Publications by year
In Press
Gambelli L, Meyer B, McLaren M, Sanders K, Quax TEF, Gold V, Albers S-V, Daum B (In Press). Architecture and modular assembly of <i>Sulfolobus</i> S-layers revealed by electron cryo-tomography.
Abstract:
Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryo-tomography
AbstractSurface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic and mechanical stability, the formation of a semi-permeable protective barrier around the cell, cell-cell interaction, as well as surface adhesion. Despite the central importance of the S-layer for archaeal life, their three-dimensional architecture is still poorly understood. Here we present the first detailed 3D electron cryo-microscopy maps of archaeal S-layers from three different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the two subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.
Abstract.
Conners R, León-Quezada RI, McLaren M, Bennett NJ, Daum B, Rakonjac J, Gold VAM (In Press). Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly.
Abstract:
Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly
AbstractPhages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ff has seen an extraordinary range of applications, including in phage display and nanotechnology. However, the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. Using cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods, we have determined the first structure of a filamentous virus, including the filament tips. Structure combined with mutagenesis was employed to identify domains of the phage that are important in bacterial attack and for release of new phage progeny. These data allow new models to be proposed for the phage lifecycle and will undoubtedly enable the development of novel biotechnological applications.
Abstract.
Troman L, Alvira S, Daum B, Gold VAM, Collinson I (In Press). INTERACTION OF THE PERIPLASMIC CHAPERONE SURA WITH THE INNER MEMBRANE PROTEIN SECRETION (SEC) MACHINERY.
Abstract:
INTERACTION OF THE PERIPLASMIC CHAPERONE SURA WITH THE INNER MEMBRANE PROTEIN SECRETION (SEC) MACHINERY
ABSTRACTGram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against the physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC – aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with a recruited chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.
Abstract.
Alvira S, Watkins DW, Troman L, Allen WJ, Lorriman J, Degliesposti G, Cohen EJ, Beeby M, Daum B, Gold VAM, et al (In Press). Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.
Abstract:
Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis
SUMMARYThe outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrelOuter-MembraneProteins (OMPs) – are secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperonese.g.SurA, which prevent aggregation. OMPs are then offloaded to the β-BarrelAssemblyMachinery (BAM) in the outer-membrane for insertion and folding. We show theHolo-TransLocon (HTL: an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC) contacts SurA and BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Our results show the trans-membrane proton-motive-force (PMF) acts at distinct stages of protein secretion: for SecA-driven translocation across the inner-membrane through SecYEG; and to communicate conformational changesviaSecDF to the BAM machinery. The latter presumably ensures efficient passage of OMPs. These interactions provide insights of inter-membrane organisation, the importance of which is becoming increasingly apparent.
Abstract.
Knapp-Wilson A, Pereira GC, Buzzard E, Richardson A, Corey RA, Neal C, Verkade P, Halestrap AP, Gold VAM, Kuwabara P, et al (In Press). Maintenance of Complex I and respiratory super-complexes by NDUF-11 is essential for respiratory function, mitochondrial structure and health in<i>C. elegans</i>.
Abstract:
Maintenance of Complex I and respiratory super-complexes by NDUF-11 is essential for respiratory function, mitochondrial structure and health inC. elegans
ABSTRACTMitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identifiedB0491.5(NDUF-11) as theC. eleganshomologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing expression by RNAi allowed development to the adult stage, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, resulting in excessive detrimental ROS production. Meanwhile, electron cryo-tomography highlight aberrant cristae morphology and widening of the inter-membrane space and cristae junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development highlights the importance of complex I/ super-complex maintenance. Their perturbation by this, or other means, is likely to be the cause of metabolic stress and disease.
Abstract.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B (In Press). New insights into the architecture and dynamics of archaella.
Abstract:
New insights into the architecture and dynamics of archaella
AbstractArchaea 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 Å 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.
Abstract.
Gambelli L, McLaren M, Conners R, Sanders K, Gaines MC, Clark L, Gold V, Kattnig D, Sikora M, Hanus C, et al (In Press). Structure of the two-component S-layer of the archaeon <i>Sulfolobus acidocaldarius</i>.
Abstract:
Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius
AbstractSurface 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.
Abstract.
2023
Buzzard E, McLaren M, Bragoszewski P, Brancaccio A, Ford H, Daum B, Kuwabara P, Collinson I, Gold VAM (2023). Cryo-electron tomography of<i>C. elegans</i>mitochondria reveals how the ATP synthase dimer interface shapes crista membranes.
