title: Calcium-dependent dimerization of human soluble calcium activated nucleotidase: characterization of the dimer interface.
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.
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati College of Medicine, Ohio 45267-0575, USA.
authors.forename: Mingyan
authors.initials: M
authors.lastname: Yang
authors.forename: Katsunori
authors.initials: K
authors.lastname: Horii
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Herr
authors.forename: Terence L
authors.initials: TL
authors.lastname: Kirley
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D003460:Crystallization; D019281:Dimerization; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D009708:Nucleotidases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009708:Nucleotidases; C030287:nucleotidase; D002118:Calcium