2H2N: Crystal Structure Of Human Soluble Calcium-activated Nucleotidase (scan) With Calcium Ion

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.
PDB ID: 2H2NDownload
MMDB ID: 40506
PDB Deposition Date: 2006/5/19
Updated in MMDB: 2006/11
Experimental Method:
x-ray diffraction
Resolution: 2.3  Å
Source Organism:
Similar Structures:
Biological Unit for 2H2N: dimeric; determined by author
Molecular Components in 2H2N
Label Count Molecule
Proteins (2 molecules)
Soluble Calcium-activated Nucleotidase 1(Gene symbol: CANT1)
Molecule annotation
Chemicals (3 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB