The inhibitory mechanism of aurintricarboxylic acid targeting serine/threonine phosphatase Stp1 in Staphylococcus aureus: insights from molecular dynamics simulations

Abstract

Serine/threonine phosphatase (Stp1) is a member of the bacterial Mg 2+-or Mn 2+-dependent protein phosphatase/protein phosphatase 2C family, which is involved in the regulation of Staphylococcus aureus virulence. Aurintricarboxylic acid (ATA) is a known Stp1 inhibitor with an IC50 of 1.03 μM, but its inhibitory mechanism has not been elucidated in detail because the Stp1-ATA cocrystal structure has not been determined thus far. In this study, we performed 400 ns molecular dynamics (MD) simulations of the apo-Stp1 and Stp1-ATA complex models. During MD simulations, the flap subdomain of the Stp1-ATA complex experienced a clear conformational transition from an open state to a closed state, whereas the flap domain of apo-Stp1 changed from an open state to a semi-open state. In the Stp1-ATA complex model, the hydrogen bond (H-bond) between D137 and N142 disappeared, whereas critical H-bond interactions were formed between Q160 and H13, Q160/R161 and ATA, as well as N162 and D198. Finally, four residues (D137, N142, Q160, and R161) in Stp1 were mutated to alanine and the mutant enzymes were assessed using phosphate enzyme activity assays, which confirmed their important roles in maintaining Stp1 activity. This study indicated the inhibitory mechanism of ATA targeting Stp1 using MD simulations and sheds light on the future design of allosteric Stp1 inhibitors.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus

“…S11C), which is in line with previously published data (51). Phosphorylation of ribosomal proteins such as RplS ( pThr 11 ), RpsC ( pThr 176 ), and RpsP ( pThr 70 ); translation elongation factors EF-Tu ( pThr 34 ) and EF-G ( pThr 34 , pThr 43 , and pThr 424 ); and cell division proteins such as FtsZ ( pThr 333 , pSer 338 , and pThr 367 ) were prominently overrepresented in the stp deletion mutant as compared with the wild type (Fig. 6, D to F).…”

“…Here, we show that deletion of stp leads to increased threonine phosphorylation ( pThr 34 , pThr 43 , and pThr 424 ) of EF-G and extensive phosphorylation of ribosomal proteins in acid stress-exposed S. aureus. Together with reduced protein synthesis, low ATP concentration, and a delayed onset of growth after stress removal, this correlated with increased antibiotic tolerance in S. aureus.…”

“…In S. aureus, the conserved eukaryotic-type serine-threonine (Ser-Thr) kinase STK (alternatively named PknB or Stk1) and the cognate phosphatase Stp affect bacterial cell signaling, central metabolism (24)(25)(26), stress response (26,27), antibiotic resistance (26,(28)(29)(30), and virulence (28,29,(31)(32)(33). Stp has been suggested as a potential target for anti-virulence agents because stp mutation or absence decreases S. aureus virulence factor production (34,35). Ser-Thr phosphorylation is also involved in the regulation of protein synthesis, the most energy-consuming cellular process, and the initiation of translation decreases in response to nutrient limitation (36).…”

Fgd5 is a Rac1-specific Rho GEF that is selectively inhibited by aurintricarboxylic acid

“…It has since been shown to be an active inhibitor of viral pathogenesis [37,38] and expression of adhesion molecules [39]; however, the specific targets for these effects are unknown, although they may be mediated through inhibition of nucleases. Nucleaseindependent targets of aurintricarboxylic acid include reports of binding and inhibition of phospholipase C [40] and more recently, inhibition of Staphylococcus aureus phosphatases involved in the regulation of their virulence [41]. Therefore, aurintricarboxylic acid may serve to inhibit multiple processes, with identification of its interactors being key to the design of more specific inhibitors.…”

Molecular mechanism of green tea polyphenol epicatechin gallate attenuating Staphylococcus aureus pathogenicity by targeting Ser/Thr phosphatase Stp1

“…Strikingly, the binding of ECG to Stp1 led to the conformational change of the flap subdomain from a horizontal position to an almost vertical position. According to the literature, [47][48][49] the flexible flap subdomain located to the catalytic core (four metal ions binding region) of Stp1 is critical in its activity and substrate binding. Thus, in this paper, the flap subdomain in the vertical position was treated as the closed state (incapable of binding to the substrate), whereas that in the horizontal position was treated as the open state of the catalytic active region.…”