User:Kinkreet/SYL/StructureofAmantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: The Role of Ser31 in Amantadine Binding

Influenza A viruses are negative-sense, single-stranded, segmented RNA viruses. There are 17 different H antigens (H1 to H17) and nine different N antigens (N1 to N9). They are endocytosed into the cell into vesicles; these vesicles becomes acidified and this lowering of pH triggers protein-protein dissociation and virion uncoating, allowing the viral genetic components to replicate and eventually cause infection. About 36,000 people die each year of problems from the flu.

The M2 transmembrane domain of Influenza A is a pH-activated proton channel required for the acidification process and thus, ultimately, virus replication . It is also of interest on a separate note to viral replication - it is the smallest and simplest ion channel capable of selectivity and able to be activated.

There are currently four antiviral agents approved by the FDA - oseltamivir, zanamivir, amantadine, and rimantadine, with the latter two binding to the M2 transmembrane peptide (M2TMP) of Influenza A to prevent its action. The structure of amantadine and rimantadine are based on the structure of adamantane, with an amino group or ethanamine group substitution, respectively, at one of the four methyne positions. It was found that a S31N mutation at the M2TMP prevented amantadine and rimantadine from binding, thus conferring resistance to these drugs. Currently, resistance of common influenza viruses to amantadine and rimantadine is very high, and so new drugs must be developed against the influenza A virus; understanding the interaction between amantadine and the M2TMP will aid in future drug development.

Previous groups have identified conflicting drug binding sites and no consensus was reached; in this paper, Mei Hong and group uses magic-angle-spinning solid-state NMR to determine the structure of Ser31 and surrounding residues, in order to determine the role of Ser31 in the binding of amantadine.

Preparation of sample
M2TMP was reconstituted into DLPC bilayers by detergent dialysis.

Large unilamellar DLPC vesicles were prepared by dissolving powdered DLPC lipids in a phosphate buffer (10 mM Na2-HPO4/NaH2PO4, 1mM ethylenediaminetetraacetic acid, 0.1mM NaN3, pH 7.5, 30 °C) or in a 10 mM citrate buffer (pH 4.5, 30 °C). Unilamellar vesicles are synthesized via 8 freeze-thaw cycles - dipping in liquid nitrogen followed by boiling water. M2TMP is then dissolved into the solution at a peptide-to-lipid ratio of 1:15, which is sufficiently high for the M2TMP to tetramerize. The solution was vortex for 1 hour and incubated at room temperature for 6 - 8 hours, and then dialysed with a 3.5 kDa-cutoff membrane against phosphate buffer for 3 days to remove detergents. The dialysed M2TMP/DLPC solution (without detergent) is ultracentrifuged at 150 000 g for 3 h at 10 °C. This yielded a 90% reconstitution of the peptide. The pellet containing the reconstituted peptide-lipid is spun on a 4 mm rotor with borosilicate glass spacers and incubated at 30 °C for 2 days, after which NMR was performed.

Magic Angle Spinning Solid-state NMR
Magic angle spinning (MAS) solid-state NMR is a technique which involves spinning the sample at a high frequency (1-70kHz) at the magic angle (θm (ca. 54.74°, where cos2θm=1/3)) with respect to the direction of the magnetic field. This technique removes the effects of chemical shift anisotropy and homonuclear dipolar coupling for spin-½ nuclei, thus increasing the resolution of the spectrum. MAS is able to analyse intact tissue samples at high resolution.