Browsing by Author "Woodward, Jeremy"

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    Cryo-electron microscopy of HPV16 pseudovirions reveal changes in capsid conformation upon furin cleavage
    (2021) Marx, Melissa Lauren; Schafer, Georgia; Woodward, Jeremy
    Persistent infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, a leading cause of cancer deaths in women worldwide. There are no treatments for HPV infection, and although prophylactic vaccines are effective and safe, they are HPV type specific, provide little therapeutic benefit and developing countries often have limited access to these. Therefore, additional measures against HPV infection are urgently needed. Preventing HPV entry into host cells is an attractive option for therapeutic intervention. The HPV capsid is icosahedral and consists of two proteins, L1 and L2, which participate in entry and infection of host cells. During entry, the virus capsid attaches to the cell surface via binding to heparan sulphate proteoglycans (HSPGs). Cleavage of L2 by a host protease, furin, is necessary for infection and is thought to facilitate a conformational change in the virus capsid. Furin cleavage may affect the ability of HPV to bind to sulphated glycoproteins and a HSPG substitute, heparin. Understanding these proposed structural changes may aid in the development of therapeutics targeting virus entry. Here, we directly visualize the conformation changes to HPV16 pseudovirions (HPV16 PsVs) resulting from cleavage of L2 by exogenous furin using cryoelectron microscopy (cryo-EM). At 5 Å resolution, we observed that furin-cleaved HPV16 PsVs capsids display widespread changes in the arrangement of capsomeres relative to uncleaved control virions. This structural change is relevant because heparin has previously been observed to bind to the HPV16 capsid in the canyon surrounding the capsomere at the five-fold icosahedral symmetry axis, but not in other canyons between capsomeres, related by pseudo-symmetry. This suggests that differences in the relative orientations of the surrounding capsomeres to each other either prevent or allow heparin binding. We observed a narrowing of the putative heparin binding site by 0.4 Å after furin cleavage and propose that this change may be responsible for the transfer of HPV from cell-surface HSPGs to the unknown entry receptor(s) by a yet unidentified mechanism.
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    Engineering nitrilases for enhanced thermostability
    (2025) Dlamini, Lenye Sebenzile; Sewell, Bryan; Woodward, Jeremy; Sturrock, Ed
    Cyanide dihydratase from Bacillus pumilus C1 (CynDpum) catalyses the hydrolysis of cyanide to formic acid and ammonia. CynDpum has the potential to remediate cyanide-containing wastewater. However, two obstacles hinder the commercial use of recombinantly expressed CynDpum as a biocatalyst: reduced activity at pH>8 which is typical of cyanide-rich environments, and inactivation at temperatures above 42 °C. Several variants with either enhanced thermostability or activity at alkaline pH have been discovered by random mutagenesis and directed evolution; however, these methods are slow and limited in their explorable sequence space. The aim of this project is to investigate the structural and chemical determinants of thermostability in cyanide dihydratases. This was achieved through rational site-saturation mutagenesis, followed by experimental validation using nanoscale differential fluorimetry, negative stain and cryogenic electron microscopy, and single-particle analysis. In this study, atomic resolution structures of wild-type CynDpum and its variant CynDpum (Q86R/H305K/H308K/H323K) were employed to predict, using empirical force fields, the change in Gibb's free energy resulting from mutating each amino acid in the atomic resolution structure of the variant (amino acids 3-319) to each of the remaining 19 residues. Favourable Gibb's free energy changes were used as indicators of the thermostability of CynDpum variants and validated experimentally using chemical denaturation monitored by nanoscale differential scanning fluorimetry. The thermodynamic favourability of six variants (S29A, T217I, T260I, Q86M, N119R, and E155R) was successfully validated. Negative stain electron microscope micrographs of these variants revealed that these variants formed helical fibres with increased length relative to wild-type CynDpum, indicating a positive correlation between a favourable change in Gibb's free energy of protein unfolding and fibre length. The observed favourable Gibb's free energy change of the surface variants (S29A, T217I, and T260I) is attributed to the reduced number of solvent-accessible amino acid sidechains, promoting protein folding and oligomerisation. Variants N119R and E155R, located along the groove of the helical assembly, form electrostatic interactions across the grooves, thereby enhancing the structural rigidity of the quaternary structure and leading to a favourable Gibb's free energy change. These interactions were visualised in the 2.78 Å resolution atomic model of the E155R variant of CynDpum and the 3.26 Å resolution structure of CynDstu solved by cryo-electron microscopy. Analyses of the conventional interfaces (named A-, C-, D-, and E-) through which the CynDpum helix and cyanide dihydratase from Pseudomonas stutzeri AK61 (CynDstu) spiral are formed, revealed that the helical and spiral assemblies are predominantly stabilised through hydrophobic and electrostatic interactions occurring at the A and C interfaces, respectively. Additionally, examination of the C-terminal tail regions of the atomic-resolution structures of wild-type CynDpum, its variants (Q86R/H305K/ H308K/H323K, and E155R), and CynDstu revealed that the C-terminal tail stabilises all interfacial regions through specific interactions, demonstrating its critical structural and functional role in assembly formation and thermostability.
