X-ray crystal structure of voltage-gated proton channel

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.

ASAXS measurements with soft (tender) X-rays to control the contrast

The structure and properties of thin soft matter materials might be better examined in detail by scattering measurements when the contrast between the materials and penetration depth are tunable. In the so-called tender X-ray region, there are several elements of interest for small-angle scattering measurements, such as Mg, Al, Si, P, and S. We worked on GISAXS measurements at K absorption edges of Si and P to control contrast and penetration depth for thin block copolymer (SEBS) samples and phospholipid alloy systems comprising of phospholipid (PC, DPPC), cholesterol, and ganglioside, and SAXS at Al for Al alloys. The photon energy used in the present measurements is soft enough to give large anomalous effect in the real part of refractive indices for light elements, yet hard enough so that the scattering vector required for nanostructure analysis can be covered. By choosing the photon energy at the K absorption edge of Si, the contrast between Si substrate and polymer thin film can be suppressed, thereby suppressing dynamical effect in GISAXS intensity caused by reflected beam[1]. Combined with the depth sensitive analysis, this is useful to examine the nanostructures that are changing with the distance from the surface. Concerning the contrast change inside the thin films, change of the GISAXS intensities from PC (phosphatidylcholine)-cholestrol-ganglioside (GD1) films spin-cast on Si substrates have been examined at the K absortpion edge of P [2]. ASAXS for P has been examined by Stuhrmann and coworkers[3], but the number of works after them is not large. Present measurements using image plates showed that the hexagonal phase appearing in dried phospholipid alloy films suggests a contrast reversal with the photon energy.