ABSTRACT
Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20° and 31° tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.
INTRODUCTION
Influenza is a worldwide epidemic that causes substantial morbidity and mortality. Of the three types of influenza viruses-A, B, and C-only Influenza A and B can cause epidemic diseases. Amantadine (1-adamantanamine hydrochloride) and its analog rimantadine (Fig. 1) are licensed drugs in the United States and Europe. Both drugs have been used in the prophylaxis and treatment of influenza A viral infections. Unlike zanamivir and oscltamivir, which are neuraminidase inhibitors, amantadine and rimantadine act on the M2 proton channel in the membrane of the influenza A virus. Amantadine is generally believed to block the M2 channel in a manner similar to the interaction of quaternary ammonium blockers with various ion channels (1), and consequently stunts the replication of the Influenza A viruses in host cells. During the past influenza season, 94% of Influenza A mutated to an amantadine resistant (S31N) form (2).
The M2 proton channels function as pH modulators at two stages in viral replication. Initially, viruses enter cells via endocytosis, i.e., the host cell membrane engulfs a virus and forms an endosome. In this acidic compartment (pH 5-6), the opening of the M2 channel imports protons into the viron, triggering a change in protein-protein and protein-membrane interactions that leads to the uncoating of the viral particle. In a late stage of infection, newly synthesized M2 proteins form channels in the trans Golgi network and balance the pH gradient across the membrane. In this case, the channel exports protons from the trans Golgi lumen to the cytoplasm. The inhibitory efficacy of amantadine is directly associated with the function of the M2 channel in that the presence of amantadine results in the failure of viral uncoating (the early stage) and the premature conformational change of hemagglutinin (the late stage).
The M2 protein (97 amino-acid residues) is an integral membrane protein with a single trans-membrane (TM) helix. The functional M2 channel is a homotetramer (3) stabilized in part by disulfide bonds linked between the N-terminal cysteines near the membrane interface. The M2 protein exhibits proton conductivity in a variety of artificial and natural membrane systems such as oocytes (3), mammalian cells (4), and even lipid bilayers (5). Consistently, the proton conductance is inhibited by a few µm amantadine or rimantadine, except in very low pH lipid bilayer preparations. Measurement of the proton current decay as a function of the amantadine concentration suggests that one drug molecule binds to one M2 tetramer with an apparent Kd of 0.3 µm (3).
The functional core of the channel is a TM domain (TMD) consisting of four α-helices. Evidence shows that the 25-residue M2-TMD polypeptides (S22SDP-LVVAASIIGILHLILWILDRL46) spontaneously form amantadine-sensitive proton channels once they are incorporated into lipid bilayers (6-9). The M2-TMD structure in lipid bilayers determined by solid-state NMR spectroscopy clearly displays an aqueous pore in the center that is most likely responsible for the proton conduction (10-14). In this structure, four helices tilt at ~38° with respect to the bilayer normal and form a lefthanded bundle with polar residues (e.g., His^sup 37^ and Trp^sup 41^) oriented toward the channel lumen (PDB code 1nyj). This structure is consistent with the cysteine scanning mutagenesis and electrophysiological studies of the M2 protein (15,16). The TM helices of the intact M2 protein, however, appear to orient in lipid bilayers with a somewhat smaller tilt angle of ~25° (17).
The first M2/amantadine model was proposed by Sugrue and Hay (18,19). It was based on an analogy of the distribution of the amantadine-resistant M2 mutations with that of the mutations in the nicotinic acetylcholine receptor. The key feature of this model emphasizes the interaction between the amantadine amino group and the Ser^sup 31^ hydroxyl group. This interaction was adopted later in the molecular modeling of the M2-TMD/amantadine complex (20). Recent structures of the M2-TMD provide more insight into amantadine binding, particularly that the pore volume is sufficient to accommodate an amantadine molecule. Taking advantage of these structures and analytical ultracentrifugation results for the M2-TMD mutants, Stouffer et al. (21) constructed a recent model that was very similar to that of Sugrue and Hay. Another model, proposed by Gandhi et al. (23), focuses on the possible H-bond interaction between the amantadine ammonium group and the nonprotonated nitrogen atoms on the His^sup 37^ side chains. In all of these models, the adamantyl group of amantadine is believed to reside closer to the external surface of the viral membrane, consistent with the map of the amantadine-resistant mutations. Additionally, Astrahan et al. (24) suggested a model based on the Nishimura model (11) and surface plasma resonance spectroscopy of amantadineinsensitive mutants to explore the resistance mechanism of the M2 mutants.
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