Biology subjects

Gerle, C.

Publications and source records attributed to Gerle, C..

12 recordsLinked to original sources

bioRxiv2026

Polymorphic Structures of an Artificial Nucleocapsid Evolved from a Circularly Permuted Bacterial Protein Cage

Molecular self-assembly provides a route to nanoscale structures with defined sizes and shapes. Protein cages assembled from repeated subunits form compartments that can package molecular cargoes. Although amino acid substitutions can alter intersubunit interactions and cage architecture, how protein backbone connectivity relates to the cage architectures accessible during laboratory evolution remains unclear. Here we evolved an artificial nucleocapsid from a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS). Laboratory evolution increased the yield of purified nucleocapsids relative to the progenitor, and the evolved variant, NC119-3, packaged its encoding mRNA. Cryo-electron microscopy revealed a heterogeneous population of cages, within which we resolved 180- and 210-subunit cage families composed of 36 and 42 pentamers, respectively. Within each resolved family, particles with the same subunit stoichiometry differed in the arrangement and orientation of their pentameric building blocks. A 2.90 [A] reconstruction of a tetrahedral 180-subunit cage further revealed that sequence-identical subunits adopt distinct conformations depending on their positions within the assembly. These pentamer-based architectures differ from the domain-swapped T = 4 cage previously reported for nucleocapsids evolved from an AaLS variant with a different circular permutation site. Together with previous work, these findings show that RNA-packaging nucleocapsids evolved from scaffolds with different backbone connectivity can adopt distinct cage architectures.

synthetic biology↗

bioRxiv2025

A planar dimer of bovine ATP synthase

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by antiparallel arrangement of two ATP synthase complexes linked by a straight conformation of the inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent subunit. These structural divergences suggest a specialized functional role and localization distinct from the canonical sharply membrane bending ATP synthase oligomers, providing structural support for a model of membrane curvature-driven division of labor within mitochondrial ATP synthase populations.

biophysics↗

bioRxiv2025

Mrx6 binds the Lon protease Pim1 N-terminal domain to confer selective substrate specificity and regulate mtDNA copy number

Mitochondrial DNA (mtDNA) copy number regulation remains incompletely understood, despite its importance in cellular function. In Saccharomyces cerevisiae, Mrx6 belongs to the Pet20-domain-containing protein family, consisting of Mrx6, Pet20, and Sue1. Notably, absence of Mrx6 leads to increased mtDNA copy number. Here, we identify the C-terminus of Mrx6 as essential for its stability and interaction with the mitochondrial matrix protein Mam33. Deletion of Mam33 mimics the effect of Mrx6 loss, resulting in elevated mtDNA copy number. Bioinformatics, mutational analyses, and immunoprecipitation studies reveal that a subcomplex of Mam33 and Mrx6 trimers interacts with the substrate recognition domain of the conserved mitochondrial Lon protease Pim1 through a bipartite motif in the Pet20 domain of Mrx6. Loss of Mrx6, its paralog Pet20, Mam33, or mutations disrupting the interaction between Mrx6 and Pim1 stabilize key proteins required for mtDNA maintenance, the RNA polymerase Rpo41 and the HMG-box-containing protein Cim1. We propose that Mrx6, alongside Pet20 and Mam33, regulates mtDNA copy number by modulating substrate degradation through Pim1. Additionally, Mrx6 loss alters Cim1s function, preventing the detrimental effect on mtDNA maintenance observed upon Cim1 overexpression. The presence of three Pet20-domain proteins in yeast implies broader roles of Lon protease substrate recognition beyond mtDNA regulation.

cell biology↗

bioRxiv2024

High-resolution Cryo-EM visualises hydrated type I and IV pilus structures from enterotoxigenic Escherichia coli

