Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein

The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Although free-standing PilZ proteins are by far the most prevalent among c-di-GMP effectors, their physiological functions remain largely unknown. In the present study, I found that the free-standing PilZ protein PA2799 (named as HapZ - histidine kinase associated PilZ) from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the phosphoreceiver (REC) domain of the hybrid histidine kinase SagS. The interaction between SagS and HapZ is further enhanced at elevated c-di-GMP levels. I demonstrated that binding of HapZ to SagS inhibits the phosphotransfer from SagS to the downstream protein HptB in a c-di-GMP-dependent manner. Consistent with the role of SagS as a motile-sessile switch and biofilm growth factor, I found that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. These findings suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signalling through a PilZ adaptor protein. To understand the molecular basis of the protein-protein interaction between the receiver domain of SagS (RECSagS) and both HptB and HapZ, we determined the solution structure of RECSagS by NMR spectroscopy to show that that RECSagS adopts a typical CheY-like protein fold. By structural modelling and NMR titration, we found that the interaction between RECSagS and HptB involves a buried hydrophobic interface and potentially several specific hydrogen bonding residues. As some of the interacting residues are different in the homologous proteins HptA and HptC, the findings provide a mechanistic explanation for the specific recognition of SagS by HptB, but not HptA and HptC. To understand the interaction between SagS and HapZ, systematic site-directed mutagenesis and bacterial two-hybrid binding assays were performed to identify key residues involved in the protein-protein interaction. The results from the two-hybrid binding assays suggest that the only essential residues for protein-protein binding are the c-di-GMP binding residues. Based on the observations, we proposed a model where dimeric c-di-GMP mediates the interaction between HapZ and RECSagS by functioning as a molecular glue. Together, the research work reveals a novel mechanism used by c-di-GMP to regulate two-component signalling through a PilZ adaptor protein and advance our understanding of the molecular mechanism of c-di-GMP signalling.DOCTOR OF PHILOSOPHY (SBS

A cyclic di-GMP-binding adaptor protein interacts with histidine kinase to regulate two-component signaling

The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.MOE (Min. of Education, S’pore)Published versio