Biology subjects

Hirst, J.

Publications and source records attributed to Hirst, J..

18 recordsLinked to original sources

bioRxiv2026

A modular mechanism determines the proton-pumping stoichiometry of respiratory complex I

Respiratory complex I, a crucial metabolic enzyme, uses the reducing potential of NADH to pump protons across energy-transducing membranes, but its mechanism of catalysis remains unknown. Here, guided by detailed structural information, we combine targeted mutagenesis and biophysical analyses to define the proton-pumping pathways in the three antiporter-like (proton-pumping) subunits of complex I from Paracoccus denitrificans and Escherichia coli. In Pd-CI, a four-proton pump, one of the three proton-uptake pathways is inactive, reconciling the four-proton/three-subunit mismatch. In Ec-CI two pathways are inactive - and Ec-CI pumps only two protons. We define the structural determinants for redox-coupled proton uptake, establish a mechanistic framework for proton pumping, and reveal how the proton-pumping stoichiometry has been adapted to deliver efficient energy conservation in diverse bioenergetic environments.

biochemistry↗

bioRxiv2026

Conformational gating at histidine junctions coordinates proton translocation in respiratory complex I

Respiratory complex I, a central enzyme in cellular metabolism, converts the free energy of NADH oxidation into a transmembrane proton-motive force to drive ATP synthesis, but the molecular mechanisms by which it couples redox catalysis to vectorial proton translocation remain unresolved. Here, we present high-resolution cryo-EM structures of complex I from Bos taurus captured under conditions designed to change the protonation states of residues in the membrane domain. Our structures reveal conformational rearrangements at key pathway junctions that reconfigure proton-transfer connections. In ND5, helical rearrangements switch the connectivity of histidine-248 between proton-uptake and proton-output pathways. In ND4, rotameric changes of histidine-220 alternately enable proton uptake or lateral proton transfer along the membrane domain. Combined with molecular simulations, our structures define gating mechanisms that impose directionality on proton transfer reactions and provide a framework for proton-coupled energy transduction in complex I.

biochemistry↗

bioRxiv2026

Rational scaffold design mitigates mitochondrial complex I off-target inhibition in bifunctional degraders

Chemical inducers of proximity have transformed small-molecule pharmacology, but the large, bifunctional architectures they often employ introduce new and poorly understood off-target risks. During a targeted protein degrader synthesis project, we identified a subset of compounds that cause rapid and unexpected ATP depletion in cells. Mechanistic studies traced this effect to inhibition of mitochondrial complex I, a central component of oxidative phosphorylation. This inhibition does not stem from off-target binding by either of the two target ligands, but from the overall long, linear architecture of the bifunctional molecules, which renders them effective ligands of the narrow ubiquinone binding tunnel of complex I. Strikingly, this liability extends to structurally unrelated bifunctional molecules, including six androgen receptor PROTACs including the clinical candidate ARV-110, which inhibits complex I at low nanomolar concentrations. To mitigate complex I inhibition, we established a generalizable design strategy to disrupt linear molecular geometry through the introduction of structural "bumps" or "kinks". In a proof-of-concept study, we successfully apply this strategy to the ARV-110 scaffold, discovering potent AR-degrading ARV-110 analogs that do not inhibit complex I. These findings uncover a previously underappreciated structural determinant of off-target mitochondrial toxicity and establish new design principles for safer proximity-inducing therapeutics.

cancer biology↗

bioRxiv2024

Structure of the turnover-ready state of an ancestral respiratory complex I

Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-[A] resolution structure of complex I from the -proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.

biochemistry↗

bioRxiv2023

Structural basis of a regulatory switch in mammalian complex I

Respiratory complex I powers oxidative phosphorylation in mammalian mitochondria by using the reducing potential of NADH to reduce ubiquinone-10 and drive protons across the inner mitochondrial membrane. High-resolution cryoEM structures have provided a molecular framework for complex I catalysis, but controversies about how to assign functional properties to the states identified in single-particle analyses are preventing progress on its energy-converting mechanism. Here, we combine precise biochemical definition with high-resolution cryoEM structures in the phospholipid bilayer of coupled vesicles and show that the closed and open states observed in mammalian complex I preparations are components of the deactive transition that occurs during ischaemia. Populations of the cryoEM open state and biochemical deactive state match exactly. Deactivation switches the enzyme off, converting the closed state that is capable of rapid, reversible catalysis into an open, dormant state that is unable to start up in reverse. The deactive state is switched back on by slow priming reactions with NADH and ubiquinone-10. Thus, by developing a versatile membrane system to unite structure and function, we define the role of large-scale conformational transitions in complex I and establish a new gold standard for structure-based investigations of catalysis by energy-coupled proteins.

