The goal of this project is to determine the structures of two signal transduction proteins, Che-Y and VHR, that are potentially relevant to human diseases. The unique strategy of this work is the use of chemical modification of the proteins to create stable analogs of transient, phosphorylated forms of each protein that would be otherwise difficult to study. Specifically, a cysteine residue is modified with a phosphonomethyl group, CH2PO3. The presence of the phosphonomethylcysteine residue will be confirmed by liquid chromatography and mass spectrometry. The structures of the proteins will be determined by x-ray crystallography. The first protein is CheY, a protein necessary for bacteria to swim toward a more desirable environment, a process known as chemotaxis. Phosphono-CheY was synthesized from a mutant form of CheY and phosphonomethytrifluoromethanesulfonate. The structure of phosphono-CheY will be determined in complex with its partner, FliM. The structure of phosphono-CheY complexed with phosphatases designed to return CheY to its dephosphorylated state may also be determined to obtain insight into the signal termination process. Disruption of chemotaxis may render an organism less pathogenic. The second protein is VHR, a dual-specificity phosphatase. Dysregulation of protein tyrosine phosphatases plays a role in many human diseases, including cancer and diabetes. Protein tyrosine phosphatase enzymes break down phosphoproteins via the creation of a cysteinyl phosphate intermediate that is subsequently hydrolyzed. VHR is inactivated when the cysteine residue is modified with a phosphonomethyl group to become phosphonomethylcysteine. Reversible inhibitors protect against inactivation. Determining the structure of a stable analog of this phosphonomethylcysteine intermediate may provide information about the mechanism of this reaction and may provide a target for structure-based drug design.
This project may lead to a better understanding of how to decrease bacterial pathogenesis by interfering with bacterial motility. Further, this project will provide insights into how cellular growth signals are turned on and off. Errors in these signaling processes underlie many human diseases, including diabetes.
| Lookadoo, Daniel B; Beyersdorf, Matthew S; Halkides, Christopher J (2018) Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY. Methods Mol Biol 1729:337-343 |
| Sircar, Ria; Borbat, Peter P; Lynch, Michael J et al. (2015) Assembly states of FliM and FliG within the flagellar switch complex. J Mol Biol 427:867-886 |
| McAdams, Kenneth; Casper, Eric S; Matthew Haas, R et al. (2008) The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides. Arch Biochem Biophys 479:105-13 |
| Halkides, Christopher J; Bottone, Cory J; Casper, Eric S et al. (2007) Synthesis of a stable analog of the phosphorylated form of CheY: phosphono-CheY. Methods Enzymol 422:338-51 |
