The architectural complexity of the human PDC core assembly

The mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly linking the glycolytic pathway to the TCA cycle via the specific conversion of pyruvate to acetyl CoA and, as such, is responsible for the maintenance of glucose homeostasis in humans. PDC comprises a central pentagonal dodecahedral core of 60 dihydrolipoamide acetyltransferase (E2) and 12 E3 binding protein (E3BP) subunits. Presently, two conflicting models of PDC (E2+E3BP) core organisation exist: the ‘addition’ (60+12) and ‘substitution’ (48+12) models. In addition to its catalytic role, the multi-domain E2/E3BP core provides the structural framework to which 30 pyruvate decarboxylase (E1) heterotetramers and 6-12 dihydrolipoamide dehydrogenase (E3) homodimers are proposed to bind at maximal occupancy. The formation of specific E2:E1 and E3BP:E3 subcomplexes are characteristic of eukaryotic PDCs and are critical for normal complex function. Despite the availability of limited structural data, the exact subunit organisation and mechanism of operation of the mammalian E2/E3BP core remains unknown. This thesis describes the large-scale purification of tagged, recombinant human PDC cores, full-length rE2 and rE2/E3BP, truncated E2/E3BP, peripheral rE3 enzyme as well as native E2/E3BP core (bE2/E3BP) purified from bovine heart. The ability to purify large amounts of pure protein has enabled the characterisation of the individual cores as well as the E2/E3BP:E3 complex using a variety of biochemical and biophysical techniques. Full-length rE2/E3BP, rE2, bE2/E3BP, truncated E2/E3BP (tLi19/tLi30) and rE2/E3BP:E3 were analysed in solution by analytical ultracentrifugation (AUC). While AUC of the cores supported the substitution model of core organisation, the stoichiometry of interaction was determined to be 2:1 (rE2/E3BP:E3). This was further complemented by gel filtration chromatography (GFC) and small angle neutron scattering (SANS), implying the possible existence of a network of E3 ‘cross-bridges’ linking pairs of E3BP molecules across the surface of the E2 core assembly. Low resolution solution structures obtained for rE2/E3BP, bE2/E3BP and tLi19/tLi30 by small angle x-ray scattering (SAXS) and SANS revealed the presence of icosahedral cores with open pentagonal faces favouring the substitution model of core organisation. These solution structures also indicated high structural similarity between the recombinant and native cores, as well as with the crystal structure obtained previously for the truncated bacterial E2 core. In addition, homology modelling and superimpositions of high- and low-resolution structures of the core revealed conservation of the overall pentagonal dodecahedral morphology despite evolutionary diversity. Evidence for the substitution model of core organisation was further substantiated by negative stain EM of the recombinant and bovine E2/E3BP cores. SANS stoichiometry data indicated the binding of 10 E3 dimers per E2/E3BP core. Although this could correspond to approximately 1:1 stoichiometry between E2/E3BP:E3, subsequent radiolabelling studies suggested possible variation in core subunit composition between the native and recombinant E2/E3BP cores. Therefore, as opposed to the 48E2+12E3BP substitution model based on AUC and SAXS studies with the recombinant E2/E3BP core, rE2/E3BP cores produced in this study indicated a higher level of incorporation of E3BPs with a maximum core composition of 40E2+20E3BP. On the basis of this new finding we have proposed the ‘variable E3BP substitution model’, wherein the number of E3BPs within the core can range from 0 to a maximum of 20, thus resulting in variable populations of E2/E3BP cores. Despite this core variability, the highly controlled regulatory mechanisms in vivo may bias the core composition towards an average of 48E2+12E3BP. However, as the over-expression of the recombinant E2/E3BP core in our study is not as tightly regulated as in vivo, higher number of E3BPs (>12) is observed to be integrated into the core. This new level of architectural complexity and variable subunit composition in mammalian PDC core organisation is likely to have important implications for the catalytic mechanism, overall complex efficiency and tissue-specific regulation by the intrinsic PDC kinases (PDKs) in normal and disease states. The E2 cores of the PDC family are known to be highly flexible, exhibiting inherent size variability reflective of the ‘breathing’ of the core. Integration of E3BP into the E2 core assembly would then be expected to have significant consequences for the structural assembly, affecting the ‘breathing’ and in turn the function and regulation of the complex. Unfolding studies to assess core stability via circular dichroism (CD) and tryptophan fluorescence revealed lower stability of the rE2/E3BP core as compared to cores composed exclusively of rE2 subunits, thus implying the contribution of E3BP towards core destabilisation. In addition, crosslinking studies indicated weak dimerisation of rE3BP, which may be a key factor promoting core destabilisation. The lower stability of the E2/E3BP core may be of benefit in mammals where sophisticated fine tuning is required to obtain cores with optimal catalytic and regulatory efficiencies. SAXS solution structures of E2/E3BP cores obtained were unable to locate the exact positions of E3BP within the core. However, SANS in combination with contrast matching of selectively deuterated components as well as cryo-EM, EM tomography and single molecule studies could be used in future for determination of the exact locations of E3BP, and validating the importance of E2/E3BP core organisation and subunit composition for overall PDC function and regulation