To efficiently colonise intestinal epithelium, enteric pathogens utilise a wide variety of pilus filaments, including those synthesised by the chaperone-usher or type IV pilus assembly pathway. Despite the importance of these filaments as potential drug and vaccine targets, their large size and dynamic nature present challenges for atomic-resolution structural determination. Here, we used cryo-electron microscopy (cryo-EM) and whole genome sequencing to identify and determine the structures of type I and IV pili expressed in enterotoxigenic Escherichia coli. The well-defined cryo-EM maps at resolutions of 2.20 and 1.78 [A] for Type I and Type IV pilus, respectively, facilitated de novo structural modelling for these filaments, revealing side chain structures in detail. Further, we resolved thousands of hydrated water molecules around and within the inner core of the filaments, functioning as a buffer to stabilise the otherwise metastable subunit assembly. The high-resolution structures offer novel insights into the subunit-subunit interactions, providing important clues to understand pilus assembly, stability, and flexibility.

microbiology↗

bioRxiv2024

Fc-independent SARS-CoV-2 infection-enhancing antibodies decouple N-terminal and receptor-binding domains by cross-linking neighboring spikes

Antibody dependent enhancement (ADE) is a serious concern in vaccine development. The canonical ADE pathways are dependent on the fragment crystallizable (Fc) region of the antibody. In SARS-CoV-2 several antibodies have been discovered that inflict ADE in vitro. These antibodies target the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. We previously proposed that these NTD-targeting infection-enhancing antibodies (NIEAs) cross-link neighboring spike proteins via their NTDs, and that this results in a decoupling between the NTD and receptor binding domain (RBD), facilitating the "RBD down" to "up" transition. In this study we present a combination of molecular dynamics simulations and cryogenic electron microscopy data that, together, demonstrate that NIEAs are indeed able to cross-link neighboring SARS-CoV-2 spike proteins, and that this cross-linking results in a decoupling of the NTD and RBD domains. These findings provide support for an Fc region independent ADE pathway that is not only relevant for SARS-CoV-2 but also for other viruses of which the spike proteins undergo a conformational change upon host cell entry.

molecular biology↗

bioRxiv2024

Monomer-dimer structural comparison in quinol-dependent nitric oxide reductase reveals a functional basis for superior enzymatic activity in the dimer

The leading cause of bacterial meningitis, Neisseria meningitidis, deploys a quinol-dependent nitric oxide reductase (NmqNOR), belonging to the heme-copper oxidase superfamily. By detoxifying NO, an antimicrobial gas produced by hosts immune system, qNOR enables pathogen survival within hosts. Here, we determined cryoEM structures of the less active monomer and highly active dimer of NmqNOR at resolutions of 2.25 and 1.89 [A], respectively, showing the structural elements responsible for effective NO reduction. Helical disorder at the dimer interface, associated with an altered conformation of the critical Glu563 near the heme/non-heme Fe active site, was observed in the monomer. These findings suggest that dimerization stabilizes the active conformation of Glu563 through the structural network between the dimerization site and the active site. Since other members of the heme-copper oxidases exhibit dimerization, the current data on qNOR helps us understand a regulatory mechanism related to the function of heme-copper oxidases upon oligomerization. TeaserCryoEM structures unveil a functional rationale for dimerization in nitric oxide detoxifying enzyme from a pathogen

biochemistry↗

bioRxiv2023

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation

Pyrenoids are subcompartments of algal chloroplasts that concentrate Rubisco enzymes and their CO2 substrate, thereby increasing the efficiency of carbon fixation. Diatoms perform up to 20% of global CO2 fixation, but their pyrenoids remain poorly characterized at a molecular level. Here, we used in vivo photo-crosslinking to catalogue components of diatom pyrenoids and identified a pyrenoid shell (PyShell) protein, which we localized to the pyrenoid periphery of both the pennate diatom, Pheaodactylum tricornutum, and the centric diatom, Thalassiosira pseudonana. In situ cryo-electron tomography (cryo-ET) revealed that the pyrenoids of both diatom species are encased in a lattice-like protein sheath. Disruption of PyShell expression in T. pseudonana resulted in the absence of this protein sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with impaired growth and reduced carbon fixation efficiency under standard atmospheric conditions. Pyrenoids in mutant cells were fragmented and lacked the thylakoid membranes that normally traverse the Rubisco matrix, demonstrating how the PyShell plays a guiding role in establishing pyrenoid architecture. Recombinant PyShell proteins self-assembled into helical tubes, enabling us to determine a 3.0 [A]-resolution PyShell structure. We then fit this in vitro structure into an in situ subtomogram average of the pyrenoids protein sheath, yielding a putative atomic model of the PyShell within diatom cells. The structure and function of the diatom PyShell provides a new molecular view of how CO2 is assimilated in the ocean, a crucial biome that is on the front lines of climate change.

