Structure Bases Of RTA Interaction With P2 And Rot And SaeR^DBD In Staphylococcus Aureus

Ribosome-inactivating proteins(RIPs)are N-glycosidases,which could depurinates a conserved adenine of 28S ribosomal RNA,thus inactivating the ribosome and inhibiting protein synthesis.Ricin has been isolated from the seeds of Ricinus communis and is a member of the type ? RIPs family.The A chain of ricin(RTA)has a molecular mass of 32 kDa and exhibits RNA N-glycosidase activity.RTA inactivates ribosomes by removing the conserved adenine in the sarcin-ricin loop(SRL)of 28S rRNA.In humans,the ribosomal stalk consists of PO and two copies of P1/P2 heterodimers that form a pentameric complex.It was reported that stalk proteins contain a highly conserved set of 11 residues(SDDDMGFGLFD)at the C-terminal tail that recruits elongation factors and interacts with several RIPs.RTA interacts with stalk proteins by the conserved 11 residues at the C-terminal tail was also confirmed,however,the details of the molecular interaction remain unknown.In this study,we demonstrate that RTA interacts with the human ribosomal stalk protein P2 and mainly recognises the highly conserved C-terminal tail of P2 by ITC assays.The crystal structure of RTA in a complex with the C-terminal peptide of P2(RTA-C10-P2)is determined at 1.50 A resolution.The structure shows that C10-P2 inserts into a hydrophobic pocket of RTA.The key residues in RTA and P2 for complex formation were determined by pull-down assays.The results of cell-free translation assays further confirmed that the interaction with stalk proteins is essential for the inhibition of protein synthesis by RTA.By comparing the structure of RTA-C10-P2 with the structure of trichosanthin(TCS)in a complex with the C-terminal peptide of P(TCS-C11-P),we found that though the conformation of the C-terminal peptide bound to RTA differs significantly from the conformation of C-terminal peptide bound to TCS,the conserved LF motif at the C-terminal tail is vital for binding RTA or TCS.Further analysis found that the LF motif is the unique element that is conserved in both eukaryotic and achaea ribosomal stalk proteins,and has been shown to be necessary for the binding of translation factors to ribosome.These results indicate that RTA,TCS and translation factors all interact with the highly conserved LF motifs by hydrophobic interactions.Staphylococcus aureus(S.aureus)is an important human pathogen capable of causing a wide range of diseases such as pneumonia,endocarditis,and sepsis and so on,and these diseases become more complicated by the emergence of methicillin-resistant and vancomycin-resistant S.aureus.Diversity and severity of these diseases depend on the expression of virulence factors in S.aureus.The expression and secretion of these virulence factors are tightly regulated by many regulatory elements.SarA is a global transcriptional regulator in S.aureus.SarA family has at least 11 members and regulates the transcription of various genes involved in virulence by binding to the target promoter regions.Rot belongs to the single-domain subfamily of SarA family and could affect the transcription of 168 genes.However,because of a lack of the structural information,the details of how Rot binds to the promoter regions of target genes remains unknown.In this study,the X-ray crystal structure of Rot is reported at 1.77 A resolution.The structure reveals that there are two Rot molecules in one asymmetric unit,which form a homodimer.Each of Rot contains a typical winged-helix motif constituted by the helix-turn-helix motif and a ?-hairpin motif.The results of fluorescence polarization indicate that Rot preferentially recognizes 30 bp AT-rich dsDNA.It was confimed that the vital role of several conserved positively charged residues on the winged-helix motif of Rot in binding to dsDNA both in vitro and in vivo.Therefore,we proposed that the DNA-recognition model of Rot may be similar to that of SarA,SarR and SarS,in which the helix-turn-helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves.It was also confirmed that Rot functions as a dimer by size-exclusion chromatography and fluorescence polarization assays.In addition,our structure also shows that the pattern of dimerization of Rot is significantly different from that of the reported SarA homologues,hinting at a divergence between Rot and other SarA homologues in the transcription regulation process.Two-component regulatory system(TCS)exhibits as a signal transduction mechanism by which bacteria,lower eukaryotes and plants monitor and respond to environmental conditions.S.aureus harbours 16 conservative TCSs.SaeR/S is one of the TCSs and plays a key regulatory role in the expression of many virulence factors including some secreted proteins,cell wall proteins,and cell wall-associated proteins.SaeR belongs to the OmpR/PhoB subfamily of response regulators.It contains an N-terminal receiver domain(RD)and a C-terminal DNA-binding domain(DBD).Though it has been confirmed that SaeR binds to the promoter regions of target genes via DBD,the details of the molecular interaction remain unknown.In this study,we determined the crystal structure of the DNA-binding domain of SaeR(referred to as SaeRDBD)at 2.50 A resolution.The structure reveals that SaeRDBD exists as a monomer and has the typical winged-helix motif constituted by the helix-turn-helix motif and a ?-hairpin motif.The results of size-exclusion chromatographic assay showed that both SaeR and SaeRDBD existed as a monomer in solution.The EMSA experiments showed that both SaeR and SaeRDBD could bind to P1 promoter,but SaeR had a higher binding affinity than that of SaeRDBD.We also confirmed the vital role of 5 residues on the winged-helix motif of SaeR in binding to P1 promoter in vitro and physiological function of SaeR in vivo.Therefore,we proposed that the DNA-recognition model of SaeRDBD may be similar to that of PhoBDBD,in which the helix-turn-helix motifs interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves.