Biology subjects

Savva, C. G.

Publications and source records attributed to Savva, C. G..

9 recordsLinked to original sources

bioRxiv2025

Structure and function of the EDEM:PDI ERAD checkpoint complex

The ERAD-L checkpoint complex de-mannosylates misfolded glycoproteins with lumenal defects, targeting them to retrotranslocation, ubiquitination, and proteasomal degradation. Commitment to ERAD-L requires an Endoplasmic Reticulum-Degradation Enhancing alpha-Mannosidase-like protein (EDEM) and its associated Protein Disulfide Isomerase (PDI). We determined Cryo-EM structures of the Chaetomium thermophilum EDEM:PDI heterodimer, both by itself and in complex with a classic ERAD substrate, the alpha1-antitrypsin Null Hong Kong mutant (A1AT-NHK). The EDEM catalytic domain nestles within the PDI arc. One intermolecular disulfide, between the a' domain of PDI and the first conserved cysteine of the EDEM linker (Cys A), stably links the two proteins. A second intermolecular disulfide, between the second conserved cysteine of the EDEM linker (Cys B) and the PDI a domain, stabilises the compact apo conformation. In the substrate-bound complex, the Cys B intermolecular disulfide is reduced and the catalytic CXXC motif of the PDI a domain oxidised. Release of the Cys B linkage increases the conformational freedom of the EDEM linker, allowing the EDEM C-terminal domains to adopt both proximal and distal conformations relative to the catalytic domain. The structures provide a framework for discovery of EDEM:PDI modulators, with potential applications in virology, rare genetic disease and cancer, and as reagents for glycoprotein quality-control engineering.

biochemistry↗

bioRxiv2024

Mechanism of cooperative strigolactone perception by the MAX2 ubiquitin ligase-receptor-substrate complex

Strigolactones (SLs) are a group of plant hormones that regulate various aspects of plant growth and development. Additionally, SLs exuded into the soil promote symbiotic relationships with arbuscular mycorrhizal fungi and stimulate the germination of parasitic plants such as Striga hermonthica. The binding and hydrolysis of SLs by their receptors (D14 in Arabidopsis and HTL in Striga) promote the ubiquitination of transcriptional repressors by Skp1-cullin-F-box (SCF)-type E3 ubiquitin ligases. The mechanistic link between SL perception by D14/HTL and substrate recognition by the E3 remains unclear. We identified an E3-HTL-substrate complex that is sufficiently stable for cryogenic electron microscopy. This complex, composed of SKP1 (ASK1) and substrate (SMAX1) from Arabidopsis, and Striga F-box (MAX2) and SL receptor (HTL7), reveals that the substrate engages in a bidentate association through its N and D2 domains. This interaction, which is both conformationally and compositionally dynamic, directly and allosterically stabilises the MAX2-(SL)HTL7 complex and affects the positioning of ASK1 relative to MAX2. This dynamic positioning influences the proximity between the substrate D2 domain and the ubiquitin-conjugated E2 enzyme. This work advances our understanding of how E3 ligases in plants translate hormone perception into genetic adaptations.

biophysics↗

bioRxiv2024

Structure of the human CTF18 clamp loader bound to PCNA

Sliding clamps like PCNA are crucial processivity factors for replicative polymerases, requiring specific clamp loaders for loading onto DNA. The human alternative clamp loader CTF18-RFC interacts with the leading strand polymerase Pol {varepsilon} and loads PCNA onto primer/template DNA using its RFC pentameric module. Here, we provide a structural characterization of the human CTF18-RFC complex and its interaction with PCNA. Our cryo-EM data support that the Ctf8 and Dcc1 subunits of CTF18-RFC, which form the regulatory module interacting with Pol {varepsilon}, are flexibly tethered to the RFC module. A 2.9 [A] cryo-EM structure shows the RFC module bound to PCNA in an auto-inhibited conformation similar to the canonical RFC loader, marking the initial step of the clamp-loading reaction. The unique RFC1 (Ctf18) large subunit of CTF18-RFC, which based on the cryo-EM map shows high relative flexibility, is anchored to PCNA through an atypical low-affinity PIP box in the AAA+ domain and engages the RFC5 subunit using a novel {beta}-hairpin at the disordered N-terminus. We show that deletion of this {beta}-hairpin impairs the CTF18-RFC-PCNA complex stability, slows down clamp loading, and decreases the rate of primer synthesis by Pol {varepsilon}. Our research identifies distinctive structural characteristics of the human CTF18-RFC complex, providing insights into its role in PCNA loading and the stimulation of leading strand synthesis by Pol {varepsilon}.

biochemistry↗

bioRxiv2024

CryoEM structure of the di-domain core of Mycobacterium tuberculosis polyketide synthase 13, essential for mycobacterial mycolic acid synthesis.