cell biology↗

bioRxiv2023

Large-scale column-free purification of bovine F-ATP synthase

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at sufficient quantity, stability and purity. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favouring IF1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the GDN to LMNG ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

biochemistry↗

bioRxiv2022

Autoinsertion (LAiR): rapid functional reconstitution of integral membrane proteins into lipid bilayers

Functional investigation of purified integral membrane proteins (IMPs) is hampered by the need to insert these hydrophobic proteins from the detergent-solubilized state into liposomal membranes. Here we report reintegration of IMPs into a lipid environment within minutes, an order of magnitude faster than currently used standard techniques. The new approach yielded optimal results for IMPs solubilized in the detergent lauryl-maltose neopentyl glycol (LMNG) and is therefore termed LMNG Auto-insertion Reintegration (LAiR). LAiR displays superior performance to standard methods in terms of protein activity, long-term stability and proton tightness of proteoliposomes. LAiR reconstituted vectorial control of membrane-bound activity by the transmembrane ion motive force, a property particularly important in mitochondrial function, which was undetectable by standard reintegration methods. LAiR also preserved fragile IMP properties that are prone to disruption upon reintegration, including long-term multi-subunit integrity, inhibitor susceptibility, and higher-order oligomeric states. LAiR proved suitable for reintegration into liposomes as well as into surface-tethered membrane bilayers, and was compatible with IMPs and lipids from prokaryotic and eukaryotic sources. We anticipate a broad scope for LAiR as a powerful tool in fundamental research, pharmaceutical applications, and biotechnology.

biochemistry↗

bioRxiv2022

Structure of cyanobacterial photosystem I complexed with Cytochrome c6 and Ferredoxin at 1.97 A resolution

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning the binding of Fd and the bound Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and Cyt c6. In our structure Cyt c6 is loosely bound in a non-productive site on the periphery of the complex. Side chain interactions at the PSI:Fd interface are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

plant biology↗

bioRxiv2021

TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

cell biology↗

bioRxiv2017

Structure and function of the intermembrane space domain of mammalian FoF1 ATP synthase

Mitochondrial FoF1 ATP synthase is a membrane bound molecular machine central to cellular energy conversion and cristae architecture. Recently, a novel domain has been visualized in the intermembrane space region of mammalian ATP synthase. The complete three-dimensional (3D) structure, composition and function of this domain - which we term intermembrane space domain (IMD) - are unknown. Here, we present two distinct 3D structures of monomeric bovine FoF1 ATP synthase by single particle cryo-electron microscopy (cryo-EM) that differ by the presence and absence of the IMD. Comparison of both structures reveals the IMD to be a bipartite and weakly associated domain of FoF1 ATP synthase. The tubular sub-domain of the IMD appears to contact the rotor-ring region, its globular sub-domain is anchored in the membrane-bending kink of the ATP synthase. However, absence of the IMD does not impact the kink in the transmembrane region ruling out a functional role in membrane bending. By combining our structural analysis with chemical cross-linking and reported biochemical, genetic and structural data we identify 6.8PL and DAPIT as the subunits forming the intermembrane space domain. We compare the present structure of the mammalian IMD in the bovine FoF1 ATP synthase monomer with structures of dimeric FoF1 ATP synthase from yeast and ciliate showing that the IMD is a common, but structurally divergent feature of several mitochondrial ATP synthases. On the basis of our analysis we discuss potential functions of the novel domain in rotary catalysis, oligomerization and mitochondrial permeability transition.

biophysics↗