Structural basis of staphylococcal Stl inhibition on a eukaryotic dUTPase.

dUTPases are key enzymes in all life kingdoms. A staphylococcal repressor protein (Stl) inhibited dUTPases from multiple species to various extents. Understanding the molecular basis underlying the inhibition differences is crucial to develop effective proteinaceous inhibitors of dUTPases. Herein, we report the complex structure of Stl N-terminal domain (Stl) and Litopenaeus vannamei dUTPase domain (lvDUT). Stl inhibited lvDUT through its N-terminal domain. The lvDUT-Stl complex structure revealed a heterohexamer encompassing three Stl monomers bound to one lvDUT trimer, generating two types of Stl-dUTPase interfaces. Interface I is formed by Stl interaction with the lvDUT active-site region that is contributed by motifs I-IV from its two subunits; interface II results from Stl binding to the C-terminal motif V of the third lvDUT subunit. Structural comparison revealed both conserved features and obvious differences in Stl-dUTPase interaction patterns, giving clues about the inhibition differences of Stl on dUTPases. Noticeably, interface II is only observed in lvDUT-Stl. The Stl-interacting residues of lvDUT are conserved in other eukaryotic dUTPases, particularly human dUTPase. Altogether, our study presents the first structural model of Stl interaction with eukaryotic dUTPase, contributing to a more complete view of Stl inhibition and facilitating the development of proteinaceous inhibitor for eukaryotic dUTPases.