COMPUTER SIMULATION OF THE INFLUENZA M2 CHANNEL

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structural complexity of proteins that transport charge across cell walls in contemporary organisms makes it extremely difficult to dissect the molecular mechanisms of their action. It is therefore desirable to have a protein model which is small and has a well known structural motif, yet operates with the efficiency and control of more complex proteins. This has led to the study of the Influenza A M2 protein -- a small, homotetrameric, voltage-gated ion channel which self-assembles in lipid bilayers and transports protons with high efficiency and selectivity. Each monomer is built of 97 amino acids and contains a single transmembrane domain. Additionally, active channels have been reconstituted from a synthetic peptide containing only a subset of 25 amino acids, including the transmembrane region, with no loss in specificity or efficiency. The sequence of amino acids in the peptide is Ser-Ser-Asp-Pro-Leu- Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp Ile-Leu-Asp-Arg-Leu. Compared with grimicidin A, perhaps the most extensively studied model of a proton channel, the rate of proton transport across the truncated M2 channel is over 1000-fold faster. This remarkable combination of simplicity and efficiency makes M2 not only an excellent model for understanding how simple peptides can achieve high efficiency of proton transport but also an attractive, potential target for re-engineering a simple proton pump. While a high resolution NMR structure for a single helix is known, the structure of the tetrameric bundle has not been determined. In line with experimental and theoretical studies of proton transport in gramicidin, it has been suggested that proton transport occurs via translocation along a transient chain of water molecules that span the pore of the channel. The channel is gated by four histidine residues which occlude the lumen. This mechanism of gating can explain why M2 is impermeable to alkali ions. However, understanding the complete process of proton conductance through the channel requires additional studies. Cysteine scanning mutagenesis has shown that replacement of the pore-lining residues results in a large perturbation of the properties of the channel, indicating that these residues are essential for channel efficiency. The identities of other residues play a smaller role. How the pore-lining residues influence proton transport is not known, as none of these residues is highly polar or capable of forming particularly strong hydrogen bonds with the hydronium ion. The M2 channel is pH gated. At basic and neutral pH, it appears to be closed. Below a pH of 5.5 (which is also the pKa of histidine), the channel opens and proton transport is observed. It has therefore been argued that four neutral histidine residues from the gate, and that opening the channel involves protonating one (or more of the histidine residues). Recent NMR work by Cross and co-workers has suggested that the tryptophan residues are close to the histidine residues, and might also participate in channel gating. They constructed a model structure based on these results (PDB designation 1NYJ). Additionally, their newer NMR results indicate that the actual gate might consist of two His-His+ hydrogen bonding pairs. Based on this result, they have argued that at neultral pH, two of the histidine residues are protonated, and that the gate opens when three (or four) histidines become protonated at lower pH.