skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Characterization and Importance of the Dimer Interface of Human Calcium-Activated Nucleotidase

Abstract

Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical asmore » the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
980052
Report Number(s):
BNL-92970-2010-JA
TRN: US201015%%1437
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 47; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AMINO ACIDS; ASPARTIC ACID; BIOLOGICAL FUNCTIONS; CRYSTAL STRUCTURE; DIMERS; ENDOPLASMIC RETICULUM; GLUTAMIC ACID; HUMAN POPULATIONS; INTERFACES; NUCLEOTIDASES; national synchrotron light source

Citation Formats

Yang, M, Horii, K, Herr, A, and Kirley, T. Characterization and Importance of the Dimer Interface of Human Calcium-Activated Nucleotidase. United States: N. p., 2008. Web. doi:10.1021/bi701578m.
Yang, M, Horii, K, Herr, A, & Kirley, T. Characterization and Importance of the Dimer Interface of Human Calcium-Activated Nucleotidase. United States. doi:10.1021/bi701578m.
Yang, M, Horii, K, Herr, A, and Kirley, T. Tue . "Characterization and Importance of the Dimer Interface of Human Calcium-Activated Nucleotidase". United States. doi:10.1021/bi701578m.
@article{osti_980052,
title = {Characterization and Importance of the Dimer Interface of Human Calcium-Activated Nucleotidase},
author = {Yang, M and Horii, K and Herr, A and Kirley, T},
abstractNote = {Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical as the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.},
doi = {10.1021/bi701578m},
journal = {Biochemistry},
number = 2,
volume = 47,
place = {United States},
year = {2008},
month = {1}
}