Structural analysis of the transmembrane domain of the M(2) protein of influenza A virus

The M₂ protein of influenza A virus is a proton-selective ion channel whose activity is essential for efficient viral replication. The active form of the channel is a homotetramer. Each subunit possesses a single transmembrane domain, which come together to form the ion-conducting pathway. We have undertaken a characterization of the transmembrane domain of the M ₂ protein of influenza A virus using substituted cysteine accessibility analysis, inhibition by the transition metal Cu2+, and site-directed spin labeling of purified protein. We identified residues Ala-30 and Gly-34 as lining the channel pore near the extracellular side, while Trp-41 lines the pore near the intracellular side. His-37 is located at the narrowest portion of the ion-conducting pathway and forms a high affinity binding site for Cu 2+ ions. This is consistent with our hypothesis that His-37 forms the selectivity filter for the channel. We purified several cysteine substitution mutants to which we attached a spin label, as verified using a mass spectrometry assay. Future experiments examining sufficient quantities of these labeled proteins using electron paramagnetic resonance (EPR) spectroscopy should yield even more insight into the structural features of the M₂ transmembrane domain.