Two-component signal transduction pathways and expanded multi-step His-Asp phosphorelay signaling pathways control how bacteria and fungal organisms respond and adapt to environmental stress. The His-Asp phosphorelay pathways found in eukaryotes frequently feature multiple upstream sensor kinases (HKs) and downstream response regulator (RR) proteins, yet nearly all depend on a single intermediate histidine phosphotransfer (HPt) protein for phosphoryl group transfer. Although several thousands of HK-RR cognate pairs have been identified in bacteria and fungi, very little is understood about protein-protein interactions that govern specificity within a particular pathway and prevent cross-talk within a single organism.
The relative simplicity of the signal transduction pathway (one HK, one HPt and two RRs) in the model yeast Saccharomyces cerevisiae, together with recent X-ray crystallographic studies of the Ypd1 HPt protein in complex with the Sln1 receiver domain, provides an excellent foundation for the investigation of molecular interactions within a phosphorelay signaling system. The long-term goal of this research is to understand regulation of phosphate flow from Ypd1 to the downstream response regulators, Ssk1 and Skn7, as a function of environmental stress. The main objective of this project is to achieve a detailed understanding of the structural, biochemical and functional implications of Ypd1 interactions with the three homologous response regulator domains associated with Sln1, Ssk1 and Skn7.
The specific aims are designed to test the hypothesis that molecular interactions within the yeast phosphorelay signaling pathway are influenced by external environmental signals and the phosphorylation state of the interacting signaling partners. A multidisciplinary approach using structural, biochemical and genetic approaches will be taken. Specific Aim 1 is structural characterization of Ypd1 (HPt) protein complexes with cognate RR domains. Specific Aim 2 is to determine the effect of site-specific mutations in Ypd1 and/or RR domains on protein binding affinity, phosphotransfer and specificity of interaction. Specific Aim 3 is to determine the in vivo consequences of mutations that affect Ypd1-RR interactions in the SLN1 pathway. The proposed research has broad significance with respect to how signaling partners interact with each other and influence fidelity of signal transduction. The results are expected to reveal, for the first time, key structural features that contribute to HPt-RR interactions and signaling specificity within multistep phosphorelay systems from all three domains of life.
Broader Impacts: Results from this project are expected to provide significant new insight into the principles that govern signal transduction pathways, specifically, the role of protein phosphorylation and its impact on regulating protein-protein interactions. The integration of research and student training is an important aspect of this proposal. A summer exchange program for undergraduate students at the University of Oklahoma and the University of Iowa will provide cross-disciplinary training in structural, biochemical and in vivo genetic approaches. In addition, a hands-on laboratory-based X-ray crystallography course will be developed and offered at the graduate-level or senior undergraduate capstone level at University of Oklahoma. Students will have the opportunity to access state-of-the-art (NSF-funded) crystallization robotics instrumentation and apply X-ray diffraction techniques to solve the three-dimensional structure of biomacromolecules.
