Final Activity Report Summary - FAPR (Global regulation of lipid biosynthesis in Gram-positive bacteria: structural and biochemical characterization of FapR)
This project comprised the functional and structural characterisation of FapR, a global regulator of lipid synthesis in bacteria. FapR is largely conserved in Gram positive organisms, including human pathogens as bacillus anthracis, listeria monocytogenes and staphylococcus aureus.
The main objective of this project was to understand in detail the mechanism of action of FapR and the way its activity was regulated. Malonyl-CoA acted as a signalling molecule modulating FapR activity. When malonyl-CoA was bound to its C-terminal domain, FapR did not bind to deoxyribonucleic acid (DNA) and hence could not repress the transcription of its target genes. We proposed an interdisciplinary and multidisciplinary approach so as to further characterise the binding of the malonyl-CoA effector molecule to the C-terminal domain of FapR and to firmly understand the way this binding was transmitted and affected the DNA binding affinity of the N-terminal domain. This was a crucial point, since inhibitors of the interaction of FapR with malonyl-CoA could represent a new type of antibacterial compounds.
The major aim of the first part of the project was to characterise the molecular architecture and organisation of the full-length FapR and the FapR-DNA complex. In this regard, we obtained the crystal structure of full length FapR from s. aureus, both alone and in complex with malonyl-CoA. Moreover, we obtained the structure of this protein in complex with a DNA fragment containing a FapR operator site. In this way, we succeeded in obtaining a snapshot of different steps during the FapR regulation cycle. The analysis and comparison of these conformational states, which was still in progress by the time of the project completion, would provide key elements and information to understand in detail the mechanism of action of FapR and the modulation of its activity.
We also proposed to perform structural studies of the effector-binding domain of FapR in complex with the specific ligand, malonyl-CoA. This was the second aim of the project. We obtained the atomic resolution structures of the C-terminal domain of FapR from s. Aureus, both alone and in complex with the effector molecule. We also obtained the crystal structure of the effector-binding domain of FapR from l. monocytogenes, another important human pathogen. The comparison of these two structures, together with that of b. subtilis FapR which was previously solved, was anticipated to allow us to establish whether FapR regulation of fatty acid synthesis took place through a common conserved mechanism across different Gram-positive bacterial species. Furthermore, the atomic resolution structures of s. aureus FapR were used to carry out in silico screening of chemical libraries to search for potential inhibitors of malonyl-CoA binding. Some 700 compounds corresponding to the top hits were selected and would be tested for their activity both in vivo and in vitro. The finding of effective inhibitors of the FapR-effector interaction could provide lead compounds for the development of a new type of antibacterial agents.
In summary, all the aims proposed in the first part of the project were successfully achieved. We obtained the crystal structure of:
1. full-length FapR from s. aureus (SaFapR)
2. full length SaFapR in complex with malonyl-CoA
3. full length SaFapR in complex with DNA
4. the atomic resolution structures of the malonyl-CoA binding domain of the truncated protein SaFapR, alone and in complex with the effector molecule and
5. the structure of the C-terminal malonyl-CoA binding domain of FapR from L. monocytogenes. These results opened new perspectives for the continuation of the project.
The main objective of this project was to understand in detail the mechanism of action of FapR and the way its activity was regulated. Malonyl-CoA acted as a signalling molecule modulating FapR activity. When malonyl-CoA was bound to its C-terminal domain, FapR did not bind to deoxyribonucleic acid (DNA) and hence could not repress the transcription of its target genes. We proposed an interdisciplinary and multidisciplinary approach so as to further characterise the binding of the malonyl-CoA effector molecule to the C-terminal domain of FapR and to firmly understand the way this binding was transmitted and affected the DNA binding affinity of the N-terminal domain. This was a crucial point, since inhibitors of the interaction of FapR with malonyl-CoA could represent a new type of antibacterial compounds.
The major aim of the first part of the project was to characterise the molecular architecture and organisation of the full-length FapR and the FapR-DNA complex. In this regard, we obtained the crystal structure of full length FapR from s. aureus, both alone and in complex with malonyl-CoA. Moreover, we obtained the structure of this protein in complex with a DNA fragment containing a FapR operator site. In this way, we succeeded in obtaining a snapshot of different steps during the FapR regulation cycle. The analysis and comparison of these conformational states, which was still in progress by the time of the project completion, would provide key elements and information to understand in detail the mechanism of action of FapR and the modulation of its activity.
We also proposed to perform structural studies of the effector-binding domain of FapR in complex with the specific ligand, malonyl-CoA. This was the second aim of the project. We obtained the atomic resolution structures of the C-terminal domain of FapR from s. Aureus, both alone and in complex with the effector molecule. We also obtained the crystal structure of the effector-binding domain of FapR from l. monocytogenes, another important human pathogen. The comparison of these two structures, together with that of b. subtilis FapR which was previously solved, was anticipated to allow us to establish whether FapR regulation of fatty acid synthesis took place through a common conserved mechanism across different Gram-positive bacterial species. Furthermore, the atomic resolution structures of s. aureus FapR were used to carry out in silico screening of chemical libraries to search for potential inhibitors of malonyl-CoA binding. Some 700 compounds corresponding to the top hits were selected and would be tested for their activity both in vivo and in vitro. The finding of effective inhibitors of the FapR-effector interaction could provide lead compounds for the development of a new type of antibacterial agents.
In summary, all the aims proposed in the first part of the project were successfully achieved. We obtained the crystal structure of:
1. full-length FapR from s. aureus (SaFapR)
2. full length SaFapR in complex with malonyl-CoA
3. full length SaFapR in complex with DNA
4. the atomic resolution structures of the malonyl-CoA binding domain of the truncated protein SaFapR, alone and in complex with the effector molecule and
5. the structure of the C-terminal malonyl-CoA binding domain of FapR from L. monocytogenes. These results opened new perspectives for the continuation of the project.