Biology subjects

Isupov, M. N.

Publications and source records attributed to Isupov, M. N..

14 recordsLinked to original sources

bioRxiv2025

Cryo-EM reveals multiple mechanisms of ribosome inhibition by doxycycline

Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A-site) of the small subunit. Using cryo electron-microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal unexpected binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major uncharacterised conformation of the ribosome. This fundamentally reorganises the peptidyl transferase centre and blocks tRNA binding, challenging the concept that this region is largely static. We identify a new ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics.

biochemistry↗

bioRxiv2025

New clues about ribosome hibernation and activation in microsporidia revealed by cryo electron tomography

Microsporidia are single-celled parasitic eukaryotes that alternate between a dormant environmental phase and a metabolically active, progeny-producing phase inside their animal hosts. A successful transition between these phases requires a tight regulation of translation, which must be suppressed during dormancy and rapidly reinitiated upon host ingress. Previous studies by us and others have shown that translational silencing in microsporidia is underpinned by ribosome hibernation, a process involving both the binding of hibernation factors and the formation of ribosome dimers. Using focussed ion beam milling and cryo-electron tomography to image dormant microsporidian spores, we have gained new structural insights into this hibernation mechanism. Our data reveal that ribosome dimers within the spores assemble into ordered arrays surrounding the convex surface of polar tube, a specialised organelle essential for host cell invasion. Sub-tomogram averaging resolved the structure of the spore-borne ribosomes, further extending our understanding of ribosome hibernation in microsporidia.

cell biology↗

bioRxiv2025

Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

Extremolytes - unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their promise, only a few extremolytes, such as ectoine and hydroxyectoine, are commercially established, primarily due to the lack of efficient production strategies for other compounds. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2 U mg-1, with KM values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 minutes, using just 0.5 U of recombinant MfcDPGS at 55{degrees}C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value-added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

bioengineering↗

bioRxiv2025

Structure and catalytic mechanism of methylisocitrate lyase, a potential drug target against Coxiella burnetii

We present a comprehensive investigation into the catalytic mechanism of methylisocitrate lyase, a potential drug target candidate against the zoonotic pathogen Coxiella burnetii, the causative agent of Q fever and a federal select agent. Current treatment regimens are prolonged, often with incomplete clearance of the pathogen. We utilised a structure-based bioinformatics pipeline to identify methylisocitrate lyase as a candidate therapeutic target against C. burnetii from a list of essential genes. Wild-type C. burnetii methylisocitrate lyase has a kcat of 32,000 s-1 (compared to 105 s-1 for Salmonella enterica) and isocitrate inhibits with a KI of 6 mM. We have determined the previously uncharacterised substrate-bound structure of this enzyme family, alongside product and inhibitor-bound structures. These structures of wild-type enzyme reveal that in the active state the catalytic C118 is positioned 2.98 [A] from O5 of methylisocitrate and Arg152 moves towards the substrate relative to the inhibitor bound structure. Analysis of structure-based mutants reveals that Arg152 and Glu110 are both essential for catalysis. We suggest that Arg152 acts as the catalytic base that initiates the methylisocitrate lyase reaction. These results deepen our understanding of the catalytic mechanism of methylisocitrate lyase and could aid the development of new therapeutics against C. burnetii.

biochemistry↗

bioRxiv2024

Cryo-EM resolves the structure of the archaeal dsDNA virus HFTV1 from head to tail

