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. 2017 Dec 19;114(51):13357-13362.
doi: 10.1073/pnas.1705624114. Epub 2017 Aug 23.

XFEL Structures of the Influenza M2 Proton Channel: Room Temperature Water Networks and Insights Into Proton Conduction

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Free PMC article

XFEL Structures of the Influenza M2 Proton Channel: Room Temperature Water Networks and Insights Into Proton Conduction

Jessica L Thomaston et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

Keywords: XFEL; influenza; membrane protein; proton channel.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
(Top) The gating His37 tetrad of the M2 channel can have a charge state ranging from neutral to +4. Shown here is the transition from the 2+ to the 3+ charge state, which is hypothesized to occur during the proton transport cycle. (Bottom) The charge states of the His tetrad are in equilibrium, and the Inwardopen conformation is in equilibrium with the Inwardclosed conformation.
Fig. 1.
Fig. 1.
Low-pH (pH 5.5) structures of M2TM under cryogenic synchrotron (4QKC, Left), room temperature XFEL (5JOO, Center), and room temperature (r.t.) synchrotron (4QKM, Right) diffraction conditions. (Top) The front helix of each tetramer has been removed; waters are modeled as spheres, with red spheres representing full-occupancy waters and light and dark blue spheres as half-occupancy waters in alternate-occupancy networks A and B. Waters within hydrogen-bonding distance of each other are connected by sticks. The number of ordered waters decreases moving from Left to Right across the figure. (Bottom) Electron density for the pore solvent network (blue mesh) is shown to a contour of 0.5 σ. The largest amount of ordered density is present in the cryogenic synchrotron data collection condition. The volume and shape of the solvent density for the room temperature structures collected using XFEL and synchrotron sources are similar.
Fig. 2.
Fig. 2.
High-pH (pH 8.0) structures of M2TM under cryogenic synchrotron (4QK7, Left), room temperature (r.t.) XFEL (5TTC, Center), and room temperature synchrotron (4QKL, Right) diffraction conditions. Waters are shown as spheres (red, full occupancy; light and dark blue, half-occupancy); potential hydrogen bonds are shown as sticks. (Top) The largest number of ordered waters is found in the cryogenic synchrotron diffraction condition; fewer ordered waters are present at room temperature. (Bottom) Electron density for the pore solvent network (blue mesh) is shown to a contour of 0.5 σ. The largest amount of ordered solvent electron density is observed under cryogenic diffraction conditions; the volume and shape of the solvent density for the two room temperature conditions are similar.
Fig. 3.
Fig. 3.
Alternate-occupancy water networks in the low-pH (pH 5.5) room temperature XFEL structure (5JOO). Full-occupancy waters are red, waters from alternate-occupancy network A are cyan, and waters from alternate-occupancy network B are dark blue. Waters within hydrogen-bonding distance of each other are connected by sticks; yellow sticks indicate hydrogen bonds that can be made between the solvent network and protein carbonyls. (Left) Side view of solvent from alternate-occupancy network A (Top) and B (Bottom), with top-down views of the three layers of water in the pore. (Right) Top-down view of all three layers of water in alternate-occupancy network A (Top) and B (Bottom).
Fig. S2.
Fig. S2.
Possible water networks 1 and 2 form a continuous chain of hydrogen bonds from the top of the water network to the gating His37 residues.
Fig. S3.
Fig. S3.
Possible water networks 3–11 form hydrogen bonds that do not continuously link the top of the water network to the gating His37 residues.
Fig. S4.
Fig. S4.
Solvent layer above His37 in the cryogenic structure of the Inwardclosed conformation at pH 6.5 [3LBW (36), Left] and the RT XFEL structure at pH 6.5. The distance between the His37 residues increases from 4.5 to 8.0 Å, and the water network expands.
Fig. 4.
Fig. 4.
Room temperature XFEL structures of M2TM under all pH conditions: low (pH 5.5, 5JOO, red), intermediate (pH 6.5, 5UM1, green), and high (pH 8.0, 5TTC, blue). Waters are shown as spheres (red, full occupancy; light and dark blue, half-occupancy); potential hydrogen bonds are shown as sticks. (Top) The most ordered waters are observed under the low-pH condition, with fewer waters present at intermediate pH and the smallest number of ordered waters at high pH. Moving from low to high pH, the number of half-occupancy waters decreases and the hydrogen-bonding network becomes less complex. (Bottom) Electron density for the pore solvent network (blue mesh) is shown to a contour of 0.5 σ. The same trend is observed from the electron density maps; the largest volume of solvent density is at low pH, and the smallest volume is at high pH.
Fig. S5.
Fig. S5.
Distance of waters from His37 residues in XFEL structures at high (8.0), intermediate (6.5), and low (5.5) pH, measured in angstroms. (Top) Side view of one monomer; only the waters in the low-pH XFEL structure are close enough to form hydrogen bonds with His37. (Bottom) The water hydrogen bonding to the epsilon nitrogen of His37 is positioned close enough to the Trp41 of an adjacent monomer to form a cation–pi interaction.
Fig. S6.
Fig. S6.
(Left) Visible light image of square M2TM (–46) microcrystals at a concentration of over 1 × 109 per milliliter. (Right) Visible light image of glass syringes full of pooled M2 microcrystals.

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