| Matrix protein 2, M2, forms a highly selective proton channel that is an important constituent of the influenza virus. It equilibrates pH across the viral membrane during viral entry and across the trans-Golgi membrane of infected cells during viral maturation. We obtained conductance measurements for M2 and a number of M2 variants using a liposome proton flux assay. The proton transport follows Michaelis-Menten-like kinetics with two saturation steps: one pseudo-saturation at pH ∼5.5, and another at pH ∼4. The heart of the mechanism is the pore-lining His37 and Trp41. NMR measurements suggest that histidine and tryptophan act in unison to transport protons down the concentration gradient. The histidine pKa correlates with chemical shift perturbation of the Trp41 gate, indicating that histidine protonation and opening of the channel gate are synchronized events. Finally, mutagenesis and structural analysis identified key residues that affect the rate of conduction.;M2 is the target of two anti-influenza drugs amantadine and rimantadine, but emergence of drug resistant strains has severely compromised the effectiveness of these compounds. Two distinct drug-binding sites have been proposed based on experimental results. One is a lipid-facing pocket between two adjacent transmembrane helices (around Asp44) at which the drug binds and inhibits proton conductance allosterically. The other drug binding site is inside the pore (around Ser31) at which the drug directly blocks proton passage. We solved the structures of two drug resistant mutants S31N and V27A and conducted functional experiments to investigate the mechanism of drug inhibition and resistance. The solution structure of the S31N drug-resistant mutant of M2 shows that replacing Ser31 with Asn has little effect on the structure of the channel pore, but it destabilizes inter-helical packing. Mutagenesis and liposomal proton flux assays show that replacing residues in the allosteric binding pocket, e.g. Asp44, with alanine has dramatic effect on drug sensitivity but that the channel remains fully drug sensitive when replacing Ser31 with alanine. Similarly, V27A structure forms more dynamic weaker tetrameric assembly. Our experiments indicated that resistance might be a result of the weaker more dynamic inter-helical packing that disrupts protein drug interaction. |