Outnumbering their hosts by at least a factor of 10, viruses are the most common biological entity on Earth, are major drivers of evolution, and greatly impact on the dynamics of our planets ecosystems. While viruses infecting bacteria and eukaryotes have been extensively studied, the viruses roaming the archaeal domain remain largely unexplored. In recent years, a growing number of archaeal viruses have been described, revealing a stunningly diverse range of morphologies that appear unique to archaea. Detailed structural studies are paramount to fully understand how archaeal viruses infect their hosts. However, no complete atomic models of archaeal viruses are available to date. Using electron cryo-microscopy, we investigated the structure of the archaeal virus Haloferax tailed virus 1 (HFTV1), which infects the halophile Haloferax gibbonsii LR2-5 originating from the Senegalese salt lake Retba. Through single particle analysis, we achieved near-atomic resolution for the entire set of HFTV1s structural proteins, enabling the building of a full atomic model of the virion. Comparing the structures of DNA filled and empty capsids, we visualise structural changes occurring upon DNA ejection. By investigating the double-stranded DNA inside the capsid, we elucidate how the genome is spooled upon loading. Furthermore, our structure reveals putative cell-surface receptor-binding and catalytic roles of capsid turret, baseplate, and tail fibre proteins. Together, our data provide new insights into the mechanisms of HFTV1 assembly and infection, unveiling new perspectives on general rules of host-virus interactions in archaea and their evolutionary links to bacterial and eukaryotic viruses.

microbiology↗

bioRxiv2024

Binding of SARS-CoV-2 nucleocapsid protein to uninfected epithelial cells induces antibody-mediated complement deposition

SARS-CoV-2 infection triggers strong antibody response toward Nucleocapsid-Protein (NP), suggesting extracellular presence beyond its intra-virion RNA binding. Interestingly, NP was found to decorate infected and proximal uninfected cell-surfaces. Here, we propose a new mechanism through which extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated linear-glycosaminoglycans by spatial rearrangement of its RNA-binding sites facilitated by the flexible, positively charged, linker. Coating of uninfected lung-derived cells with purified NP attracted anti-NP-IgG from lung fluids and sera collected from COVID-19 patients. The magnitude of this immune recognition was significantly elevated in moderate compared to mild COVID-19 cases. Importantly, binding of anti-NP-IgG present in sera generated clusters that triggered C3b deposition by the classical complement pathway. Heparin analog enoxaparin outcompeted NP-binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings unveil how extracellular NP may exacerbate COVID-19 tissue damage, and suggest leads for preventative therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/585388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1084e20org.highwire.dtl.DTLVardef@4126caorg.highwire.dtl.DTLVardef@159c610org.highwire.dtl.DTLVardef@da7b7f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIgG from patients sera target NP-bound cells resulting in complement activation C_LIO_LIThe flexible linker allows NP to both bind linear sulfated GAGs and wrap around RNA C_LIO_LIHeparin analogs prevent NP surface binding and alleviate complement activation C_LIO_LICell-ELISA anti-NP IgG levels differ between mild and moderate COVID-19 C_LI

immunology↗

bioRxiv2023

CryoEM reveals the structure of an archaeal pilus involved in twitching motility

Amongst the major archaeal filament types, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus (Uvp) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Uvp in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. All previously solved Aap appear to have almost identical helical structures. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.

microbiology↗

bioRxiv2022

Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single particle cryo electron microscopy (cryoEM), cryo electron tomography (cryoET) and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesize that jackknife-like conformational changes, as well as pH-induced alterations in the surface charge of SlaA, play important roles in S-layer assembly.

microbiology↗

bioRxiv2022

In situ structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.

molecular biology↗

bioRxiv2022

Donor strand complementation, isopeptide bonds and glycosylation stabilise highly resilient archaeal thread filaments

Pili are ubiquitous filamentous surface extensions that play crucial roles for bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer to name a few. Here we report on the discovery and structure of the archaeal thread - a remarkably stable archaeal pilus that belongs to a so-far largely unknown class of protein filaments. We find that the filament is highly glycosylated and interconnected via donor strand complementation, as well as isopeptide bonds, reminiscent of bacterial type I pili. Despite striking structural similarity with bacterial type-1 pili, archaeal threads appear to have evolved independently and are likely assembled by a markedly distinct mechanism.

