Structural investigation of rimantadine inhibition of the AM2-BM2 chimera channel of influenza viruses

Abstract

The M2 channel of influenza A is a target of the adamantane family antiviral drugs. Two different drug-binding sites have been reported: one inside the pore, and the other is a lipid-facing pocket. A previous study showed that a chimera of M2 variants from influenza A and B that contains only the pore-binding site is sensitive to amantadine inhibition, suggesting that the primary site of inhibition is inside the pore. To obtain atomic details of channel-drug interaction, we determined the structures of the chimeric channel with and without rimantadine. Inside the channel and near the N-terminal end, methyl groups of Val27 and Ala30 from four subunits form a hydrophobic pocket around the adamantane, and the drug amino group appears to be in polar contact with the backbone oxygen of Ala30. The structures also reveal differences between the drug-bound and -unbound states of the channel that can explain drug resistance.

Figure 1. Functional investigation of the (AM2-BM2)_TM channel by the liposomal proton flux assay

(A) The conductance of the (AM2-BM2)_TM channel exhibits similar conductance rate (~10 H⁺/s/channel) as the wildtype AM2 channel (Pielak and Chou, 2010a). (B) The conductance of (AM2-BM2)_TM was reduced by ~95% by the addition of 50 μM rimantadine. (C) Introducing the S31N mutation to (AM2-BM2)_TM did not affect its proton conduction rate. (D) The S31N mutation however completely abolished inhibition by rimantadine.

Figure 2. Functional investigation of the (AM2-BM2)_TM channel by NMR

The ¹H-¹⁵N TROSY-HSQC spectra of the (AM2-BM2)_TM (1.4 mM monomer) reconstituted in DHPC are as follows: (A) at pH 7.5; (B) at pH 7.5 with 50 mM rimantadine; (C) with the S31N mutation at pH 7.5; (D) same as in (C) but with 50 mM rimantadine; (E) at pH 6.1; (F) at pH 6.1 with 100 mM rimantadine. The red peaks correspond to resonances that have emerged upon addition of rimantadine. Please also see Fig. S1–S4.

Figure 3. Selected regions of NOESY spectra for identifying protein-drug NOEs

Left panel: strips from the 3D ¹⁵N-edited NOESY-tr-HSQC spectrum recorded using the ¹⁵N-, ²H-labeled chimera in the presence of 50 mM rimantadine. Right panel: strips from the 3D ¹³C-edited NOESY-HSQC spectrum recorded using the ¹⁵N-, ¹³C-labled chimera in the presence of 50 mM rimantadine. The resonances of the drug-bound state are labeled in red.

Figure 4. Solution structures of the (AM2-BM2)_TM channel in the absence and presence of rimantadine

(A) Ensembles of 15 low-energy structures of the drug-free (left) and drug-bound (right) chimera channels, determined at pH 7.5. Rimantadine is highlighted in red. (B) Ribbon representation of the drug-free (AM2-BM2)_TM tetramer (left), and overlay of its AM2 and BM2 regions (green) with the corresponding regions (yellow) of the AM2 (PDB code: 2RLF) and BM2 (PDB code: 2KIX) structures (right). The backbone r.m.s. deviation for the AM2 and BM2 regions are 1.3 and 2.2 Å, respectively. (C) Overlay of the drug-free (white) and the drug-bound (cyan) chimera structures, showing substantial differences in helical packing.

Figure 5. Structural details of rimantadine binding inside the chimera channel

(A) Hydrophobic and polar interactions between rimantadine and protein. The eight methyl groups (four C^γ1H₃ from Val27 and four C^βH₃ from Ala30) that are in VDW contacts with the adamantane cage of rimantadine are shown as green balls. (B) Surface representation of the channel showing the internal hydrophobic pocket that wraps around the adamantane cage of rimantadine. One subunit of the tetramer was removed to unveil the channel interior.

Figure 6. Comparing the solution and crystal structures of drug binding

Combined surface, ribbon, and sphere representation of the pore binding sites described by (A) the solution structure of rimantadine binding to the chimeric channel (PDB code: 2LJC) and (B) the crystal structure of amantadine binding to the TM domain of AM2 channel (PDB code: 3C9J). One subunit of the tetramer was removed to unveil the channel interior. Sidechains of important residues such as Val27, Ala30, and Ser31 are shown as spheres.