Structural and dynamic mechanisms for the function and inhibition of the M2 proton channel from influenza A virus

Abstract

The M2 proton channel from influenza A virus, a prototype for a class of viral ion channels known as viroporins, conducts protons along a chain of water molecules and ionizable sidechains, including His37. Recent studies highlight a delicate interplay between protein folding, proton binding, and proton conduction through the channel. Drugs inhibit proton conduction by binding to an aqueous cavity adjacent to M2's proton-selective filter, thereby blocking access of proton to the filter, and altering the energetic landscape of the channel and the energetics of proton-binding to His37.

Figure 1

Comparison of (a) crystal (PDB: 3LBW), (b) solution NMR (PDB: 2RLF), and (c) solid state NMR (PDB: 2L0J) structures. Backbone is colored in grey, while residues 49–60 are colored in yellow. Critical residues His37 (in cyan) and Trp41 (in green) are key to the extensive hydrogen bond network in the crystal structure. Water clusters are shown in pink.

Figure 2

Motions of pore forming helices of KcsA and M2 shown by overlaying KcsA and M2 structures from different sources. a) Ten structures for KcsA (residues 86–122) (PDB codes: 1R3J, 2HVK, 3EFF, 3F5W, 3F7V, 3F7Y, 3FB5, 3FB6, 3FB7, 3FB8). b) Eight structures were analyzed for M2 (residues 25–46) (PDB codes: 2RLF, 3C9J, 2KQT, 2L0J, 3LBW, and three symmetric structures derived from three different helices of the asymmetric structure 3BKD). Figures are adapted from [130].

Figure 3

Hydrogen bonding network in the high resolution M2 crystal structure (PDB: 3LBW). Crystal waters are shown in red spheres and named entry cluster, bridging dimer and bridging cluster from Gly/Ala 34 to Asp 44. The histidine residues in the His-box stacks in an edge-to-face conformation and hydrogen bonds to crystallographically well ordered waters in both the entry cluster and bridging dimer. Trp 41 rings are stabilized in a basket-like structure via a third cluster of water molecules-bridging cluster, which bridge the indole NH of Trp41 with the carboxylate of Asp44. This structure is in the + 2 protonation state.

Figure 4

Docked conformation of four M2 inhibitors with M2 model generated from the high resolution X-ray crystal structure of M2 (PDB: 3LBW) and SSNMR structure of amantadine bound M2 (PDB: 2KQT). Poses are shown with the amine pointing downwards towards His 37. a) Amantadine b) Rimantadine c) Spiro-piperidine d) Spiran amine. e) The structure of tetrabutylammonium (TBA) bound to KcsA (PDB: 2HVK). Note TBA displaces the K⁺ hydrate near the entry of the selectivity filter, in a binding manner highly similar to the binding of M2 inhibitors (a–d) to M2, which lie proximal to or displace the top two waters in the entry cluster.