Abstract
The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the β-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage genomes and have been shown to interact with the Stl repressor protein of S. aureus pathogenicity island SaPIbov1. In the present work we set out to characterize the interactions between these proteins based on a range of biochemical and biophysical methods and shed light on the binding mechanism of the dimeric φNM1 phage dUTPase and Stl. Using hydrogen deuterium exchange mass spectrometry, we also characterize the protein regions involved in the dUTPase:Stl interactions. Based on these results we provide reasonable explanation for the enzyme inhibitory effect of Stl observed in both types of complexes. Our experiments reveal that Stl employs different peptide segments and stoichiometry for the two different phage dUTPases which allows us to propose a functional plasticity of Stl. The malleable character of Stl serves as a basis for the inhibition of both dimeric and trimeric dUTPases.
Highlights
Infections caused by Staphylococcus aureus are hazardous for both humans and livestock especially since S. aureus strains develop resistance and adapt to the new hosts rapidly via horizontal gene transfer (HGT)[1,2]
Based on our hydrogen deuterium exchange mass spectrometry (HDX-MS) results, we identify regions of both Stl and homotrimeric and homodimeric dUTPase proteins which are involved for complex formation
Based on steady-state enzymatic activity measurements of DUTφNM1 performed in the presence of Stl of different concentrations, we found that the maximal inhibition was about 40%, half of the original enzymatic activity was retained even at relatively high concentration of Stl
Summary
Infections caused by Staphylococcus aureus are hazardous for both humans and livestock especially since S. aureus strains develop resistance and adapt to the new hosts rapidly via horizontal gene transfer (HGT)[1,2]. Mobile S. aureus pathogenicity islands (SaPI) play a key role in this process since they frequently carry genes encoding toxic shock syndrome toxin, staphylococcal enterotoxin B, and other superantigens [3]. Thereafter a specific derepressor protein of the phage relieve the repression of the genes responsible for SaPI excision, replication, and packaging [4]. It has been shown that in case of SaPIbov pathogenicity island homotrimeric dUTPase enzymes of specific phages are responsible for the SaPI induction through direct interaction with Stl; the master repressor protein of the SaPI lifecycle [5,6]. It has been hypothesized that this phage-specific insert plays an important role in the SaPI induction since the pH15 phage dUTPase, which lacks this insertion region, cannot function as a SaPI-derepressor [5,7]
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