Intramembrane proteolysis is an important and widespread biochemical mechanism in cell biology: many membrane proteins undergo intramembrane proteolysis to become activated for signal transduction, or to be converted to poorly soluble amyloidal peptides that may cause human disease. The long-term objective of this proposal is to gain a deeper understanding of this mechanism through crystallographic analysis of specific membrane proteins that catalyze the reaction and of their complexes with inhibitors and substrates. Many intramembrane proteases have been recognized as novel and important drug targets for treating infectious and age-related diseases. The current application focuses on GlpG, an E. coli integral membrane protein of the rhomboid serine protease family. Biochemical, mutagenesis and crystallographic experiments are planned to study: (1) how GlpG interacts with class specific inhibitors in order to examine hypothesis that GlpG and other rhomboid proteases use a membrane-embedded Ser-His dyad to directly attack substrate, and to determine features of the protease active site that are important for catalysis;(2) how GlpG interacts with transmembrane substrates through complex structural rearrangements in both proteins, and which factors determine the specificity of this interaction;and (3) the mechanism by which an interesting structural motif regulates protease activity in the membrane. Recent breakthroughs in crystal structure determination of GlpG suggest that this bacterial membrane protein is an excellent model system for studying enzyme action in cell membranes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM082839-03S1
Application #
8141468
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2010-09-30
Project End
2011-08-31
Budget Start
2010-09-30
Budget End
2011-08-31
Support Year
3
Fiscal Year
2010
Total Cost
$46,553
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Xue, Yi; Ha, Ya (2013) Large lateral movement of transmembrane helix S5 is not required for substrate access to the active site of rhomboid intramembrane protease. J Biol Chem 288:16645-54
Ha, Ya; Akiyama, Yoshinori; Xue, Yi (2013) Structure and mechanism of rhomboid protease. J Biol Chem 288:15430-6
Xue, Yi; Ha, Ya (2012) Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate. J Biol Chem 287:3099-107
Xue, Yi; Chowdhury, Somenath; Liu, Xuying et al. (2012) Conformational change in rhomboid protease GlpG induced by inhibitor binding to its S' subsites. Biochemistry 51:3723-31
Hu, Jian; Xue, Yi; Lee, Sangwon et al. (2011) The crystal structure of GXGD membrane protease FlaK. Nature 475:528-31
Ha, Ya (2009) Structure and mechanism of intramembrane protease. Semin Cell Dev Biol 20:240-50
Wang, Yongcheng; Maegawa, Saki; Akiyama, Yoshinori et al. (2007) The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG. J Mol Biol 374:1104-13
Ha, Ya (2007) Structural principles of intramembrane proteases. Curr Opin Struct Biol 17:405-11