Abstract:
Cryo-electron tomography ofC. elegansmitochondria reveals how the ATP synthase dimer interface shapes crista membranes
Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans. We showed that the angle formed by the ATP synthase dimer is different to previously determined structures. The consequences of species-specific differences at the dimer interface were investigated by comparing mitochondrial morphology between C. elegans and S. cerevisiae. We reveal that a larger ATP synthase dimer angle in C. elegans is consequent with more lamellar (flatter) cristae compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling and comparing it to an existing S. cerevisiae structure. We reveal extensions and rearrangements of C. elegans subunits that maintain the dimer interface. We propose that increased dimer angles resulting in flatter cristae could provide an energetic advantage for species that inhabit variable-oxygen environments. Significance Statement ATP synthase, the world’s smallest rotary motor, generates the energy required for all living processes. The universal formation of ATP synthase dimers in mitochondria induces membrane curvature and cristae formation, generating the famously convoluted inner mitochondrial membrane that maximises ATP production. Intriguingly, the angle between ATP synthase dimers varies between species. Here, we report the discovery of a novel ATP synthase dimer angle in mitochondria from the nematode worm C. elegans, identify a relationship between dimer angle and crista membrane morphology, and exploit AlphaFold homology modelling to investigate associated structural changes. In summary, we have taken steps towards understanding the importance of subunit composition at the ATP synthase dimer interface, providing insights into the origins of this evolutionary divergence.
Abstract.
Troman L, Alvira S, Daum B, Gold VAM, Collinson I (2023). Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery.
Biochemical Journal,
480(4), 283-296.
Abstract:
Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery
Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein–channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG–SecDF–YidC — aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.
Abstract.
2022
McLaren M, Gil-Diez P, Isupov M, Conners R, Gambelli L, Gold V, Walter A, Connell S, Williams B, Daum B, et al (2022). <i>In situ</i> structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen.
Abstract:
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.
Abstract.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B (2022). An archaellum filament composed of two alternating subunits.
Nature Communications,
13(1).
Abstract:
An archaellum filament composed of two alternating subunits
AbstractArchaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament’s flexibility.
Abstract.
Gaines MC, Isupov MN, Sivabalasarma S, Haque RU, McLaren M, Tripp P, Neuhaus A, Gold V, Albers S-V, Daum B, et al (2022). Donor strand complementation, isopeptide bonds and glycosylation reinforce highly resilient archaeal thread filaments.
Gaines MC, Isupov MN, Sivabalasarma S, Haque RU, McLaren M, Mollat CL, Tripp P, Neuhaus A, Gold VAM, Albers S-V, et al (2022). Electron cryo-microscopy reveals the structure of the archaeal thread filament.
Nature Communications,
13(1).
Abstract:
Electron cryo-microscopy reveals the structure of the archaeal thread filament
AbstractPili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named “thread”. Here, we report on the cryo-EM structure of the archaeal thread. The filament is highly glycosylated and consists of subunits of the protein Saci_0406, arranged in a head-to-tail manner. Saci_0406 displays structural similarity, but low sequence homology, to bacterial type-I pilins. Thread subunits are interconnected via donor strand complementation, a feature reminiscent of bacterial chaperone-usher pili. However, despite these similarities in overall architecture, archaeal threads appear to have evolved independently and are likely assembled by a distinct mechanism.
Abstract.
Bakker SE, Bhella D, Brescia R, Bullough P, Clare DK, Daum B, Frank RAW, Gold VAM, Jackson Hirst I, Kühlbrandt W, et al (2022). Pushing the limits in single particle cryo-EM: general discussion. Faraday Discussions, 240, 312-322.
Al-Otaibi N, Aminian J A, Anane RF, Baatsen P, Bakker SE, Bergeron J, Bharadwaj A, Bhella D, Braun T, Brescia R, et al (2022). Sample preparation in single particle cryo-EM: general discussion.
Faraday Discuss,
240(0), 81-100.
Author URL.
2021
Conners R, McLaren M, Łapińska U, Sanders K, Stone MRL, Blaskovich MAT, Pagliara S, Daum B, Rakonjac J, Gold VAM, et al (2021). CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Nature Communications,
12Abstract:
CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Abstract.
Conners R, McLaren M, Łapińska U, Sanders K, Stone MRL, Blaskovich MAT, Pagliara S, Daum B, Rakonjac J, Gold VAM, et al (2021). CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Nature Communications,
12(1).
Abstract:
CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
AbstractThe Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Abstract.
Knapp-Wilson A, Pereira GC, Buzzard E, Ford HC, Richardson A, Corey RA, Neal C, Verkade P, Halestrap AP, Gold VAM, et al (2021). Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.
