Structural studies of the full fibronectin-binding site of pathogenic bacteria-extending and characterizing the tandem beta-zipper

An enormous range of normal processes in the human body depend on interactions between two or more proteins. Many diseases are the result of disruption of such interactions due to mutations which change the shape of the protein. Also, in order for bacteria to cause infections, they have to attach to hmuan tissue, and frequently this also occurs through protein-protein interactions (in this case between bacterial proteins and human proteins). In the past it was thought that functional protein-protein interactions depended on both proteins having a specific shape, but more recently it has been discovered that a very significant number of functional proteins are disordered and quite flexible. There is a lot of interest in discovering how these disordered proteins bind to other proteins. We recently discovered an interesting way in which this can occur and in the proposed research we plan to extend these studies. We are investigating the binding of disordered proteins from two very different types of bacteria to the human protein fibronectin. The bacteria are Streptococcus pyogenes, which causes throat infections, but also more life-threatening infections, and Borrelia burgdoerferi, which causes Lyme disease and is transmitted to humans through the bite of a tick. Fibronectin is a protein which is found in blood and in the extracellular matrix. Among other functions, the extracellular matrix provides surfaces to which cells can attach and migrate over. Fibronectin is a modular protein; it is made up of folded protein modules, like beads on a string. At one end of fibronectin there is a string of five F1 modules (1-5F1), each with the same shape, consisting of two anti-parallel beta-sheets. A beta-sheet is a type of protein structure where extended pieces (called beta-strands) of the protein chain lie anti-parallel to each other. One of the sheets has three anti-parallel beta-strands. We discovered that when the disordered bacterial protein binds to the first two F1 modules, it does so by adding an extra strand to the three-stranded sheet in each module. This is the first time this type of binding has been observed. Because the bacterial protein 'zips-up' along the fibronectin modules, we called the interaction a tandem beta-zipper. We obtained evidence which suggests that the bacterial protein may bind the full string of five F1 modules in this way. We aim to test this hypothesis in the proposed research. The bacterial proteins not only bind to 1-5F1, but they also bind to an adjacent site in fibronectin. Very little is known about this interaction so the second main aim of this work is to study it closely so that we can discover the parts of the bacterial and human proteins that are essential for the binding to occur. The proposed research is likely to reveal an exciting three-dimensional structure demonstrating a very extended tandem beta-zipper interaction. The research will also define the full binding sites involved in this host-pathogen interaction. The results of proposed research will be of value to research groups studying disordered proteins, protein-protein interactions and mechanisms of infection.

We recently reported a novel-mechanism of protein-protein recognition discovered in our studies of interactions of unstructured bacterial proteins with the human protein fibronectin. In this tandem beta-zipper interaction, the unstructured protein forms an additional beta strand on the triple-stranded beta sheet of two sequential F1 modules in the N-terminal domain of fibronectin. We have speculated that this may be a more general mechanism for the interaction of disordered regions of proteins with proteins containing tandemly arrayed modules, such as fibronectin. In particular, the disordered proteins are able to exploit the modular nature of fibronectin by assembling weakly-binding motifs in the correct order to bind sequential modules. These weakly-binding motifs together provide a high-affinity binding site. Already a second tandem beta-zipper has been discovered in a different cellular context, and given the elegant 'design' of these interactions, we predict others will be discovered as studies of unstructured proteins expand. We have evidence that the tandem beta-zipper in fibronectin-binding proteins from streptococci and spirochetes extends over at least four F1 modules, and we propose to determine the three-dimensional structures of these complexes using NMR spectroscopy and X-ray crystallography. The structures will allow the source of specificity of the single beta-zippers to be elucidated. This information will be particularly important for designing higher-affinity fibronectin ligands. The second aim of this work is to study the interaction of bacterial proteins with the collagen binding region of fibronectin. Although this interaction has been suggested to play an important role in invasion of host cells by streptococci, there is as yet, no residue-specific or structural information available. Overall, this work will both characterise an unusual and potentially very important mechanism of protein-protein recognition as well as defining the full binding site for fibronectin in the bacterial proteins. We anticipate that the results will be of interest to researchers in fields of host-pathogen interactions, disordered proteins and protein-protein interactions in general.