<p><i>Staphylococcus
aureus</i> (<i>S. aureus</i>)
is a Gram-positive pathogen that causes a wide range of infections in both
hospitals and communities, of which the total mortality rate is higher than
AIDS, tuberculosis, and viral hepatitis combined. The drug resistant <i>S. aureus </i>is a member of the “ESKAPE”
pathogens that require immediate and sustained actions of novel method to
combat. However, the current antimicrobial development against <i>S. aureus</i> is in stagnation, which underscores
the urgent need for novel antimicrobial scaffolds and targets. <i>S. aureus</i> Ribonuclease P protein (RnpA)
is an essential protein that plays important roles in both tRNA maturation and
mRNA degradation pathways. The goal of this research was to evaluate RnpA as an
antimicrobial target using biophysical methods. The crystal structures of
wild-type RnpA in three different constructs were determined, among which the
tag-free RnpA construct has a structural model of 2.0 Å resolution and R<sub>crys</sub>/R<sub>free</sub>=
0.214/0.234, and its crystals are reproducible. This crystal structure of
tag-free <i>S. aureus </i>RnpA shows a
globular representation with key structural motifs, including the “RNR”
Ribonuclease P RNA binding region and a substrate binding central cleft, which
shares high similarity to previously solved RnpA structures from other species
despite of their low sequence identity. Meanwhile, in a screen of <i>S. aureus </i>RnpA mutants performed by our
collaborator, RnpA<sup>P89A</sup> was found lacking the mRNA degradation
activity while retaining the tRNA maturation function, and causing defects in
cell viability. We therefore studied this mutant using differential scanning
fluorimetry, crystallography, and circular dichroism. It was shown that RnpA<sup>P89A</sup>
is thermally less stable than wild-type RnpA by ~2.0 ˚C,
but no secondary structural or 3D conformational differences were found between
the two proteins. Although the mutant RnpA<sup>P89A</sup> requires further
characterization, the results of the studies in this thesis have begun to shed
light on the relatively new role of <i>S.
aureus </i>RnpA in mRNA degradation, and will serve as useful tools in future
structure-based drug discovery for multi-drug resistant <i>S. aureus </i>treatment. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/9033764 |
| Date | 13 August 2019 |
| Creators | Lisha Ha (5930654) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/EVALUATION_OF_STAPHYLOCOCCUS_AURUES_RNPA_PROTEIN_AS_AN_ANTIBACTERIAL_TARGET/9033764 |
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