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The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

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.

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.

The voltage-gated proton channel, Hv1 (VSOP) has a voltage-sensor domain (VSD) but lacks an authentic pore domain, and the VSD of Hv1 plays dual roles of voltage sensing and proton permeation. Hv1 is required for high-level superoxide production by phagocytes through its tight functional coupling with NADPH oxidase to eliminate pathogens. Hv1 is also expressed in human sperm and has been suggested to regulate motility through activating pH-sensitive calcium channels. The activities of Hv1 also have pathological implications, such as exacerbation of ischemic brain damage and progression of cancer. In this study, our crystal structure of mouse Hv1 (mHv1) showed a "closed umbrella" shape with a long helix consisting of the cytoplasmic coiled-coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. We also found a Zn2+ion at the extracellular region of mHv1 protomer. The binding of Zn2+strongly suggested that the crystal structure of mHv1 represents the resting state, since Zn2+specifically inhibits activities of voltage-gated proton channels. Actually, two out of three arginines as sensor residues (R204 and R207) were located lower than the conserved phenylalanine, F146, on the S2 in a charge transfer center. This makes contrast with previous structures of other VSDs in the activated state where many positive residues of S4 were located upper than the conserved phenylalanine. Additionally, the crystal structure of mHv1 highlighted two hydrophobic barriers. Aspartic acid (D108), which is critical for proton selective permeation, was located facing intracellular vestibule below the inner hydrophobic barrier, thereby being accessible to water from the cytoplasm. Another hydrophobic layer of extracellular side probably ensures interruption of the proton pathway of mHv1 in resting state. These findings provide a novel platform for understanding the general principles of voltage sensing and proton permeation.

Background: Dnmt1 faithfully propagates DNA methylation patterns to the next generation. Results: The DNA methylation activity of Dnmt1 was stimulated by the direct interaction of the SRA domain of Uhrf1 and Dnmt1. Conclusion:The SRA facilitates DNA accession to the catalytic center. Significance: The RFTS and SRA interaction contributes to the correct feeding of the hemi-methylated DNA to the catalytic center of Dnmt1.

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, wildtype (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.The voltage-sensing phosphatase (VSP) 3 was discovered through the survey of the genome of the ascidian, Ciona intestinalis, as a hybrid protein that has a voltage sensor domain (VSD) consisting of four ␣-helices (S1-S4) for voltage sensing and a cytoplasmic region encoding a phosphatidylinositol phosphatase domain (1). The VSP gene is conserved from sea urchins to humans where it is expressed in the testis (2). In the recent report, C. intestinalis VSP (Ci-VSP) is expressed in the cells of the stomach, intestine, and blood of juveniles detected by whole mount in situ hybridization (3). The VSD of VSP bears homology to the VSD of voltage-gated ion channels and a recently identified voltage-gated proton channel protein that contains only the VSD without pore domain, VSOP or Hv1 (4, 5). The Ci-VSP cytoplasmic region, which consists of a phosphatase domain (PD) and a C2 domain, shares high sequence similarity with the phosphatase and tensin homolog (PTEN) (1, 6). The amino acid sequence of the Ci-VSP cytoplasmic region has similarity to that of PTEN with 36% identity. A unique feature of both invertebrate and vertebrate VSPs is that depolarization induces phosphoinositide phosphatase activity through coupling of VSD to the phosphatase region (7-9). The enzymatic activity of Ci-VSP increases in the range of membrane potentials, from Ϫ80 to 100 mV, correlating with the extent of voltage sensor movement (7-8). Ci-VSP most likely operates as a monomer as shown by a study of single molecule imaging in heterologous expression system (10). The linker region between the VSD and ...

Polymeric micelles of zinc protoporphyrin (ZnPP) with water soluble biocompatible and amphiphilic polymer, polyethylene glycol (PEG) demonstrated unique characteristics to target tumor tissues selectively based on the enhanced permeability and retention (EPR) effect. The micellar macromolecular drug of ZnPP (SMA-ZnPP and PEG-ZnPP) previously showed notable anticancer activity as a consequence of selective tumor targeting ability and its potent HO-1 inhibitory potential, resulting in suppressed biliverdin/bilirubin production in tumors thereby leading to oxystress induced tumor cell killing. Furthermore, recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light, laser, or xenon light source, which could augment its oxystress induced cell killing abilities. In the present paper, we report the synergistic effects of light induced photosensitizing capabilities and HO-1 inhibitory potentials of these unique micelles when tested in vitro and in vivo on tumor models under localized, mild illumination conditions using a tungsten-xenon light source. The results indicate that these water soluble polymeric micelles of ZnPP portend to be promising candidates for targeted chemotherapy as well as photodynamic therapy against superficial tumors as well as solid tumors located at light penetrable depths.