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ABSTRACT: Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H+ channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the voltage-gated H+ channel forms a dimeric coiled-coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structure of the coiled-coil domain in reduced, oxidized and mutated (Cys→Ser) states. The crystal structures indicated that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. Circular dichroism spectroscopy revealed that the disulfide bond increased the thermal stability of the coiled-coil protein. We also revealed that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the voltage-gated H+ channel depend on the redox condition, which may play a role in redox sensing in the phagosome.
Journal of Biological Chemistry 05/2013; · 4.77 Impact Factor
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ABSTRACT: The voltage-gated H(+) channel functions as a dimer, a configuration that is different from standard tetrameric voltage-gated channels. Each channel protomer has its own permeation pathway. The C-terminal coiled-coil domain has been shown to be necessary both for dimerization and cooperative gating in the two channel protomers. Here we report the gating cooperativity in trimeric and tetrameric Hv channels engineered by altering the hydrophobic core sequence of the coiled-coil assembly domain. Trimeric and tetrameric channels exhibited more rapid and less sigmoidal kinetics of activation of H(+) permeation than dimeric channels, suggesting that some channel protomers in trimer and tetramer failed to produce gating cooperativity observed in WT dimer. Mulitimerization of trimer and tetramer channels were confirmed by protein-biochemical analyses including crystallography. These findings indicate that the voltage-gated H(+) channel is optimally designed as a dimeric channel, on a solid foundation of the sequence pattern of the coiled-coil core, with efficient cooperative-gating that ensures sustained and steep voltage-dependent H(+) conductance in blood cells.
The Journal of Physiology 11/2012; · 4.72 Impact Factor
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ABSTRACT: Through their ubiquitin ligase activity, Cbl-family proteins suppress signaling mediated by protein-tyrosine kinases (PTKs), but can also function as adaptor proteins to positively regulate signaling. The tyrosine kinase binding (TKB) domain of this family is critical for binding with tyrosine-phosphorylated target proteins. Here, we analyzed the crystal structure of the TKB domain of Cbl-c/Cbl-3 (Cbl-c TKB), which is a distinct member of the mammalian Cbl-family. In comparison to Cbl TKB, Cbl-c TKB showed restricted structural flexibility upon phosphopeptide binding. A mutation in Cbl-c TKB augmenting this flexibility enhanced its binding to target phosphoproteins. These results suggest that proteins, posttranslational modifications, or mutations that alter structural flexibility of the TKB domain of Cbl-family proteins could regulate their binding to target phosphoproteins, and thereby affect PTK-mediated signaling.
Journal of biochemistry 08/2012; · 1.95 Impact Factor
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ABSTRACT: Hv1/VSOP is a dimeric voltage-gated H(+) channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the S4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H(+) current through Hv1/VSOP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between S4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. Our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with S4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VSOP operates.
Nature Communications 01/2012; 3:816. · 7.40 Impact Factor
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ABSTRACT: Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.
Proceedings of the National Academy of Sciences 05/2011; 108(22):9055-9. · 9.68 Impact Factor
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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.
Journal of Biological Chemistry 05/2011; 286(26):23368-77. · 4.77 Impact Factor
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ABSTRACT: Tapes japonica lysozyme (TJL) is classified as a member of the recently established i-type lysozyme family. In this study, we solved the crystal structure of TJL complexed with a trimer of N-acetylglucosamine to 1.6A resolution. Based on structure and mutation analyses, we demonstrated that Glu-18 and Asp-30 are the catalytic residues of TJL. Furthermore, the present findings suggest that the catalytic mechanism of TJL is a retaining mechanism that proceeds through a covalent sugar-enzyme intermediate. On the other hand, the quaternary structure in the crystal revealed a dimer formed by the electrostatic interactions of catalytic residues (Glu-18 and Asp-30) in one molecule with the positive residues at the C terminus in helix 6 of the other molecule. Gel chromatography analysis revealed that the TJL dimer remained intact under low salt conditions but that it dissociated to TJL monomers under high salt conditions. With increasing salt concentrations, the chitinase activity of TJL dramatically increased. Therefore, this study provides novel evidence that the lysozyme activity of TJL is modulated by its quaternary structure.
Journal of Biological Chemistry 10/2007; 282(37):27459-67. · 4.77 Impact Factor
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ABSTRACT: Tapes japonica lysozyme (TJL) is classified as a member of the recently established i-type lysozyme family. In this study, we solved the
crystal structure of TJL complexed with a trimer of N-acetylglucosamine to 1.6Å resolution. Based on structure and mutation analyses, we demonstrated that Glu-18 and Asp-30 are
the catalytic residues of TJL. Furthermore, the present findings suggest that the catalytic mechanism of TJL is a retaining
mechanism that proceeds through a covalent sugar-enzyme intermediate. On the other hand, the quaternary structure in the crystal
revealed a dimer formed by the electrostatic interactions of catalytic residues (Glu-18 and Asp-30) in one molecule with the
positive residues at the C terminus in helix 6 of the other molecule. Gel chromatography analysis revealed that the TJL dimer
remained intact under low salt conditions but that it dissociated to TJL monomers under high salt conditions. With increasing
salt concentrations, the chitinase activity of TJL dramatically increased. Therefore, this study provides novel evidence that
the lysozyme activity of TJL is modulated by its quaternary structure.
Journal of Biological Chemistry 09/2007; 282(37):27459-27467. · 4.77 Impact Factor
