Initial crystallization of GtgA from Salmonella Typhimurium

Abstract

Salmonella Typhimurium is a pathogen of some mammals including human. Its pathogenic behaviors are first emerging by delivering its own bacterial proteins, known as effector, into host, but Salmonella confronts host defense system, leading to inflammation in epithelial cells of hosts. Inflammation is one of host defense response to pathogens and thus prevents further proliferation of bacteria. In human, a nuclear factor κB (NF-κB) has been known to play a key role in regulating inflammation by mediating transcriptional activation of proinflammatory cytokine production. To circumvent or counteract the NF-κB dependent defense system in human, Salmonella deploys three different effector proteins that all belong to members of the PipA family. Proteins in this family are characterized by a zinc-dependent protease. In fact, GtgA, one of Salmonella effectors, exhibited a protease activity against NF-κB by cleaving essential residues for DNA binding. This GtgA-mediated NF-κB degradation eventually led to decreasing proinflammatory cytokines. However, molecular details regarding a mode of action by GtgA remain largely uncharacterized. In order to identify structural features of GtgA, I performed extensive experimental approaches including expression of GtgA in various forms, crystallization of those proteins, and X-ray diffraction analysis. Initial experiment indicates that GtgA in its full length remains highly soluble but resists crystallization. Therefore, its hydrodynamic properties were modified by replacing a cluster of hydrophilic residues with hydrophobic ones. Among five constructs, two produced GtgA in insoluble form, but three resulted in a soluble GtgA. Those soluble GtgAs were further subject to purification and crystallization under various conditions. Initial condition for GtgA crystallization was recently observed and the X-ray diffraction data using those crystals were successfully collected at 2.5 Å-resolution. Currently, a molecular replacement using a template structure with amino acid sequence of 20% identity however failed to deliver a GtgA structure. Now, several experiments are carrying out to determine the phase information of GtgA. Structural properties of GtgA could provide valuable information of host-pathogen interactions.