A structure-function characterization of the novel pyocin S3 DNase domain with its cognate immunity protein
Publication Date
December 15, 2022
Creator
Abstract
Pyocin S3 is an unorthodox S-type DNase pyocin produced by Pseudomonas aeruginosa. It is a multi-domain bacteriocin which comprises of an effector protein, which exerts killing activity, and an immunity component, which protects the pyocinogenic bacteria. The killing activity of pyocin S3 is due to an unique DNase domain which does not share sequence homology nor the characteristic H-N-H fold that defines the active site of all other Gram negative bacteriocin DNases such as the colicin E type family, pyocin S-type DNases and homing endonucleases. Due to its therapeutic potential as a novel narrow-spectrum antimicrobial, the DNA encoding the DNase-immunity domains of pyocin S3 produced by P. aeruginosa P12 was cloned into the expression vector, pET21a and purified following IPTG expression using metal chelate chromatography and gel filtration. The DNase was separated from its cognate Immunity protein by Guanidine-HCl denaturation. A final purification step was performed using gel filtration chromatography before the identification of DNase activity using in vivo cytotoxicity and in vitro endonuclease activities relative to the full length native pyocin S3. Further interrogation with isothermal titration calorimetry (ITC), inductively coupled plasma mass spectrometry (ICP-MS), circular dichroism (CD) and tryptophan fluorescence was conducted to provide an insight into the extent of structural integrity following refolding from Guanidine treatment and role of any divalent metal ion co-factor in the activity of pyocin S3 DNase similar to the role of divalent metal ions in the H-N-H DNase activities of other pyocin and colicin proteins.
Metal dependence assays of the purified DNase domain in the presence or absence of divalent metal ions suggested that pyocin S3 is a metal-dependent DNase. A few first row transition metals, including Mg2+, Mn2+, Ni2+, Ca2+ and Co2+ (metal ions listed from the most potent to less) could restore the activity of apo-pyocin S3 DNase in the absence of its cognate immunity protein to different extents.
X-ray crystallography revealed the structure of the purified DNase domain in complex with its cognate immunity protein. The structure is completely novel for a DNase/immunity complex, and resembles an inactivating octopus clasp by the larger immunity protein on its cognate DNase prey domain. Analysis of the X-ray structure of the DNase-Immunity protein complex combined with site directed mutagenesis of conserved residues identified two histidine residues (His26 and His66) and an aspartate residue (Asp25) as likely co-ordinating residues of a divalent metal ion within a novel 30-40 amino acid metal binding motif of a common DNA catalytic aab fold.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Associated Schools / Departments
School of Life Sciences
eprints ID
69657
UoN Repository URI
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Final Thesis- Siyu Wu 20220415.pdf
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Examined. Final thesis
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