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis, the bacterial organism causing tuberculosis (TB), catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 [A]. We observe a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predict to contain a dimeric domain with similarity to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, that combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

microbiology↗

bioRxiv2024

Internal unwinding mechanism of a replicative helicase

Ring-shaped hexameric helicases are nucleotide hydrolases that unwind double-stranded DNA into single strands, a necessary step in DNA replication. Two main questions are critical to understanding their function: the location of DNA strand separation within the helicase, and the precise dynamic linkage between nucleotide hydrolysis and DNA translocation. We explore these questions by employing cryo-EM to visualize the translocation mechanics of a AAA+ helicase, the SV40 Large Tumor Antigen (LTag), on forked DNA. We find the DNA fork nexus is positioned deep within the core helicase domain, with each DNA-binding loop from the six subunits securing the tracking strand by forming a paired staircase spiral that substitutes the passive strand. This structure forges an internal separation wedge, channeling the passive strand through a gap in the subunit C-tiers at the back of the helicase. Via cryo-EM continuous heterogeneity analysis we capture, and exhaustively model, a seamless spectrum of conformations of translocating LTag at high resolution. ATP hydrolysis at the tightest inter-subunit interface operates like an entropy switch, triggering coordinated rigid-body rotations of the subunit C-tiers and DNA-binding loops, resulting in directional escorting of the tracking strand through the central channel, concomitantly setting preparatory steps for cycle restart. Overall, we demonstrate a dynamic model for hydrolysis-coupled translocation and DNA unwinding by a model helicase active in replication, with implications for origin unwinding. High structural conservation of core helicase regions suggests this mechanism is applicable to hexameric helicases across domains.

biophysics↗

bioRxiv2024

Structure of Aquifex aeolicus Lumazine Synthase by Cryo-Electron Microscopy to 1.42 Angstrom Resolution

Single particle Cryo-Electron microscopy (Cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution at which individual atoms, including hydrogen atoms, can be resolved. Thus Cryo-EM allows not only unprecedented detail regarding the structural architecture of complexes but also a better understanding surrounding their chemical states. In this study we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5[A] or better which so far has only been achieved for the iron carrier ferritin. Using state of the art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42[A] map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48-hour microscope session. In addition to water molecules and ligands involved in AaLS function, we can observe positive density for [~]50% of hydrogen atoms. A small improvement in resolution was achieved by Ewald sphere correction which was expected to limit the resolution to [~]1.5[A] for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

biophysics↗

bioRxiv2023

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR complex

SummaryLiving organisms have developed protein sensors helping them to adapt to their environment1. The aryl hydrocarbon receptor (AHR) is an emblematic member of this class of proteins, and a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites2. However, in the absence of high-resolution structural data, a molecular understanding of how AHR is activated by such diverse compounds is lacking. Here we present a 2.85 [A] cryo-electron microscopy structure of the cytosolic complex comprising AHR bound to the ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2. The structure reveals a closed Hsp90 dimer with AHR threaded through its lumen. XAP2 directly interacts with Hsp90 and the AHR ligand-binding domain, thereby acting as a brace stabilizing the entire complex. Importantly, we provide the first experimental visualization of the AHR PAS-B domain bound to a ligand, revealing a unique organization of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing unprecedented structural details of the molecular initiating event leading to AHR activation, our study rationalizes prior biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

biochemistry↗

bioRxiv2021

In vitro evolution predicts emerging CoV-2 mutations with high affinity for ACE2 and cross-species binding

Emerging SARS CoV-2 variants are creating major challenges in the ongoing Covid-19 pandemic. Predicting CoV-2 mutations that increase transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines and prioritising viral monitoring and containment. Using in vitro evolution, we identify a double mutation in CoV-2 receptor binding domain (RBD) that increases affinity for ACE2 almost 20-fold. We determine the mutant:ACE2 structure to reveal the binding mechanism and show the main affinity driver, Q498H, boosts binding of other RBD variants. We find this mutation incompatible with the common N501Y mutation, but N501Y variants can acquire Q498R to access a similar bonding network and affinity gain. We show Q498H, and Q498R plus N501Y, enable variants to bind rat ACE2 with high affinity. These mutations are now emerging in CoV-2 variants, such as the Omicron variant, where they would be expected to drive increased human-to-human and cross-species transmission.

biochemistry↗