Structural basis of staphylococcal Stl inhibition on a eukaryotic dUTPase

https://doi.org/10.1016/j.ijbiomac.2021.06.107Get rights and content

Highlights

  • The first structure of Stl in complex with a eukayotic dUTPase is reported.

  • A more complete view of Stl inhibition on trimeric dUTPase is proposed.

  • The structure facilitates Stl optimization for a better inhibitor of eukaryotic dUTPases.

Abstract

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 (StlN-ter) and Litopenaeus vannamei dUTPase domain (lvDUT65–210). Stl inhibited lvDUT65–210 through its N-terminal domain. The lvDUT65–210-StlN-ter complex structure revealed a heterohexamer encompassing three StlN-ter monomers bound to one lvDUT65–210 trimer, generating two types of Stl-dUTPase interfaces. Interface I is formed by Stl interaction with the lvDUT65–210 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 lvDUT65–210 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 lvDUT65–210-StlN-ter. The Stl-interacting residues of lvDUT65–210 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.

Introduction

dUTPase is an essential enzyme in cellular nucleotide metabolism [1]. It catalyzes the hydrolysis of dUTP into dUMP and pyrophosphate (PPi) to prevent DNA uracilation, and supplies dUMP to synthesize dTTP for DNA building [1]. Some dUTPases also moonlight in the modulation of immune system and apoptosis [2], [3]. Based on the oligomerization state, dUTPases have been classified into three groups: homotrimeric, homodimeric and monomeric [4]. The homotrimeric group comprises the majority of the dUTPase family, including those from Homo sapiens [5], Drosophila melanogaster [6], Escherichia coli [7], Mycobacterium tuberculosis [8] and Staphylococcus aureus phage [9]. All homotrimeric dUTPases contain active sites formed by five conserved motifs (I–V) which are contributed by all three subunits [5], [10] or by two subunits only [11], [12], [13], [14]. Motifs I–IV form the majority of the active-site pocket, while motif V at the C-terminal tail is mobile and completes the active site upon substrate binding [1]. Some dUTPases contain extra motifs (VI), which are not required for catalysis but needed for other moonlighting functions [4].

dUTPases have been proposed as promising drug targets for the therapy of cancer and infectious diseases [15], [16], with many small molecule inhibitors developed, which are mainly substrate analogs [17], [18]. Recently, a proteinaceous inhibitor for the homotrimeric dUTPase has been identified during the study on the transfer of Staphylococcus aureus pathogenicity island (SaPI) [19]. A couple of S. aureus phage dUTPases mobilized SaPI transfer in the host through the interaction with the SaPI repressor protein StlSaPIBov1 (in short, Stl) of S. aureus [20], [21]. The interaction not only damages the DNA binding activity of Stl but also inhibits the dUTPase enzymatic activity, which makes Stl a proteinaceous inhibitor of the dUTPases [19], [22]. The crystal structure of Stl in complex with the dUTPase of S. aureus phage Φ11 (Φ11DUT) has been determined. It revealed that the interaction between the two proteins impedes Stl from dimerization, which is required for binding to its cognate DNA. Meanwhile, the N-terminal region of Stl blocks the active site of Φ11DUT and disrupts the enzymatic activity by precluding the nucleotide binding [23]. To be noted, Φ11DUT carries motif VI that is involved in the interaction with Stl [22].

Besides phage dUTPases, Stl showed a broad-spectrum inhibitory activity against a variety of homotrimeric dUTPases isolated from organisms like mycobacterium, fruitfly, and human, despite low sequence similarities to phage dUTPases [24], [25], [26]. However, compared to 100% inhibition rate of Stl on Φ11DUT, the maximal inhibition rates of Stl on these dUTPases are in a range from 40% to 80%. For an extreme case, Stl bound to E. coli dUTPase (EcDUT) tightly but did not inhibit its enzymatic activity [27]. Although the binding mode of Stl on dUTPase has been revealed by the structure of Φ11DUT-StlN-ter, structural information of Stl in complex with dUTPases from other species is also demanded to understand the molecular basis for the broad-spectrum inhibition activity and its species-specific inhibition rates. Indeed, a previous study has established a low-resolution structural model of Stl in complex with human dUTPase (hDUT), using small-angle x-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) [26]. This model reveals the overall interaction mode, but it is not sufficient to clarify the interaction details for understand the molecular basis of species-specific inhibition rate, which would be crucial for engineering Stl to be a better broad-spectrum inhibitor.

Herein, we reported the crystal structure of Stl N-terminal domain in complex with Litopenaeus vannamei dUTPase domain. This is the first complex structure between Stl and a eukaryotic dUTPase, contributing to a more complete view of Stl inhibition on trimeric dUTPases. The results also provide clues about Stl inhibition differences on dUTPases and facilitate Stl engineering as a better proteinaceous inhibitor for eukaryotic dUTPases, particularly for hDUT.

Section snippets

Gene cloning and protein production

The gene encoding Stl was synthesized and inserted into the vector pET-28a (+) by Beijing Genomics Institution. The gene sequence was amplified by standard PCR method using primers (forward, 5′-AAACCATGGAAGGAGCTGGTCAAATGGCAGAATTAC-3′; reverse, 5′-CCGCTCGAGTTAATTAGTGTCTTTTTCAAGTATG-3′). The PCR product was ligated into the pETM11 vector (EMBL) using the NcoI and XhoI restriction sites to express a recombinant protein containing an N-terminal 6 × His tag and a tobacco etch virus protease (TEV)

Stl inhibited the enzymatic activity of lvDUT65–210 by the N-terminal region

As Stl has showed broad-spectrum inhibitory activity on multiple homotrimeric dUTPases, we assumed that it would also inhibit lvDUT65–210. To test this hypothesis, we measured the enzymatic activity of lvDUT65–210 both in the presence and the absence of Stl using a phenol red assay. As expected, the enzymatic activity of lvDUT65–210 was inhibited by Stl in a concentration-dependent manner (Fig. 1A). The apparent IC50 was 47.45 ± 5.22 nM, and the maximal inhibition to lvDUT65–210 at a high Stl

Discussion

In this study, we showed that Stl binds to lvDUT65–210 through its N-terminal region and inhibits the enzymatic activity of lvDUT65–210. We also solved the crystal structure of StlN-ter in complex with lvDUT65–210, which reveals the inhibition mechanism of Stl on lvDUT65–210. This is the first time to report how Stl inhibits a eukaryotic dUTPase.

Remarkably, our study modifies the structural model of Stl inhibition on homotrimeric dUTPase. Stl is a broad-spectrum inhibitor of dUTPases from

Funding

This work was supported by the National Natural Science Foundation of China [31572660 and 31872600], “Qingdao Innovation Leadership Program” (No. 18-1-2-12-zhc) and the open fund of Laboratory for Marine Biology and Biotechnology, Qingdao Pilot National Laboratory for Marine Science and Technology (No. OF2015NO12).

CRediT authorship contribution statement

FW, CW, YW, KZ and XW performed experiments; FW, CL, XW, XL, SL, FL and QM analyzed data; FW, CL and QM wrote the paper. QM conceived and supervised the study. All authors have read and approved the final version of manuscript.

Declaration of competing interest

The authors declare that they have no conflicts of interest related to the contents of this article.

Acknowledgements

We thank the staff from BL19U1 beamline of National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation facility, for assistance during data collection.

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