Structure/function studies of a cyclomodulin

Proteins are a biological molecules encoded by a gene, and they are the molecular machines of life. Proteins form a complex 3-dimensional structure that frequently determines their function. A protein's function is usually dependent on interaction with another molecule and this interaction leads to an effect. The experiments described in this proposal aim to investigate the structure and function of a protein from pathogenic bacteria that is able to change the properties of the host cell during infection. Specifically this protein, called 'Cif' interferes with progression of the host cell cycle (process of growth and division), irreversibly stopping it in its tracks. Although it is not currently understood why, this presumably has some benefit for the invading bacteria. Studying proteins such as this is important for both understanding host-pathogen interactions (the molecular causes of disease) and the host cell processes themselves. For instance, a deregulated cell cycle is often linked to development of cancer, and proteins that regulate the cell cycle are one of the most targeted set of molecules by the pharmaceutical industry. How Cif actually brings about its effect in host cells is not currently understood. One powerful approach to determining how this protein functions is to look at its structure. This can be visualised using a technique called X-ray crystallography followed by reconstruction of a model using computer graphics. Once the structure is known alterations to the protein can be designed to further investigate protein function in host cells. Also important for understanding Cif function is identifying the protein's molecular targets within host cells (most likely other proteins), characterising the interaction (how strongly do they bind to each other?), and ultimately determining the structure of molecules together. This would generate a picture of a bacterial protein interacting with a host cell protein, and a complex responsible for transducing an effect ultimately leading to host cell cycle arrest. Understanding the nature of these interactions for Cif has long-term applications for development of novel therapeutics targeting host-pathogen interactions and also carcinogenesis.

Cif (cycle inhibiting factor) is a type 3 secretion dependent 'cyclomodulin' protein initially identified in the food-borne pathogens Enteropathogenic E. coli (EPEC) and Enterohaemorrhagic E. coli (EHEC). Translocation of Cif by these bacteria into model host cells induces a cytopathic effect characterised by irreversible cell cycle arrest and formation of actin stress fibres (an identical effect is also observed using an artificial lipid-based protein delivery system in the absence of bacteria). Cell cycle arrest by Cif is not achieved by activation of the DNA damage pathway but by a novel, as yet uncharacterised mechanism. To determine this mechanism of action, protein structures for members of the Cif family are urgently required, as is information on the complexes formed by Cifs in host cells that are mediating effects. To this end, building on preliminary data, the crystal structure of Cifs from EPEC and Burkholderia pseudomallei will be determined. These structures will be analysed in the context of identifying protein function. From the structures, site directed mutagenesis studies will be designed to probe function as assayed by the established lipid-based protein delivery system (plus any in vitro assay appropriate). Complexes between Cifs and target host proteins already identified in a yeast two hybrid screen will be investigated using appropriate thermodynamic and hydrodynamic techniques in solution. Complexes that form stable interactions will be the subject of structure determination by the complementary techniques of small angle X-ray scattering (SAXS) and X-ray crystallography. Interacting surfaces will be probed by site directed mutagenesis to understand their role in protein function. These studies are designed to probe the function of a bacterially derived modulator of the eukaryotic cell cycle, with implications in understanding host-pathogen interactions and in the longer term identifying novel therapeutic targets.