Molecular basis of binding between the global post-transcriptional regulator CsrA and the T3SS chaperone CesT

Nature Communications, Mar 2018

The T3SS chaperone CesT is recently shown to interact with the post-transcriptional regulator CsrA to modulate post-attachment signaling in enteropathogenic and enterohemorrhagic Escherichia coli. The molecular basis of the CesT/CsrA binding, however, remains elusive. Here, we show that CesT and CsrA both created two ligand binding sites in their homodimers, forming irregular multimeric complexes in solution. Through construction of a recombinant CsrA-dimer (Re-CsrA) that contains a single CesT binding site, the atomic binding features between CesT and CsrA are delineated via the structure of the CesT/Re-CsrA complex. In contrast to a previously reported N-terminally swapped dimer-form, CesT adopts a dimeric architecture with a swapped C-terminal helix for CsrA engagement. In CsrA, CesT binds to a surface patch that extensively overlaps with its mRNA binding site. The binding mode therefore justifies a mechanism of CsrA-modulation by CesT via competitive inhibition of the CsrA/mRNA interactions.

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Molecular basis of binding between the global post-transcriptional regulator CsrA and the T3SS chaperone CesT

Abstract The T3SS chaperone CesT is recently shown to interact with the post-transcriptional regulator CsrA to modulate post-attachment signaling in enteropathogenic and enterohemorrhagic Escherichia coli. The molecular basis of the CesT/CsrA binding, however, remains elusive. Here, we show that CesT and CsrA both created two ligand binding sites in their homodimers, forming irregular multimeric complexes in solution. Through construction of a recombinant CsrA-dimer (Re-CsrA) that contains a single CesT binding site, the atomic binding features between CesT and CsrA are delineated via the structure of the CesT/Re-CsrA complex. In contrast to a previously reported N-terminally swapped dimer-form, CesT adopts a dimeric architecture with a swapped C-terminal helix for CsrA engagement. In CsrA, CesT binds to a surface patch that extensively overlaps with its mRNA binding site. The binding mode therefore justifies a mechanism of CsrA-modulation by CesT via competitive inhibition of the CsrA/mRNA interactions. Introduction The pathogenic Escherichia coli (E. coli) strains of enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) can cause severe diseases in humans, resulting in the characteristic attaching and effacing (A/E) lesions1,2. These pathogens has evolved to acquire a wide variety of virulence genes, among which those encode for a type III secretion system (T3SS) and the related proteins are shown to play central roles in the colonization and A/E pathogenesis of the bacteria3. T3SS is widely distributed among Gram-negative pathogens and functions to translocate diverse bacterial effector proteins into host cells4. In EPEC and EHEC, a battery of up to 40 effector proteins are delivered via T3SS, leading to not only enterocyte damages but also subversions of multiple cellular signaling pathways3. It was recently shown that T3SS also participates in host cell sensing and post-attachment remodeling of gene expressions. The process was demonstrated to be mediated by the T3SS-specific chaperone CesT antagonizing the post-transcriptional regulator CsrA5. CesT was initially identified as the chaperone for E. coli secreted protein Tir (translocated intimin receptor)6. It was later shown that CesT also interacts with a variety of bacterial effectors (e.g., Map, NleA, NleG, NleH, NleH2, EspF, EspG, EspH, and EspZ) and is therefore better characterized as a multicargo T3SS chaperone7,8,9. In addition to effector binding, CesT is also shown to play a role in effector secretion, mediating hierarchical protein translocations10. It is notable that Infection studies in both cell line and animal models have demonstrated that CesT is essential for efficient host colonization6,7. The chaperone protein is, therefore, a key T3SS player involved in the pathogenesis of EPEC and EHEC. As with other T3SS chaperones, CesT exists as stable homodimers in solution11,12. A previous study has reported the atomic structure of CesT, which shows a dimeric architecture with a swapped N-terminal domain11. Nevertheless, the biological relevance of this dimer form remains to be investigated, since a majority of the homologous T3SS chaperones utilize an unswapped dimer for effector binding13,14,15,16,17,18. It is notable that it has been proposed that CesT, in the absence of a domain-swap event, might also form an unswapped dimer similar to other T3SS chaperones11. A later NMR study also indicated the presence of such a dimer form in solution12. The Csr/Rsm system (for carbon storage regulator or repressor of secondary metabolism) is a widespread regulatory system that is present in abundant bacteria species19,20,21. Functionally, the system can mediate post-transcriptional regulation of gene expression and modulate multiple physiological processes, including central carbon metabolism, secondary metabolite metabolism, motility, biofilm formation, virulence factor production, and etc.19,20,21. A central player in this system is the CsrA/RsmA protein which is able to bind the ribosome-binding site (RBS) of target messenger RNAs (mRNAs) and thereby repress translation initiation22,23. The activity of CsrA is further modulated by small non-coding RNAs (sRNAs, e.g., CsrB/C and RsmX/Y/Z24,25), which can competitively antagonize the binding of CsrA to mRNA and relive the translation repression26,27. CsrA and its homologous proteins form highly conserved domain-swapped homodimers28,29,30. The mechanism of CsrA-mediated translational modulation has been illustrated with the Pseudomonas fluorescens (P. fluorescens) CsrA-homolog of the RsmE protein. The complex structure of RsmE bound with an RBS RNA-fragment derived from the hcnA gene revealed two RNA binding sites in the protein dimer, making optimal contacts with an A/UCANGGANGU/A sequence motif23. Later studies showed that sRNAs can engage the same RNA binding site in RsmE, thereby competing against the RsmE/mRNA interactions26,27. In addition to the sRNA-dependent regulation, the (...truncated)


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Fei Ye, Fanli Yang, Ruijie Yu, Xi Lin, Jianxun Qi, Zhujun Chen, Yu Cao, Yuquan Wei, George F. Gao, Guangwen Lu. Molecular basis of binding between the global post-transcriptional regulator CsrA and the T3SS chaperone CesT, Nature Communications, 2018, Issue: 9, DOI: 10.1038/s41467-018-03625-x