Iron-sulfur cluster-containing sensor regulators: mechanistic and structural studies of DNA-binding

In order to survive, bacteria must be able to sense and adapt to their (changing) environment. This includes pathogens trying to establish infection in a human host. Regulatory proteins, which control gene transcription by specifically binding to DNA, play key roles in sensing and responding to environmental change. Many of these proteins contain a special cofactor, consisting of iron and sulfur, called an iron-sulfur cluster. In regulatory proteins, this cluster functions as the sensor, where it detects a particular signal by undergoing a reaction that leads to protein conformational changes. These changes cause DNA-binding to be turned on or off, and thus transduce the original signal to produce the necessary cellular response.
The Rrf2 family of regulators is widespread amongst bacteria and controls some of the most important cellular pathways, including iron metabolism, the biosynthesis of iron-sulfur cluster cofactors, and responses to oxidative and nitrosative stresses. Many Rrf2 family regulators bind an iron-sulfur cluster cofactor, and the reactivity of this cluster underpins the sensing function of the regulator. It turns out that, although Rrf2 proteins appear to be similar to one another in terms of sequence and overall structure, the type of cluster they bind, and the way that they bind it, varies from one Rrf2 protein to another.
Recently, we have made a lot of progress in understanding mechanistic and structural features of these regulators, including the first structures of cluster bound forms of two of them. This revealed features of how the cluster is bound to the protein not previously observed in other iron-sulfur cluster proteins. Our work has also led to detailed functional understanding of how the cluster reacts with its particular signal molecule, for example the cytotoxin nitric oxide, and how this reaction leads to changes in shape of the protein that are likely to affect the protein's ability to bind DNA.
Despite this recent progress, we still know relatively little about the interaction of Rrf2 family proteins with DNA and how this affects the response to signaling molecules. This is difficult to study in solution because of the viscosity of DNA solutions and so nearly all information currently available relates to non-DNA-bound forms. We have developed the application of an analytical technique called mass spectrometry, which provides accurate mass information for very large molecules such as proteins with their cofactors bound. This has provided unprecedented insight into the reactions of iron-sulfur cluster cofactors. Now we have succeeded in establishing conditions under which Rrf2 proteins bound to DNA can be detected, where the low concentrations necessary mean that viscosity is not a problem. This opens up the possibility to gain fundamental insight into these regulatory proteins by studying their binding to DNA and reactivity when in their DNA-bound forms. This will enable us to address questions that cannot be tackled currently by other methods. For example, we will be able to determine at what point in the sensing reaction shape changes that turn off DNA binding occur. We have also succeeded in determining 3-4 Å resolution structures of two Rrf2 regulators in DNA-bound forms, and this is beginning to reveal details of the particular shape of the protein and the points of interaction between the protein the DNA. This work, although fundamental in nature, will significantly advance understanding of how bacteria sense and overcome inhospitable conditions, including those that they encounter when trying to establish infection in a host.

The ability to sense and respond to a wide variety of environmental stimuli is essential for the survival and adaptability of many bacteria, including pathogens. The reactivity of iron-sulfur (FeS) clusters to small molecule effectors (eg. nitric oxide (NO) and reactive oxygen species) and ability to undergo redox processes is exploited in nature: many of the proteins that function as sensors of environmental change, and which coordinate the cell's response to those changing conditions, are FeS cluster proteins. In particular, many of these belong to the Rrf2 family of FeS regulators, which is widespread and of increasingly recognised importance. Although significant progress towards understanding structure/function relationships in these proteins has been made recently, there remains much to learn.
This application is focused on Rrf2 regulators representing the major classes of regulatory mechanisms that are currently known for FeS proteins: sensing via chemical reaction at the FeS cluster, e.g. with NO, resulting in cluster nitrosylation (NsrR); sensing via degradation/loss of the FeS cluster, e.g. under low iron (or low FeS cluster availability) conditions (RirA and IscR); and, sensing via cluster redox changes (RsrR). Here, we are particularly focused on Rrf2 protein-DNA interactions and their effects on cluster reactivity, and on understanding the molecular basis of the switch between DNA-binding and non-binding forms. We will exploit our recent breakthroughs in the development of novel mass spectrometry approaches, which provide information that is not accessible using other methods, and in using anaerobic X-ray crystallography, which have recently yielded the first cluster-bound structures of Rrf2 regulators. The proposed work will lead to fundamental new insight into the DNA-bound forms of Rrf2 family regulators, including the key FeS cluster sensing events that switch off DNA-binding, and how these are fine-tuned for different DNA promoter sequences.