Biology subjects

Blaza, J. N.

Publications and source records attributed to Blaza, J. N..

15 recordsLinked to original sources

bioRxiv2026

A 13-subunit c-ring in the Chlamydomonas chloroplast ATP synthase lowers the H⁺/ATP cost of carbon fixation

The chloroplast F1Fo ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H+/ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO2 fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 [A] structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c14 in a chloroplast, and with a lower predicted H+/ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching {gamma}-subunit abolishes the contact with the catalytic {beta}-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H+/ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

biochemistry↗

bioRxiv2026

Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures

Mycobacteria synthesise the unusual glycan [x1D05]-arabinan as a major component of the cell wall glycoconjugates arabinogalactan (AG) and lipoarabinomannan (LAM). We previously identified Dysgonomonas gadei, a member of the Bacteroidota, as capable of complete [x1D05]-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo--[x1D05]-arabinofuranosidase activity against AG and LAM, although their linkage specificities were unknown. Here, using defined synthetic substrates, we show that the three enzymes possess distinct linkage preferences. We also develop -[x1D05]-arabinofuranosyl cyclophellitol aziridines as covalent inhibitors and activity-based probes for GH172 enzymes. X-ray crystallography and cryo-EM to reveal strikingly different quaternary assemblies across the three homologues, while a 1.5 [A] cryo-EM structure of dodecameric Dg67 covalently modified by an aziridine inhibitor identifies the catalytic nucleophile and provides direct structural support for a retaining mechanism. A BODIPY-tagged aziridine probe selectively labelled the three GH172 enzymes in D. gadei cell lysates. Together, these findings define functional and structural diversity within GH172 and establish chemical probes for profiling -[x1D05]-arabinofuranosidase activity in complex biological samples.

biochemistry↗

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2025

Insights from aquaporin structures into drug-resistant sleeping sickness

Trypanosoma brucei is the causal agent of African trypanosomiasis in humans and animals, the latter resulting in significant negative economic impacts in afflicted areas of the world. Resistance has arisen to the trypanocidal drugs pentamidine and melarsoprol through mutations in the aquaglyceroporin TbAQP2 that prevent their uptake. Here we use cryogenic electron microscopy to determine the structure of TbAQP2 from Trypanosoma brucei, bound to either the substrate glycerol or to the sleeping sickness drugs, pentamidine or melarsoprol. The drugs bind within the AQP2 channel at a site completely overlapping that of glycerol. Mutations leading to a drug-resistant phenotype were found in the channel lining. Molecular dynamics simulations showed the channel can be traversed by pentamidine, with a low energy binding site at the centre of the channel, flanked by regions of high energy association at the extracellular and intracellular ends. Drug-resistant TbAQP2 mutants are still predicted to bind pentamidine, but the much weaker binding in the centre of the channel observed in the MD simulations would be insufficient to compensate for the high energy processes of ingress and egress, hence impairing transport at pharmacologically relevant concentrations. The structures of drug-bound TbAQP2 represent a novel paradigm for drug-transporter interactions that could provide new mechanisms for targeting drugs into pathogens and human cells.

molecular biology↗

bioRxiv2025

Bioenergetic metabolism restarts alongside germinant sensing and hydration in bacterial spore germination

Bacterial spore germination transforms spores from dormant bodies to vegetative cells. Initiation of germination or spore rehydration can proceed without exogenous energy sources, so bioenergetic processes in germination have been overlooked, despite many germinants being energy sources, such as sugars. Here, we apply remission spectroscopy to noninvasively measure the energy-transducing electron-transport chain of intact spores during germination. In Bacillus megaterium and Bacillus subtilis, we find energisation of cytoplasmic metabolism and the electron transport chain occurs early in germination, before or alongside rehydration. The aa3-type oxidases (Qox, Cta) accumulate nonradical ferryl intermediates of their catalytic cycle demonstrating that the electron-transport chain is operating in a regime of high membrane potential. The Yth isoform of the bd oxidase is found in abundance in both organisms, functioning to allow rapid electron transfer to O2 when the aa3-type oxidases are hindered, establishing a new role for this enzyme beyond O2-scavenging. Deletion of Yth slows rehydration, directly linking bioenergetic processes to germination. Our data lead us to propose a powered germination model, where the Ger-mediated signalling cascade and bioenergetic processes occur simultaneously and semi-independently.

biophysics↗

bioRxiv2024

Remission spectroscopy resolves the mode of action of bedaquiline within living mycobacteria