J Cell Sci,
134(13).
Abstract:
Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.
Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Animals homozygous for a CRISPR-Cas9-generated knockout allele of nduf-11 arrested at the second larval (L2) development stage. Reducing (but not eliminating) expression using RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its supercomplexes and perturbation of respiratory function. The loss of NADH dehydrogenase activity was compensated by enhanced complex II activity, with the potential for detrimental reactive oxygen species (ROS) production. Cryo-electron tomography highlighted aberrant morphology of cristae and widening of both cristae junctions and the intermembrane space. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I and supercomplex maintenance. This highlights the importance of respiratory complex integrity for health and the potential for its perturbation to cause mitochondrial disease. This article has an associated First Person interview with Amber Knapp-Wilson, joint first author of the paper.
Abstract.
Author URL.
2020
Neuhaus A, Selvaraj M, Salzer R, Langer JD, Kruse K, Kirchner L, Sanders K, Daum B, Averhoff B, Gold VAM, et al (2020). Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium.
Nature Communications,
11(1).
Abstract:
Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium
AbstractType IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus (‘wide’ and ‘narrow’), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Abstract.
Alvira S, Watkins DW, Troman L, Allen WJ, Lorriman JS, Degliesposti G, Cohen EJ, Beeby M, Daum B, Gold VAM, et al (2020). Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis.
eLife,
9Abstract:
Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis
The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrelOuter-MembraneProteins (OMPs) – are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the β-BarrelAssemblyMachinery (BAM) in the outer-membrane for insertion and folding. We show theHolo-TransLocon (HTL) – an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC – contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.
Abstract.
2019
Gambelli L, Meyer BH, McLaren M, Sanders K, Quax TEF, Gold VAM, Albers S-V, Daum B (2019). Architecture and modular assembly of. <i>Sulfolobus</i>. S-layers revealed by electron cryotomography.
Proceedings of the National Academy of Sciences,
116(50), 25278-25286.
Abstract:
Architecture and modular assembly of. Sulfolobus. S-layers revealed by electron cryotomography
. Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell–cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different
. Sulfolobus
. strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.
.
Abstract.
2018
Wang S, Powers R, Gold VAM, Rapoport T (2018). The ER morphology-regulating lunapark protein induces the formation of stacked bilayer discs. Life Science Alliance
Daum B, Gold V (2018). Twitch or swim: towards the understanding of prokaryotic motion based on the type IV pilus blueprint.
Biol Chem,
399(7), 799-808.
Abstract:
Twitch or swim: towards the understanding of prokaryotic motion based on the type IV pilus blueprint.
Bacteria and archaea are evolutionarily distinct prokaryotes that diverged from a common ancestor billions of years ago. However, both bacteria and archaea assemble long, helical protein filaments on their surface through a machinery that is conserved at its core. In both domains of life, the filaments are required for a diverse array of important cellular processes including cell motility, adhesion, communication and biofilm formation. In this review, we highlight the recent structures of both the type IV pilus machinery and the archaellum determined in situ. We describe the current level of functional understanding and discuss how this relates to the pressures facing bacteria and archaea throughout evolution.
Abstract.
Author URL.
2017
Gold VAM, Brandt T, Cavellini L, Cohen MM, Ieva R, van der Laan M (2017). Analysis of mitochondrial membrane protein complexes by electron cryo-tomography. In (Ed)
Methods in Molecular Biology, 315-336.
Abstract:
Analysis of mitochondrial membrane protein complexes by electron cryo-tomography
Abstract.
Gold VAM, Chroscicki P, Bragoszewski P, Chacinska A (2017). Cytosolic ribosomes on the surface of mitochondria.
Gold VA, Chroscicki P, Bragoszewski P, Chacinska A (2017). Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography.
EMBO Rep,
18(10), 1786-1800.
Abstract:
Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography.
We employed electron cryo-tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation-arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.
Abstract.
Author URL.
2016
Gold V, Chrościcki P, Brągoszewski P, Chacinska A (2016). Insights into mitochondrial protein import studied by cryoET. In (Ed) European Microscopy Congress 2016: Proceedings, 276-277.
Turakhiya U, von der Malsburg K, Gold VAM, Guiard B, Chacinska A, van der Laan M, Ieva R (2016). Protein Import by the Mitochondrial Presequence Translocase in the Absence of a Membrane Potential.
J Mol Biol,
428(6), 1041-1052.
Abstract:
Protein Import by the Mitochondrial Presequence Translocase in the Absence of a Membrane Potential.
