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Calcium-dependent dimerization of human soluble calcium activated nucleotidase: characterization of the dimer interface.

Yang M, Horii K, Herr AB, Kirley TL
J Biol Chem 281 28307-17 (2006)
Cited: 8 times
EuropePMC logo PMID: 16835225

Abstract

Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile(170), Ser(172), and Ser(226) with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys(30), located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

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Articles citing this publication (7)

  1. The first crystal structure of gluconolactonase important in the glucose secondary metabolic pathways. Chen CN, Chin KH, Wang AH, Chou SH. J Mol Biol 384 604-614 (2008)
  2. Revelation of a catalytic calcium-binding site elucidates unusual metal dependence of a human apyrase. Rooklin DW, Lu M, Zhang Y. J Am Chem Soc 134 15595-15603 (2012)
  3. A modulator domain controlling thermal stability in the Group II chaperonins of Archaea. Luo H, Robb FT. Arch Biochem Biophys 512 111-118 (2011)
  4. Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo. Yang M, Kirley TL. Thromb Res 122 541-548 (2008)
  5. Characterization and importance of the dimer interface of human calcium-activated nucleotidase. Yang M, Horii K, Herr AB, Kirley TL. Biochemistry 47 771-778 (2008)
  6. Cloning, expression and enzyme activity delineation of two novel CANT1 mutations: the disappearance of dimerization may indicate the change of protein conformation and even function. Wang HD, Guo LJ, Feng ZQ, Zhang DW, Zhang MT, Gao Y, Chen CL, Zhu BF. Orphanet J Rare Dis 15 240 (2020)
  7. Conformational requirement on peptides to exert laminin's activities and search for protein segments with laminin's activities. Umezawa K, Ikebe J, Nomizu M, Nakamura H, Higo J. Biopolymers 92 124-131 (2009)


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