Structure of the herpes-simplex virus portal-vertex

Herpesviruses include many important human pathogens such as herpes simplex virus, cytomegalovirus, varicella-zoster virus, and the oncogenic Epstein–Barr virus and Kaposi sarcoma–associated herpesvirus. Herpes virions contain a large icosahedral capsid that has a portal at a unique 5-fold vertex, similar to that seen in the tailed bacteriophages. The portal is a molecular motor through which the viral genome enters the capsid during virion morphogenesis. The genome also exits the capsid through the portal-vertex when it is injected through the nuclear pore into the nucleus of a new host cell to initiate infection. Structural investigations of the herpesvirus portal-vertex have proven challenging, owing to the small size of the tail-like portal-vertex–associated tegument (PVAT) and the presence of the tegument layer that lays between the nucleocapsid and the viral envelope, obscuring the view of the portal-vertex. Here, we show the structure of the herpes simplex virus portal-vertex at subnanometer resolution, solved by electron cryomicroscopy (cryoEM) and single-particle 3D reconstruction. This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures that occupy the sites normally taken by the penton and the Ta triplex. Our data revealed that the PVAT is composed of 10 copies of the C-terminal domain of pUL25, which are uniquely arranged as two tiers of star-shaped density. Our 3D reconstruction of the portal-vertex also shows that one end of the viral genome extends outside the portal in the manner described for some bacteriophages but not previously seen in any eukaryote viruses. Finally, we show that the viral genome is consistently packed in a highly ordered left-handed spool to form concentric shells of DNA. Our data provide new insights into the structure of a molecular machine critical to the biology of an important class of human pathogens

In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging

Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly

The final version of this article is available at the link below.Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an ‘average’ 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.This work was partly supported by EPSRC (under grants GR/R99393/01 and EP/C015452/1)

Cryotomography of budding influenza a virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end

Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process

Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly

Multiplexed biosensing of proteins and virions with disposable plasmonic assays

Our growing ability to tailor healthcare to the needs of individuals has the potential to transform clinical treatment. However, the measurement of multiple biomarkers to inform clinical decisions requires rapid, effective, and affordable diagnostics. Chronic diseases and rapidly evolving pathogens in a larger population have also escalated the need for improved diagnostic capabilities. Current chemical diagnostics are often performed in centralized facilities and are still dependent on multiple steps, molecular labeling, and detailed analysis, causing the result turnaround time to be over hours and days. Rapid diagnostic kits based on lateral flow devices can return results quickly but are only capable of detecting a handful of pathogens or markers. Herein, we present the use of disposable plasmonics with chiroptical nanostructures as a platform for low-cost, label-free optical biosensing with multiplexing and without the need for flow systems often required in current optical biosensors. We showcase the detection of SARS-CoV-2 in complex media as well as an assay for the Norovirus and Zika virus as an early developmental milestone toward high-throughput, single-step diagnostic kits for differential diagnosis of multiple respiratory viruses and any other emerging diagnostic needs. Diagnostics based on this platform, which we term “disposable plasmonics assays,” would be suitable for low-cost screening of multiple pathogens or biomarkers in a near-point-of-care setting

Cation-mediated interplay of loops in chaperonin-10

The ubiquitously occurring chaperonins consist of a large tetradecameric Chaperonin-60, forming a cylindrical assembly, and a smaller heptameric Chaperonin-10. For a functional protein folding cycle, Chaperonin-10 caps the cylindrical Chaperonin-60 from one end forming an asymmetric complex. The oligomeric assembly of Chaperonin-10 is known to be highly plastic in nature. In Mycobacterium tuberculosis, the plasticity has been shown to be modulated by reversible binding of divalent cations. Binding of cations confers rigidity to the metal binding loop, and also promotes stability of the oligomeric structure. We have probed the conformational effects of cation binding on the Chaperonin-10 structure through fluorescence studies and molecular dynamics simulations. Fluorescence studies show that cation binding induces reduced exposure and flexibility of the dome loop. The simulations corroborate these results and further indicate a complex landscape of correlated motions between different parts of the molecule. They also show a fascinating interplay between two distantly spaced loops, the metal binding "dome loop" and the GroEL-binding "mobile loop", suggesting an important cation-mediated role in the recognition of Chaperonin-60. In the presence of cations the mobile loop appears poised to dock onto the Chaperonin-60 structure. The divalent metal ions may thus act as key elements in the protein folding cycle, and trigger a conformational switch for molecular recognition

The structure of the portal-vertex interior.

<p>A central slice through the C5 reconstruction of the HSV-1 virion reveals the internal features of the portal-vertex (a). Notably, a strong linear density is seen to run through the portal-vertex that we attribute to genomic DNA (white arrow). The outermost feature, the PVAT, is weakly resolved as fuzzy density, suggesting that this feature is not well constrained. Isosurface representation of the unsharpened map presents a clearer representation of the PVAT (b), while in the sharpened density map, the packaged DNA is not seen, revealing the interior features of the capsid shell (c). A clipped, close-up view of the portal-vertex (boxed in c) highlights the morphology of the portal (pUL6) and, lying between the portal and the PVAT, the pentameric portal-vertex protein (wall-eyed stereo pair view, d). A close-up stereo view of the pentameric portal-vertex protein (boxed in d) clearly shows the density that is consistent with a two-helix coiled-coil motif (pink arrow, e). The density running through the centre of the portal-vertex that we attribute to DNA is also clearly visible (blue arrow). The density map was segmented to highlight three features: the portal (mauve), the pentameric portal-vertex protein (purple), and the periportal triplex–like density (magenta). The segmented portal-vertex is presented as stereo views both perpendicular to (e) and along (f) the portal axis. In panel e, the capsid and triplex-like assemblies are clipped to expose the pentameric portal-vertex protein; this and the portal are not clipped. In panel f, the pUL25/PVAT component is clipped away to expose the underlying features. HSV, Herpes Simplex Virus; PVAT, portal-vertex–associated tegument.</p