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

ABSTRACT: 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.

SUBMITTER: Ptacek J

PROVIDER: S-EPMC6194262 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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

Ptacek Jakub J Nedvedova Jana J Navratil Michal M Havlinova Barbora B Konvalinka Jan J Barinka Cyril C

Protein science : a publication of the Protein Society 20180901 9

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 Ca<sup>2+</sup> -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 Ca<sup>2+</sup> coordination sphere of GCPII and analyzed their structural and enzymatic properties usin ...[more]

PMID: 30168215

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Project description:Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics. Our data show that higher binding affinities of phosphoramidates, compared to matching phosphonates, are linked to the presence of additional hydrogen bonds between Glu424 and Gly518 of the enzyme and the amide group of the phosphoramidate. While the positioning of the P1' glutamate-derived module within the S1' pocket of GCPII is invariant, interaction interfaces between effector functions and residues lining the entrance funnel are highly varied, with the positively charged arginine patch defined by Arg463, Arg534 and Arg536 being the only 'hot-spot' common to several studied complexes. This variability stems in part from the fact that the effector/GCPII interfaces generally encompass isolated areas of nonpolar residues within the entrance funnel and resulting van der Waals contacts lack the directionality typical for hydrogen bonding interactions. The presented data unravel a complexity of binding modes of inhibitors within non-prime site(s) of GCPII and can be exploited for the design of novel GCPII-specific compounds.Atomic coordinates of the present structures together with the experimental structure factor amplitudes were deposited at the RCSB Protein Data Bank under accession codes 4P44 (complex with JRB-4-81), 4P45 (complex with JRB-4-73), 4P4B (complex with CTT54), 4P4D (complex with MP1C), 4P4E (complex with MP1D), 4P4F (complex with NC-2-40), 4P4I (complex with T33) and 4P4J (complex with T33D).

Dataset Information

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

Publications

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

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