microbiology↗

bioRxiv2021

Spinning sugars in antigen biosynthesis: a direct study of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalysed by the enzymes RmlA/RmlB/RmlC/RmlD. Streptose and DHHS biosynthesis unusually require a ring contraction step that might be performed by the orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii proposed the StrM and CBU1838 proteins respectively as RmlC orthologues. Here, we demonstrate through both coupled and direct observation studies that both enzymes can perform the RmlC 3,5 double epimerisation activity; and that this activity supports TDP-rhamnose biosynthesis in vivo. We demonstrate that proton exchange is faster at the 3 position than the 5 position, in contrast to a previously studied orthologue. We solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to previously characterised enzymes. These results further support the hypothesis that streptose and DHHS are biosynthesised using the TDP pathway and are consistent with the ring contraction step being performed on a double epimerised substrate, most likely by the RmlD paralogue. This work will support the determination of the full pathways for streptose and DHHS biosynthesis.

biochemistry↗

bioRxiv2021

New insights into the architecture and dynamics of archaella

Archaea swim by means of a unique molecular machine called the archaellum. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament, allowing the cell to rapidly propel itself through liquid media. The archaellum filament comprises multiple copies of helically organised subunits named archaellins. While in many species several archaellin homologs are encoded in the same operon, structural studies conducted to date have suggested that archaella consist of only one protein species. Thus, the role of the remaining archaellin genes remains elusive. Here we present the structure of the Methanocaldococcus villosus archaellum filament at 3.08 [A] resolution. We find that the filament is composed of two alternating archaellins - ArlB1 and ArlB2, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify two major structural elements that enable the archaellum filament to move. Our findings provide new insights into archaeal motility and challenge the current view on the archaellum architecture and assembly.

microbiology↗

bioRxiv2021

Structure and function of N-acetylglucosamine kinase illuminates the catalytic mechanism of ROK kinases

N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolise environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase NagK performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analogue AMP-PNP. Surprisingly, PsNagK showed two conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Molecular dynamics modelling of catalytic ion binding confirmed the location of the essential catalytic metal. Site-directed mutagenesis confirmed the catalytic base, and that the metal coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.

biochemistry↗

bioRxiv2020

The Structural Basis for SARM1 Inhibition, and Activation Under Energetic Stress

SARM1 is a central executor of axonal degeneration (1). Mechanistically, SARM1 contains NADase activity, which, in response to nerve injury, depletes the key cellular metabolite, NAD+ (2-5). Interestingly, SARM1 knockout mouse models do not present any apparent physiological impairment. Yet, the lack of SARM1 protects against various neuropathies (6, 7), thereby highlighting SARM1 as a likely safe and effective drug target (8). However, the absence of a SARM1 structure, in its active or inhibited form, makes it impossible to understand the molecular basis of SARM1 inhibition, and its activation under stress conditions. In this study we present two cryo-EM maps of SARM1 (at 2.6 [A] and 2.9 [A] resolution). We show that the inhibited SARM1 homo-octamer assumes a packed conformation with well-ordered inner and peripheral rings. Here the catalytic TIR domains are held apart from each other and are unable to form dimers, which is a prerequisite for NADase activity. More importantly, after screening several cellular metabolites we discovered that the inactive conformation is stabilized by the binding of SARM1s own substrate: NAD+. The NAD+ inhibitory allosteric site is located away from the NAD+ catalytic site of the TIR domain. Site-directed mutagenesis of the allosteric site leads to constitutive active SARM1. Based on our data we propose that a reduction of cellular NAD+ concentrations (an early indication of disease-associated and age-related neurodegeneration (9)) disassemble SARM1s peripheral ring, which allows NADase activity. This leads to an energetic catastrophe and eventually cell death. The discovery of the allosteric inhibitory site opens the door for the development of effective drugs that will prevent SARM1 activation, rather than compete for binding to the NADase catalytic site. Brief descriptionIt is not known how NAD+ depletion brings about neurodegeneration. Here, we show that the intrinsic NADase activity of SARM1 is allosterically inhibited by physiological concentrations of NAD+. NAD+ stabilizes a compact, auto-inhibited conformation of the SARM1 octamer. Once NAD+ levels are depleted, the allosteric inhibition is released, enabling SARM1s NADase activity, which eventually leads to energetic catastrophe and cell death.

biochemistry↗