biochemistry↗

bioRxiv2023

Structural insights into complex I deficiency and assembly from the disease-related ndufs4-/- mouse

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4-/- mouse, to define the structural, biochemical and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of mouse-heart ndufs4-/- complex I revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12 (that replaces its paralogue NDUFAF2 in mature complex I) is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free but otherwise complete enzyme. We propose NDUFAF2 as the recruiter of the NADH dehydrogenase module during assembly of the complex. Our results provide new molecular level understanding of the ndufs4-/- mouse model and complex I-linked mitochondrial disease.

biochemistry↗

bioRxiv2023

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III2 and IV (CI, CIII2 and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI- CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knock-in mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in the mouse, but raises questions about their alternate functions, such as relating to regulation of protein stability and prevention of age-associated protein aggregation.

molecular biology↗

bioRxiv2023

The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors

Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent third-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in [~]5-15% of patients. However, it is not fully understand how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.

pharmacology and toxicology↗

bioRxiv2022

Cryo-EM structures of mitochondrial respiratory complex I from Drosophila melanogaster

Respiratory complex I powers ATP synthesis by oxidative phosphorylation, exploiting the energy from NADH oxidation by ubiquinone to drive protons across an energy-transducing membrane. Drosophila melanogaster is a candidate model organism for complex I due to its high evolutionary conservation with the mammalian enzyme, well-developed genetic toolkit, and complex physiology for studies in specific cell types and tissues. Here, we isolate complex I from Drosophila and determine its structure, revealing a 43-subunit assembly with high structural homology to its 45-subunit mammalian counterpart, including a hitherto unknown homologue to subunit NDUFA3. The major conformational state of the Drosophila enzyme is the mammalian-type ready-to-go active resting state, with a fully ordered and enclosed ubiquinone-binding site, but a subtly altered global conformation related to changes in subunit ND6. The mammalian-type deactive pronounced resting state is not observed: in two minor states the ubiquinone-binding site is unchanged, but a deactive-type{pi} -bulge is present in ND6-TMH3. Our detailed structural knowledge of Drosophila complex I provides a foundation for new approaches to disentangle mechanisms of complex I catalysis and regulation in bioenergetics and physiology.

biochemistry↗

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↗

bioRxiv2022

Accurate tropical forest individual tree crown delineation from RGB imagery using Mask R-CNN

Tropical forests are a major component of the global carbon cycle and home to two-thirds of terrestrial species. Upper-canopy trees store the majority of forest carbon and can be vulnerable to drought events and storms. Monitoring their growth and mortality is essential to understanding forest resilience to climate change, but in the context of forest carbon storage, large trees are underrepresented in traditional field surveys, so estimates are poorly constrained. Aerial photographs provide spectral and textural information to discriminate between tree crowns in diverse, complex tropical canopies, potentially opening the door to landscape monitoring of large trees. Here we describe a new deep convolutional neural network method, Detectree2, which builds on the Mask R-CNN computer vision framework to recognise the irregular edges of individual tree crowns from airborne RGB imagery. We trained and evaluated this model with 3,797 manually delineated tree crowns at three sites in Malaysian Borneo and one site in French Guiana. As an example application, we combined the delineations with repeat lidar surveys (taken between 3 and 6 years apart) of the four sites to estimate the growth and mortality of upper-canopy trees. Detectree2 delineated 65,000 upper-canopy trees across 14 km2 of aerial images. The skill of the automatic method in delineating unseen test trees was good (F1 score = 0.64) and for the tallest category of trees was excellent (F1 score = 0.74). As predicted from previous field studies, we found that growth rate declined with tree height and tall trees had higher mortality rates than intermediate-size trees. Our approach demonstrates that deep learning methods can automatically segment trees in widely accessible RGB imagery. This tool (provided as an open-source Python package) has many potential applications in forest ecology and conservation, from estimating carbon stocks to monitoring forest phenology and restoration. Python package available to install at https://github.com/PatBall1/Detectree2

