| dc.description.abstract |
Structural biology is a branch of science dedicated to the determination and analysis
of the shapes of biological molecules. This work describes two projects aiming to
determine the structures of complexes formed by proteins from pathogens.
Project 1 – MurNAc kinases of Tannerella forsythia
T. forsythia is a Gram-negative bacterium associated with late-stage periodontitis,
unable to synthesise the sugar N-acetylmuramic acid (MurNAc), required for the
synthesis of the peptidoglycan (PGN) layer. The sugar kinase murein kinase (MurK)
has been previously shown to 6-phosphorylate MurNAc after import into the cytoplasm.
Here, the crystal structures of MurK and its paralogue K1058, in complex with their
MurNAc substrate, are presented, leading to the identification of the basis of MurNAc
specificity in sugar kinases. This is mediated by an absence of bulky residues
introducing steric hindrance against the lactyl ether group. Results of kinetic assays of
the kinases agreed with previously published MurK apparent kinetic parameters (KM
180 μM, Vmax 80 μmol min-1 mg-1) and showed that K1058 had a higher binding affinity
for MurNAc than MurK (apparent KM 30 μM), but much lower efficiency (apparent Vmax
0.34 μmol min-1 mg-1). Hence, the natural substrate of K1058 remains to be
determined. While the potential of MurK as a druggable target for periodontitis is
uncertain, these first structures of MurNAc kinases in complex with their substrate
provide new insights into their specificity, which could be applied to drug design or
research on glycan processing in other organisms.
Project 2 – Human adenovirus and its integrin receptor
Human adenoviruses (HAdV) are double-stranded DNA viruses, responsible for a wide
range of symptomatic diseases such as keratoconjunctivitis, respiratory, and
gastrointestinal illnesses. Their broad tropism and large carrying capacity also make
them attractive gene delivery vectors for vaccine or gene therapy applications.
To initiate infection, HAdV bind to an attachment receptor on the host cell surface, then
enter the cell by engaging a transmembrane integrin protein. This work aimed to
structurally characterise the complex between the HAdV penton base (pb) capsid
protein from HAdV-C5, HAdV-D9, and HAdV-B35, and the αvβ3 integrin entry receptor.
The purification of both complex components from insect cells was optimised
successfully, but preparations of pb formed higher-order oligomers and had to be
maintained at a low concentration. Complex formation was confirmed with negative
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stain electron microscopy (EM) and cryoelectron microscopy (CryoEM). CryoEM 3D
reconstructions of the HAdV-C5 penton base in complex with αvβ3 included a
pentameric pb structure at resolution below 5 Å and pb at lower resolution (below 10
Å) with separate, featureless electron density, in some cases linked to the pb by an
elongated bridge. The density could not be identified as the integrin with certainty due
to the lack of detail. Initial attempts to purify the complex and remove the unbound
pentamers that weaken the signal, failed. Future work will include purification of
optimised pb constructs and structural characterisation of the complex. |
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