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...
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| Format: | Thesis |
| Language: | English |
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2016
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| Online Access: | https://hdl.handle.net/10356/69390 |
| _version_ | 1811682754645983232 |
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| author | Venkataramani, Prabhadevi |
| author2 | Liang Zhao-Xun |
| author_facet | Liang Zhao-Xun Venkataramani, Prabhadevi |
| author_sort | Venkataramani, Prabhadevi |
| collection | NTU |
| description | 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. |
| first_indexed | 2024-10-01T04:01:52Z |
| format | Thesis |
| id | ntu-10356/69390 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T04:01:52Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | ntu-10356/693902023-02-28T18:36:30Z Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein Venkataramani, Prabhadevi Liang Zhao-Xun School of Biological Sciences DRNTU::Science::Biological sciences 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) 2016-12-23T03:52:41Z 2016-12-23T03:52:41Z 2016 Thesis Venkataramani, P. (2016). Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/69390 10.32657/10356/69390 en 155 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences Venkataramani, Prabhadevi Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein |
| title | Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein |
| title_full | Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein |
| title_fullStr | Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein |
| title_full_unstemmed | Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein |
| title_short | Regulation of two-component signalling by a cyclic di-GMP-binding PilZ protein |
| title_sort | regulation of two component signalling by a cyclic di gmp binding pilz protein |
| topic | DRNTU::Science::Biological sciences |
| url | https://hdl.handle.net/10356/69390 |
| work_keys_str_mv | AT venkataramaniprabhadevi regulationoftwocomponentsignallingbyacyclicdigmpbindingpilzprotein |