In all organisms, DNA transcription is controlled by proteins called transcription factors (TFs) that recognize and bind to specific promoter DNA sequences. Many microbes have TFs that contain either cubane [4Fe-4S] or rhombic [2Fe-2S] clusters capable of sensing changes in their environment. Although iron-sulfur clusters are well-known for their importance in electron transfer reactions, transition metal-based Lewis chemical reactions and radical based catalysis, their use in gene transcription regulation has been less well characterized. In collaboration with our English partner, we intend to extend our understanding of the cluster-effector interactions and the ensuing protein conformational changes that modulate TF binding to DNA.
In general, bacteria use [Fe-S]-containing TFs to regulate the transcription of genes to fight environmental stress. Stress signals sensed by [Fe-S] clusters include the presence of effectors as molecular oxygen (O2), nitric oxide (NO), an unbalanced redox state of the cell and low levels of Fe or [Fe-S] clusters. The mechanisms of how these signals are transmitted to the DNA binding region of [Fe-S]-TFs are currently not well understood. Our project focuses on four structurally related TFs of the Rrf2 family: the NO sensing [4Fe-4S]-NsrR, the Fe sensing [4Fe-4S]-RirA, the redox sensing [2Fe-2S]-RsrR and the [Fe-S] sensing [2Fe-2S]-IscR. Apart from RsrR, these TFs are also very sensitive to O2 levels and each of them has different DNA binding specificities. As mentioned above, we propose to study, both functionally and structurally, (i) protein-bound [Fe-S] cluster degradation or modification by environmental stress signals and (ii) the way this process modulates TF binding to DNA and, by extension, gene expression. Studying four [Fe-S]-binding TFs that have a closely related fold but recognize different signals provides a unique opportunity to compare their regulatory mechanisms. Because these [Fe-S]-TFs are essential for many pathogens but absent in humans, they are also potential targets for the design of novel antibiotics.
The two project partners are well equipped for anaerobic protein production and purification and they have already solved the first crystal structures of cluster-bound forms of two of the Rrf2 family proteins, [4Fe-4S]-NsrR (doi: 10.1038/ncomms15052) and [2Fe-2S]-RsrR (unpublished results). Conversely, no structures are known yet for the labile holo forms of RirA and IscR. Our goals are to solve crystal structures of: (i) uncomplexed [4Fe-4S]-RirA and [2Fe-2S]-IscR, (ii) at least one DNA complex for each of the four studied TFs, (iii) [Fe-S]-TF intermediates formed after reaction with various effector molecules and (iv) selected variants to check the role of specific amino acid residues. Furthermore, we want to correlate the obtained structural information with biophysical characterizations of DNA binding affinity and effector-induced [Fe-S] cluster changes. In order to attain these goals, the main obstacles are to produce stable forms of the various TF states and to find the conditions for growing high-quality crystals. In order to overcome these potential obstacles, we will set up thousands of different crystallization conditions for each stable [Fe-S]-TF form using our advanced anaerobic automated setup. In addition, advanced biophysical techniques including mass spectrometry under non-denaturing conditions, Resonance Raman, infrared and nuclear resonance vibrational spectroscopy will be used by our collaborator to characterize the various [Fe-S]-TFs forms and find conditions where they are most stable in solution. This approach should help crystallizing at least some of the intermediate forms because stability is a crucial aspect in this process. Although we have already made some progress, funding of our project should boost our understanding on the mechanisms of gene transcription regulation through [4Fe-2S] and [2Fe-2S] cluster signaling.
Monsieur Anne Volbeda (INSTITUT DE BIOLOGIE STRUCTURALE)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IBS INSTITUT DE BIOLOGIE STRUCTURALE
Help of the ANR 238,002 euros
Beginning and duration of the scientific project: January 2019 - 36 Months