Structural studies of Arabidopsis Chloroplast Glutamyl Endopeptidase (CGEP)

Abstract: Peptidases play an essential role in cell machinery by regulating protein turnover and processing synthesized proteins for cellular transport. The chloroplast is a complex organelle present in plants that host several peptidases. The maintenance of the chloroplast proteome is regulated through proteolysis by peptidases to ensure optimum metabolic and photosynthetic activity. Chloroplast Glutamyl Endopeptidase (CGEP) (~97 kDa; AT2G47390) is one such peptidase recently purified from the chloroplast stroma of Arabidopsis thaliana by the Van Wijk group at Cornell University. CGEP was assigned to the S9D sub-family of Ser peptidase and exhibits both endo- and exopeptidase activity. Interestingly, in-vitro cleavage assays revealed that CGEP also shows autocatalytic processing of the C-terminal tail, which could possibly regulate substrate access to the enzyme’s catalytic pocket. Crystal structures of the S9 family (S9A, S9B, and S9C) bacterial peptidases have been solved and deposited in the PDB, but so far, no structures exist for plant glutamyl peptidases in the S9 family or S9D members. This thesis provides the groundwork for obtaining an X-ray crystal structure of Arabidopsis CGEP that would help to understand the complex CGEP catalytic mechanism. Characterization of Arabidopsis CGEP and a mutant variant (“CGEP-C2”) revealed that it forms a myriad of oligomeric states (hexamer, tetramer, trimer, and dimer). The CGEP oligomeric state may be in equilibrium and be protein concentration-dependent. While homology modeling of CGEP relied on threading templates with low sequence identity, the resulting models had convincing confidence scores. Analyses of the position of the autocatalytically cleaved GCEP suggested that these homology models had an unlikely domain architecture for CGEP to allow substrate access to the enzyme’s active site without substantial structural re-arrangement. This further emphasizes the need for a crystal structure that would ultimately reveal important structural features for CGEP’s function.