ecology↗

bioRxiv2022

Superinfection exclusion creates spatially distinct influenza virus populations

Influenza viruses can interact during coinfections, allowing viral fitness to be altered by genome complementation and competition, and increasing population diversity through reassortment. However, opportunities for these interactions are limited, as coinfection is blocked shortly after primary infection by a process known as superinfection exclusion (SIE). We asked whether SIE, which occurs at the level of individual cells, could limit within-host interactions between populations of influenza viruses as they spread across regions of cells. We first created a simplified model of within-host spread by infecting monolayers of cells with two isogenic influenza A viruses, each encoding a different fluorophore, and measuring the proportion of coinfected cells. In this system SIE begins within 2-4 hours of primary infection, with the kinetics of onset defined by the dose of primary virus. We then asked how SIE controls opportunities for coinfection as viruses spread across a monolayer of cells. We observed that viruses spreading from a single coinfected focus continued to coinfect cells as they spread, as all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before SIE blocked further coinfection. This patterning was recapitulated in the lungs of infected mice and is likely to apply to other viruses that exhibit SIE. It suggests that the kinetics of SIE onset separate a spreading infection into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked. ImportanceViral fitness and diversity are altered by genome interactions, which occur when multiple viruses coinfect a cell. This has been extensively studied for influenza A viruses (IAV), which use genome reassortment to adapt to new hosts and create pandemic strains, and whose replication can be compromised by the acquisition of defective-interfering RNAs. Coinfection of an individual cell by IAV is restricted by the gradual onset of superinfection exclusion (SIE). Replication of IAVs within host organisms involve the asynchronous replication of viruses as they spread to infect multiple cells. We found that under these circumstances, SIE creates spatially separated sub-populations of IAV, between which there are limited opportunities for genome interactions. Our work suggests SIE will cause many viruses to segregate into distinct subpopulations within their hosts, constraining the effects of genome interactions on their fitness and evolution.

microbiology↗

bioRxiv2022

Stable and ancient endocytic structures navigate the complex pellicle of apicomplexan parasites

Apicomplexan parasites have an immense impact on humanity, but their basic cellular processes are often poorly understood. The sites of endocytosis, the conservation of this process with other eukaryotes, and its functions across Apicomplexa are major unanswered questions. Yet endocytosis in Plasmodium is implicated in antimalarial drug failure. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. Moreover, conserved molecular adaptation of this structure is seen in Apicomplexa, including the kelch-domain protein K13 central to malarial drug-resistance. We determine that an essential function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata, likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

cell biology↗

bioRxiv2022

Cryo-EM structures reveal ubiquinone-10 binding to mitochondrial complex I and conformational transitions associated with Q-site occupancy

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, structures of Q10-bound states have remained elusive. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the active (ready-to-go) resting state (plus a matching substrate-free structure) and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the deactive resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.

biochemistry↗

bioRxiv2020

Rag GTPases and Phosphatidylinositol 3-Phosphate Mediate Recruitment of the AP-5/SPG11/SPG15 Complex

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells, and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which on its own localises to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15 while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway, and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation. SummaryThe AP-5/SPG11/SPG15 complex is recruited onto late endosomes/lysosomes, and contributes to lysosomal homeostasis and autophagic lysosome reformation. Hirst et al. show that recruitment is by coincidence detection, requiring both phosphatidylinositol 3-phosphate and Rag GTPases, thus uncovering a link between AP-5/SPG11/SPG15 and the mTORC1 pathway.

cell biology↗

bioRxiv2020

Complexome profile of Toxoplasma gondii mitochondria identifies a divergent cytochrome bc1 complex

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their role as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC complexes and sets the stage for future structural and drug discovery studies. Author SummaryApicomplexan parasites, such as Toxoplasma and Plasmodium, cause diseases of global importance, such as toxoplasmosis and malaria. The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase, which provide the parasite with energy and important metabolites, are essential for parasite function. Here, using a proteomic technique called complexome profiling, we report the composition of the Toxoplasma mETC and F1Fo-ATP synthase. In particular, we reveal the compositions of complexes II and III for the first time. Complex III is an important drug target, yet its full protein composition was unknown. We identify new parasite-specific complex III subunits and demonstrate that they are essential for parasite survival and for proper functioning of the mETC. Our study highlights the divergent nature of the apicomplexan mETC and F1Fo-ATP synthase.

cell biology↗

bioRxiv2017

AP-4 vesicles unmasked by organellar proteomics to reveal their cargo and machinery

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including Dynamic Organellar Maps, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the \"ATG9A reservoir\" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

cell biology↗

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↗