The highly organized mitochondrial inner membrane harbors enzymes that produce the bulk of cellular ATP via oxidative phosphorylation. The majority of inner membrane protein precursors are synthesized in the cytosol. Precursors with a cleavable presequence are imported by the presequence translocase (TIM23 complex), while other precursors containing internal targeting signals are imported by the carrier translocase (TIM22 complex). Both TIM23 and TIM22 are activated by the transmembrane electrochemical potential. Many small inner membrane proteins, however, do not resemble canonical TIM23 or TIM22 substrates and their mechanism of import is unknown. We report that subunit e of the F1Fo-ATP synthase, a small single-spanning inner membrane protein that is critical for inner membrane organization, is imported by TIM23 in a process that does not require activation by the membrane potential. Absence of positively charged residues at the matrix-facing amino-terminus of subunit e facilitates membrane potential-independent import. Instead, engineered positive charges establish a dependence of the import reaction on the electrochemical potential. Our results have two major implications. First, they reveal an unprecedented pathway of protein import into the mitochondrial inner membrane, which is mediated by TIM23. Second, they directly demonstrate the role of the membrane potential in driving the electrophoretic transport of positively charged protein segments across the inner membrane.
Abstract.
Author URL.
Gold V, Kudryashev M (2016). Recent progress in structure and dynamics of dual-membrane-spanning bacterial nanomachines.
Curr Opin Struct Biol,
39, 1-7.
Abstract:
Recent progress in structure and dynamics of dual-membrane-spanning bacterial nanomachines.
Advances in hard-ware and soft-ware for electron cryo-microscopy and tomography have provided unprecedented structural insights into large protein complexes in bacterial membranes. Tomographic volumes of native complexes in situ, combined with other structural and functional data, reveal functionally important conformational changes. Here, we review recent progress in elucidating the structure and mechanism of dual-membrane-spanning nanomachines involved in bacterial motility, adhesion, pathogenesis and biofilm formation, including the type IV pilus assembly machinery and the type III and VI secretions systems. We highlight how these new structural data shed light on the assembly and action of such machines and discuss future directions for more detailed mechanistic understanding of these massive, fascinating complexes.
Abstract.
Author URL.
Salzer R, D'Imprima E, Gold VAM, Rose I, Drechsler M, Vonck J, Averhoff B (2016). Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.
J Biol Chem,
291(28), 14448-14456.
Abstract:
Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.
Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique βββαβ fold is essential for the formation of gate 2. Furthermore, we identified four βαββα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.
Abstract.
Author URL.
2015
Gold VAM, Salzer R, Averhoff B, Kühlbrandt W (2015). Structure of a type IV pilus machinery in the open and closed state.
Elife,
4Abstract:
Structure of a type IV pilus machinery in the open and closed state.
Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ~30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.
Abstract.
Author URL.
2014
Stockburger C, Gold VAM, Pallas T, Kolesova N, Miano D, Leuner K, Müller WE (2014). A cell model for the initial phase of sporadic Alzheimer's disease.
J Alzheimers Dis,
42(2), 395-411.
Abstract:
A cell model for the initial phase of sporadic Alzheimer's disease.
Recent data suggest that the combined effect of oxidative stress due to aging and slightly elevated amyloid-β (Aβ) levels initiate Alzheimer's disease (AD) long before the clinical onset. Investigations of this early phase are hampered by the lack of cellular or animal models reflecting this scenario. We used SH-SY5Y cells stably transfected with an additional copy of the human AβPP gene and artificial aging by complex I inhibition. These cells show slightly elevated Aβ levels, moderately decreased ATP levels, impaired mitochondrial membrane potential, and decreased mitochondrial respiration. Assessing mitochondrial dynamics with three different methods reveals a distinct shift toward mitochondrial fission and fragmentation in SH-SY5Y AβPPwt cells. We also performed electron cryo-tomography of isolated mitochondria to reveal that there were no major differences between SH-SY5Y control and SH-SY5Y AβPPwt mitochondria with respect to swelling or loss of cristae. Dystrophic neurites are an early pathological feature of AD. Interestingly, SH-SY5Y AβPPwt cells exhibit significantly longer neurites, likely due to substantially elevated levels of sAβPPα. Complex I inhibition also shows substantial effects on mitochondrial dynamics, impairs neuritogenesis, and elevates Aβ levels in both cell types. In SH-SY5Y AβPPwt cells, these defects were more pronounced due to a relatively elevated Aβ and a reduced sAβPPα production. Our findings suggest that the progression from low Aβ levels to the beginning of AD takes place in the presence of oxidative stress during normal aging. This mechanism not only results from additive effects of both mechanisms on mitochondrial function but might also be additionally aggravated by altered amyloidogenic processing.
