The long-term objective of the application is to elucidate the catalytic mechanism of GxGD membrane protease, which differs in structure and mechanism from the soluble aspartyl proteases. Presenilin and type 4 prepilin peptidase (TFPP) are both members of the GxGD protease family: mutations in presenilin can cause familial Alzheimer's disease, whereas TFPPs are involved in bacterial pathogenesis. FlaK is an archaeal homolog of TFPP, and its crystal structure, solved in the preliminary study, is presently the only atomic resolution structure in the family. The crystal structure provides an overview of the membrane protein's fold, and suggests that the protease must undergo conformational changes upon substrate binding to move the uncoupled aspartyl residues together for catalysis.
Three specific aims are proposed in the application.
In specific aim 1, mutations that allosterically stimulate FlaK's enzymatic activity will be generated and studied by x-ray crystallography to help explain the nature of the conformational change.
In specific aim 2, transition state analog inhibitors incorporating hydroxyethylene or (hydroxyethyl)urea isosteres will be synthesized to co- crystallize with FlaK. The substrate binding subsites and the allosteric model will be tested by mutagenesis.
In specific aim 3, substrate-protease fusion proteins will be generated to evaluate the role of substrate's TM domain, which is downstream of the cleavage site, in the binding to the protease. Full-length substrate with an intact TM domain is more efficiently cleaved by FlaK than short peptide. Crystallographic and mutagenesis experiments are planned to characterize the fusion proteins.

Public Health Relevance

The GxGD membrane protease family contains many members that are important in medicine. The catalytic mechanism for the enzyme family is poorly understood. Building on the recent breakthrough in determining the first crystal structure in the family, experiments are proposed to study substrate binding and conformational change in a prokaryotic GxGD protease employing techniques of biochemistry, chemical biology and x-ray crystallography.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chin, Jean
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Yale University
Schools of Medicine
New Haven
United States
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Kuo, Ivana Y; Hu, Jian; Ha, Ya et al. (2015) Presenilin-like GxGD membrane proteases have dual roles as proteolytic enzymes and ion channels. J Biol Chem 290:6419-27
Hu, Jian; Xue, Yi; Lee, Sangwon et al. (2011) The crystal structure of GXGD membrane protease FlaK. Nature 475:528-31