda} BRCT:Ku70/80 interface. Using these mutants in two orthogonal assays in cells (live protein recruitment at biphoton laser-damaged nuclear sites and transfection with an original gap-filling reporter plasmid) defines the molecular basis and essentiality of the BRCT domain for the recruitment and activity of the Pol {lambda} within the NHEJ complex. Ultimately, these data explain the role of this interaction in cell survival to DSBs. Finally, we propose a unified model for the interaction of the three Pol X family members bearing BRCT domains with the same site of Ku70/80.

biochemistry↗

bioRxiv2023

Structural basis for human mitochondrial tRNA maturation

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), mt-RNase P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for a tRNA methyltransferase complex in mt-RNase P cleavage activity is not fully understood. Moreover, the mechanisms of pre-tRNA 3-cleavage and CCA addition have also yet to be uncovered. Here we report cryo-EM structures that visualise these events, revealing the molecular basis for the first steps of RNA-transcript processing in human mitochondria. Altogether, the structures visualise four steps of mitochondrial tRNA maturation: 5 and 3 tRNA-end processing, methylation and 3-CCA addition, and explain the defined sequential order of tRNA processing. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA processing and 3-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-variable mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes.

biochemistry↗

bioRxiv2022

Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis

The biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled through intricate post-transcription networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthetase in Escherichia coli, but when bound by the protein RapZ, it becomes inactivated through cleavage by the endoribonuclease RNase E. Here we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the protein tetrameric quaternary structure to an imperfect structural repeat in the RNA. The RNA is contacted mostly through a highly conserved domain of RapZ that shares deep evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a pre-cleavage encounter intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised for cleavage. The structures suggest how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors are recognised for processing.

molecular biology↗

bioRxiv2021

Viral tolerance in Aedes aegypti relies on the negative cooperativity between Toll5A and the cytokine ligand Spz1C

Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone duplication which may allow neofunctionalization and adaptation. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A complexes that display transient but specific interactions with Spaetzle1C, forming asymmetric complexes, with only one ligand clearly resolved. Loop structures of Spaetzle1C and Toll5A intercalate, temporarily bridging the receptor C-termini to promote signalling. By contrast unbound receptors form head-to-head homodimers that keep the juxtamembrane regions far apart in an inactive conformation. Interestingly the transcriptional signature of Spaetzle1C differs from other Spaetzle cytokines and controls genes involved in innate immunity, metabolism and tissue regeneration. Taken together our results explain how upregulation of Spaetzle1C in the midgut and Toll5A in the salivary gland shape the concomitant immune response.

immunology↗

bioRxiv2021

Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with Pol α - Primase

The molecular mechanisms that drive the infection by the SARS-CoV-2 coronavirus - the causative agent of the COVID-19 (Coronavirus disease-2019) pandemic - are under intense current scrutiny, to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent cell-based analyses of SARS-CoV-2 protein - protein interactions have mapped the human proteins targeted by the virus. The DNA polymerase - primase complex or primosome - responsible for initiating DNA synthesis in genomic duplication - was identified as a target of nsp1 (non structural protein 1), a major virulence factor in the SARS-CoV-2 infection. Here, we report the biochemical characterisation of the interaction between nsp1 and the primosome and the cryoEM structure of the primosome - nsp1 complex. Our data provide a structural basis for the reported interaction between the primosome and nsp1. They suggest that Pol - primase plays a part in the immune response to the viral infection, and that its targeting by SARS-CoV-2 aims to interfere with such function.

biophysics↗

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

CryoEM structures of human CMG - ATPγS - DNA and CMG - AND-1 complexes

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 [A] of human CMG bound to fork DNA and the ATP-analogue ATP{gamma}S. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATP{gamma}S or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one {beta}-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.

biochemistry↗