Abstract.
Author URL.
Schulze RJ, Komar J, Botte M, Allen WJ, Whitehouse S, Gold VAM, Nijeholtb JALA, Huard K, Berger I, Schaffitzel C, et al (2014). Membrane protein insertion and proton-motive-force-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA,
111(13), 4844-4849.
Author URL.
Davies KM, Daum B, Gold VAM, Mühleip AW, Brandt T, Blum TB, Mills DJ, Kühlbrandt W (2014). Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography. Journal of Visualized Experiments(91).
Davies KM, Daum B, Gold VAM, Mühleip AW, Brandt T, Blum TB, Mills DJ, Kühlbrandt W (2014). Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.
J Vis Exp(91).
Abstract:
Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.
Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.
Abstract.
Author URL.
Gold VAM, Ieva R, Walter A, Pfanner N, van der Laan M, Kühlbrandt W (2014). Visualizing active membrane protein complexes by electron cryotomography.
Nat Commun,
5Abstract:
Visualizing active membrane protein complexes by electron cryotomography.
Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.
Abstract.
Author URL.
2013
Gold VAM, Whitehouse S, Robson A, Collinson I (2013). The dynamic action of SecA during the initiation of protein translocation.
BIOCHEMICAL JOURNAL,
449, 695-705.
Author URL.
2012
Gold V, Ieva R, van der Laan M, Pfanner N, Kühlbrandt W (2012). An electron dense substrate to study mitochondrial import sites in situ. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1817
Whitehouse S, Gold VAM, Robson A, Allen WJ, Sessions RB, Collinson I (2012). Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex.
JOURNAL OF CELL BIOLOGY,
199(6), 919-929.
Author URL.
Hizlan D, Robson A, Whitehouse S, Gold VA, Vonck J, Mills D, Kühlbrandt W, Collinson I (2012). Structure of the SecY complex unlocked by a preprotein mimic.
Cell Rep,
1(1), 21-28.
Abstract:
Structure of the SecY complex unlocked by a preprotein mimic.
The Sec complex forms the core of a conserved machinery coordinating the passage of proteins across or into biological membranes. The bacterial complex SecYEG interacts with the ATPase SecA or translating ribosomes to translocate secretory and membrane proteins accordingly. A truncated preprotein competes with the physiological full-length substrate and primes the protein-channel complex for transport. We have employed electron cryomicroscopy of two-dimensional crystals to determine the structure of the complex unlocked by the preprotein. Its visualization in the native environment of the membrane preserves the active arrangement of SecYEG dimers, in which only one of the two channels is occupied by the polypeptide substrate. The signal sequence could be identified along with the corresponding conformational changes in SecY, including relocation of transmembrane segments 2b and 7 as well as the plug, which presumably then promote channel opening. Therefore, we propose that the structure describes the translocon unlocked by preprotein and poised for protein translocation.
Abstract.
Author URL.
2011
Deville K, Gold VAM, Robson A, Whitehouse S, Sessions RB, Baldwin SA, Radford SE, Collinson I (2011). The Oligomeric State and Arrangement of the Active Bacterial Translocon.
JOURNAL OF BIOLOGICAL CHEMISTRY,
286(6), 4659-4669.
Author URL.
2010
Gold VAM, Robson A, Bao H, Romantsov T, Duong F, Collinson I (2010). The action of cardiolipin on the bacterial translocon.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA,
107(22), 10044-10049.
Author URL.
2009
Robson A, Gold VAM, Hodson S, Clarke AR, Collinson I (2009). Energy transduction in protein transport and the ATP hydrolytic cycle of SecA.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA,
106(13), 5111-5116.
Author URL.
2007
Robson A, Booth AEG, Gold VAM, Clarke AR, Collinson I (2007). A large conformational change couples the ATP binding site of SecA to the SecY protein channel.
JOURNAL OF MOLECULAR BIOLOGY,
374(4), 965-976.
Author URL.
Gold VAM, Robson A, Clarke AR, Collinson I (2007). Allosteric regulation of SecA - Magnesium-mediated control of conformation and activity.
JOURNAL OF BIOLOGICAL CHEMISTRY,
282(24), 17424-17432.
Author URL.
Gold VAM, Duong F, Collinson I (2007). Structure and function of the bacterial Sec translocon.
MOLECULAR MEMBRANE BIOLOGY,
24(5-6), 387-394.
Author URL.
