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The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity
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SYSNO ASEP 0495871 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity Author(s) Ptáček, Jakub (BTO-N) RID
Nedvědová, Jana (BTO-N)
Navrátil, Michal (UOCHB-X) RID
Havlínová, Barbora (BTO-N)
Konvalinka, Jan (UOCHB-X) RID, ORCID
Bařinka, Cyril (BTO-N) RID, ORCIDNumber of authors 6 Source Title Protein Science. - : Wiley - ISSN 0961-8368
Roč. 27, č. 9 (2018), s. 1575-1584Number of pages 10 s. Language eng - English Country US - United States Keywords prostate-specific membrane antigen ; NAALADase ; folate hydrolase Subject RIV EB - Genetics ; Molecular Biology OECD category Biochemistry and molecular biology Subject RIV - cooperation Institute of Organic Chemistry and Biochemistry - Genetics ; Molecular Biology R&D Projects GBP208/12/G016 GA ČR - Czech Science Foundation (CSF) ED2.1.00/19.0390 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) EF16_013/0001776 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) GA18-14167S GA ČR - Czech Science Foundation (CSF) Method of publishing Open access Institutional support BTO-N - RVO:86652036 ; UOCHB-X - RVO:61388963 UT WOS 000447779000004 EID SCOPUS 85055040824 DOI 10.1002/pro.3460 Annotation 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. Workplace Institute of Biotechnology Contact Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Year of Publishing 2021 Electronic address https://onlinelibrary.wiley.com/doi/full/10.1002/pro.3460
Number of the records: 1