Fig 7. Ammonium accumulation and possible proton pathway.

(A) MD simulations showing ammonium accumulation (blue mesh) at aspartate residues (yellow sticks) at vacuolar (top) and cytosolic (bottom) side of AtTIP2;1. Water density (purple mesh) outlines the vertical main pore of the monomer and confirms existence of a water-filled side pore beneath loop C. Residues of the extended selectivity filter are depicted as sticks (H2^P-His 63 (blue), LC^P-His 131 (red), LE^P-Gly 194 (green), and HE^P-Arg 200 (brown)). (B) and (C) MD simulations demonstrating flexibility of His 131 at position LC^P being neutral (B) and positively charged (C). Color code as in (A). (D) Surface representation of the crystal structure depicting the water-filled side pore beneath loop C. Hydrogen bonds of water 10 (Wat10) as well as between Arg 200 at position HE^P in helix E and His 63 at position H2^P in helix 2 are indicated by dashed orange lines. (E) Tentative working model of ammonia-permeating AtTIP2;1. Ammonium may contribute to ammonia permeation by accumulating on the vacuolar protein surface and by possibly having its protons shuttled back into the acidic vacuole by His 131 (red) at position LC^P in loop C via a water-filled side pore.