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    Real-time investigation of tuberculosis transmission: developing the Respiratory Aerosol Sampling Chamber (RASC)
    (Public Library of Science, 2016) Wood, Robin; Morrow, Carl; III, Clifton E Barry; Bryden, Wayne A; Call, Charles J; Hickey, Anthony J; Rodes, Charles E; Scriba, Thomas J; Blackburn, Jonathan; Issarow, Chacha; Mulder, Nicola; Woodward, Jeremy; Moosa, Atica; Singh, Vinayak; Mizrahi, Valerie; Warner, Digby F
    Knowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis ( Mtb ) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb , as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.
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    The 2.7 resolution cryo-EM reconstruction of Mycobacterium tuberculosis encapsulin nanocompartment containing DyP peroxidase
    (2024) Willmore, Rhys; Woodward, Jeremy
    Mycobacterium tuberculosis has evolved many persistence factors in response to the host generated immune response as a means of survival. One such immune response generated by humans is the use of reactive oxygen species (ROS) such as H2O2 to cause damage to M. tuberculosis. Encapsulin (from here on referred to as Enc) nanocompartments and the cargo proteins within have been implicated as persistence factors and decreased viability of cells has been shown when they are knocked out. Previous research has found that dye-decolorizing peroxidase (DyP) is encapsulated by these Enc nanocompartments, as has been shown in Mycobacterium smegmatis. This is done by way of a C-terminal targeting peptide, with Enc and DyP also being part of the same operon in the genome of M. tuberculosis. However, not much is understood about the structure and function of this system. Both encapsulin and DyP were expressed and purified recombinantly in E. coli. Cryo- EM particle processing and EM map reconstruction was carried in an attempt to generate a density map of encapsulated DyP along with model building. Native expression and purification were carried out followed by negative-stain EM on this sample.Successful recombinant expression and purification of DyP and Enc was achieved, with a high-resolution 2.7 Å cryo-EM structure of the Enc nanocompartment obtained, but no encapsulated DyP was visualized. A model of both the Enc monomer and multimer was built, with comparisons to M. smegmatis in charge around the fivefold pore showing differences. The purification of Enc from M. tuberculosis was successful and a negative stain reconstruction of the nanocompartment was obtained. 2D classes showed what could have been DyP but it did not show up in any 3D models. Ferritin was shown to only be outside of the nanocompartment. The high-resolution map showed high similarity to other T=1 Enc nanocompartments. The multimer model built called into question the exact function of this nanocompartment as the charge distribution differed from closely related M. smegmatis. When these charge changes are correlated to findings of catalytic analysis done in the same lab, it indicates that more research is needed to understand the exact function of this system in M. tuberculosis. The 2D classes showed that DyP is the only cargo protein that appears to be present in M. tuberculosis nanocompartments, with ferritin being identified outside the nanocompartment.