title: Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.
abstract: Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.
authors.affiliation: Institute for Protein Research, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Matsuda
authors.forename: Kohei
authors.initials: K
authors.lastname: Takeshita
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Kurokawa
authors.forename: Souhei
authors.initials: S
authors.lastname: Sakata
authors.forename: Mamoru
authors.initials: M
authors.lastname: Suzuki
authors.forename: Eiki
authors.initials: E
authors.lastname: Yamashita
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okamura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakagawa
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002938:Ciona intestinalis / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D051059:PTEN Phosphohydrolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D051059:PTEN Phosphohydrolase