2h2u
From Proteopedia
(New page: 200px<br /> <applet load="2h2u" size="450" color="white" frame="true" align="right" spinBox="true" caption="2h2u, resolution 2.400Å" /> '''Crystal structure ...) |
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| - | [[Image:2h2u.gif|left|200px]]<br /> | + | [[Image:2h2u.gif|left|200px]]<br /><applet load="2h2u" size="350" color="white" frame="true" align="right" spinBox="true" |
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caption="2h2u, resolution 2.400Å" /> | caption="2h2u, resolution 2.400Å" /> | ||
'''Crystal structure of the E130Y mutant of human soluble calcium-activated nucleotidase (SCAN) with calcium ion'''<br /> | '''Crystal structure of the E130Y mutant of human soluble calcium-activated nucleotidase (SCAN) with calcium ion'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Mammals express a protein homologous to soluble nucleotidases used by | + | 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. |
==About this Structure== | ==About this Structure== | ||
| - | 2H2U is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Nucleoside-diphosphatase Nucleoside-diphosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.1.6 3.6.1.6] Full crystallographic information is available from [http:// | + | 2H2U is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Nucleoside-diphosphatase Nucleoside-diphosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.1.6 3.6.1.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2H2U OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Nucleoside-diphosphatase]] | [[Category: Nucleoside-diphosphatase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Herr, A | + | [[Category: Herr, A B.]] |
[[Category: Horii, K.]] | [[Category: Horii, K.]] | ||
| - | [[Category: Kirley, T | + | [[Category: Kirley, T L.]] |
[[Category: Yang, M.]] | [[Category: Yang, M.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
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[[Category: nucleotide-binding]] | [[Category: nucleotide-binding]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:37:37 2008'' |
Revision as of 15:37, 21 February 2008
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Crystal structure of the E130Y mutant of human soluble calcium-activated nucleotidase (SCAN) with calcium ion
Overview
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.
About this Structure
2H2U is a Single protein structure of sequence from Homo sapiens with as ligand. Active as Nucleoside-diphosphatase, with EC number 3.6.1.6 Full crystallographic information is available from OCA.
Reference
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. 2006 Sep 22;281(38):28307-17. Epub 2006 Jul 11. PMID:16835225
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