Bedaquiline, an ATP synthase inhibitor, is the spearhead of transformative therapies against drug-resistant Mycobacterium tuberculosis. Here, remission spectroscopy is used to measures the energy-transducing cytochromes within unperturbed, respiring suspensions of mycobacterial and human cells, allowing spectroscopic measurements of electron transport chains as they power living cells and respond to bedaquiline. No evidence is found for protonophoric or ionophoric uncoupling. Rather, by directly inhibiting ATP synthase, bedaquiline slows the respiratory supercomplex (Qcr:Cta; bcc:aa3) by increasing the proton-motive force, causing sub-second redirection of electron flux through the cytochrome bd oxidase (Cyd) to O2. Electron flux redirection explains the idiosyncratic bedaquiline-induced increase in O2 consumption rates previously observed. Redirection occurs as Cyd is present even in cells grown in plentiful O2. Applying the same approach to human cells did not detect bedaquiline-induced inhibition of mitochondrial function despite such inhibition being seen in isolated systems. Overall, we clarify how bedaquiline works, why different models for its action developed, and the mechanisms underlying the synergy of bedaquiline in combination regimes.

biophysics↗

bioRxiv2024

Structural unification of diverse membrane-boundacyltransferases reveals a conserved fold that defines the Transmembrane Acyl Transferase (TmAT) superfamily

The movement of acyl groups across biological membranes is essential for many cellular processes. One major family of proteins catalysing this reaction are the acyl transferase family 3 (AT3) proteins, which form a pore to allow acyl-CoA to penetrate the membrane for transfer onto an extracytosolic acceptor molecule. Recent structures of the sequence-unrelated human heparan--glucosaminide N-acetyltransferase (HGSNAT) support a similar transmembrane acyl-group transfer mechanism. Here we demonstrate that both protein families contain a conserved 10-transmembrane helical fold with high structural and detectable sequence conservation around the acyl-CoA pore, supporting the previously proposed Transmembrane Acyl Transferase (TmAT) protein superfamily. In addition, we identify TmAT proteins, including the human Golgi sialate-O-acetyltransferase (CASD1), the human/fungal PIG-W/GWT1 enzymes and the bacterial vancomycin resistance protein VanTG, where the TmAT domains function has been largely unrecognised. We conclude that the TmAT fold represents an ancient architecture for transmembrane acyl-group transfer with important roles in the dynamic modification of glycans in diverse processes across the three domains of life.

biochemistry↗

bioRxiv2024

A stargate mechanism of Microviridae genome delivery unveiled by cryogenic electron tomography

Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.

microbiology↗

bioRxiv2024

A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage

CO2 fixation is commonly limited by inefficiency of the CO2-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO2 in phase-separated condensates called pyrenoids, which complete up to one-third of global CO2 fixation. Condensation of Rubisco in pyrenoids is dependent on interaction with disordered linker proteins that show little conservation between species. We developed a sequence-independent bioinformatic pipeline to identify linker proteins in green algae. We report the linker from Chlorella and demonstrate that it binds a conserved site on the Rubisco large subunit. We show the Chlorella linker phase separates Chlamydomonas Rubisco and that despite their separation by [~]800 million years of evolution, the Chlorella linker can support the formation of a functional pyrenoid in Chlamydomonas. This cross-species reactivity extends to plants, with the Chlorella linker able to drive condensation of some native plant Rubiscos in vitro and in planta. Our results represent an exciting frontier for pyrenoid engineering in plants, which is modelled to increase crop yields.

plant biology↗

bioRxiv2023

Cryo-EM structure of the CDK2-cyclin A-CDC25A complex

The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich motif of cyclin-dependent protein kinases (CDKs) to activate CDK activity. Here, we present the cryogenic-electron microscopy (cryo-EM) structure of CDK2-cyclin A in complex with CDC25A at 2.91 [A] resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further reveal an unanticipated CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25- targeting anticancer strategies.

biochemistry↗

bioRxiv2023

Tumour mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade

The mitochondrial genome encodes essential machinery for respiration and metabolic homeostasis but is paradoxically among the most common targets of somatic mutation in the cancer genome, with truncating mutations in respiratory complex I genes being most over-represented1. While mitochondrial DNA (mtDNA) mutations have been associated with both improved and worsened prognoses in several tumour lineages1-,3, whether these mutations are drivers or exert any functional effect on tumour biology remains controversial. Here we discovered that complex I-encoding mtDNA mutations are sufficient to remodel the tumour immune landscape and therapeutic resistance to immune checkpoint blockade. Using mtDNA base editing technology4 we engineered recurrent truncating mutations in the mtDNA-encoded complex I gene, Mt-Nd5, into murine models of melanoma. Mechanistically, these mutations promoted utilisation of pyruvate as a terminal electron acceptor and increased glycolytic flux without major effects on oxygen consumption, driven by an over-reduced NAD pool and NADH shuttling between GAPDH and MDH1, mediating a Warburg-like metabolic shift. In turn, without modifying tumour growth, this altered cancer cell-intrinsic metabolism reshaped the tumour microenvironment in both mice and humans, promoting an anti- tumour immune response characterised by loss of resident neutrophils. This subsequently sensitised tumours bearing high mtDNA mutant heteroplasmy to immune checkpoint blockade, with phenocopy of key metabolic changes being sufficient to mediate this effect. Strikingly, patient lesions bearing >50% mtDNA mutation heteroplasmy also demonstrated a >2.5-fold improved response rate to checkpoint inhibitor blockade. Taken together these data nominate mtDNA mutations as functional regulators of cancer metabolism and tumour biology, with potential for therapeutic exploitation and treatment stratification.

cancer biology↗

bioRxiv2023

Sulfolactaldehyde dehydrogenase of Rhizobium leguminosarum: Kinetic and structural analysis

Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle. Graphical abstractSulfolactate is an important species in the biogeochemical sulfur cycle. Herein we report the 3D cryo-EM structure and kinetics of its biosynthetic enzyme, sulfolactaldehyde dehydrogenase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/532361v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f3d1d3org.highwire.dtl.DTLVardef@548615org.highwire.dtl.DTLVardef@12efc0eorg.highwire.dtl.DTLVardef@1a6145c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Structure of dormant bacterial ribosomes reveals an elusive translation factor with evolutionary connections to eukaryotic protein synthesis factors

During starvation and stress, virtually all organisms arrest protein synthesis to conserve energy. Inactive ribosomes are converted into a dormant state, in which they are protected from damage by hibernation factor proteins. In bacteria, two major families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats, and environmental stressors has confounded their discovery. In this study, using proteomics and cryo-EM, we identify a new dormancy factor from the psychrophilic bacterium Psychrobacter urativorans. By isolating ribosomes under cold-shock conditions, we observe a previously unknown protein bound to the ribosomal A site, protecting critical elements of both the decoding and peptidyl transferase centers. We show that this new factor, which we term Balon, is a homolog of the archaeo-eukaryotic translation factor aeRF1, providing a long-predicted evolutionary "missing link" between the eukaryotic and bacterial translation machinery. Our structures reveal that Balon is delivered to both vacant and actively translating ribosomes by EF-Tu, highlighting an unexpected and previously unknown role for this elongation factor in the bacterial stress response. We describe several unique structural motifs that allow Balon to bind ribosomes in an mRNA-independent manner, initiating a new mode of ribosome dormancy that can commence while ribosomes are still engaged in protein synthesis. Our bioinformatic analysis shows that putative Balon-encoding genes can be found within stress-response operons in nearly 20 % of all known bacterial species, including many human pathogens. Taken together, our work suggests that Balon/EF-Tu regulated ribosome dormancy is likely to be a ubiquitous stress-response mechanism throughout the bacterial kingdom. These findings call for a revision of our model of bacterial translation inferred from common model organisms and hold numerous implications for how we understand and study ribosome dormancy.

molecular biology↗

bioRxiv2022

Structural Basis of Mammalian Respiratory Complex I Inhibition by Medicinal Biguanides

The molecular mode of action of metformin, a biguanide used widely in the treatment of diabetes, is incompletely characterized. Here we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo-electron microscopy and enzyme kinetics. We explain the unique selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel and an additional binding site is in a pocket on the intermembrane space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable rational design of novel medicinal biguanides. One-Sentence SummaryBiguanides inhibit complex I by binding in the quinone channel, and exert an independent localized chaotropic effect.

biochemistry↗

bioRxiv2017

Structure of the deactive state of mammalian respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly-active preparation of Bos taurus complex I into the biochemically-defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 [A] resolution. The deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding templates their reordering. Our structure both rationalizes biochemical data on the deactive state, and offers new insights into its physiological and cellular roles.

biochemistry↗