The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity

0495871 - BTÚ 2021 RIV US eng J - Journal Article
Ptáček, Jakub - Nedvědová, Jana - Navrátil, Michal - Havlínová, Barbora - Konvalinka, Jan - Bařinka, Cyril
The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity.
Protein Science. Roč. 27, č. 9 (2018), s. 1575-1584. ISSN 0961-8368. E-ISSN 1469-896X
R&D Projects: GA ČR(CZ) GBP208/12/G016; GA MŠMT(CZ) ED2.1.00/19.0390; GA MŠMT(CZ) EF16_013/0001776; GA ČR(CZ) GA18-14167S
Institutional support: RVO:86652036 ; RVO:61388963
Keywords : prostate-specific membrane antigen * NAALADase * folate hydrolase
OECD category: Biochemistry and molecular biology; Biochemistry and molecular biology (UOCHB-X)
Impact factor: 2.420, year: 2018
Method of publishing: Open access
https://onlinelibrary.wiley.com/doi/full/10.1002/pro.3460

Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+-binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca2+-binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca2+-binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca2+ for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.
Permanent Link: http://hdl.handle.net/11104/0288755