2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

Abstract

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs. © 2014 AIP Publishing LLC. [http://dx

Infrared and fluorescence assessment of the hydration status of the tryptophan gate in the influenza A M2 proton channel

“…DPC micelles have been used in many previous 2D IR studies of similar nature. 41,43 As shown (Figure S2, ESI), the C≡N stretching band of M2TM-W CN in DPC micelles was almost identical to that obtained in membranes composed of POPC/POPG/cholesterol (4:1:2), suggesting that the use of DPC micelles in the current case has not significantly changed the assembly and function of the M2TM-W CN peptide.…”

Watching Proteins Wiggle: Mapping Structures with Two-Dimensional Infrared Spectroscopy

“…This result shined light on how the water structure in the channel changes from “icelike” to “liquidlike” in response to the conformational transition of the tetramer, a conclusion that has also been supported by MD simulations and demonstrated that conformational dynamics of a protein can be interpreted through the ultrafast relaxation of water associated with it. The authors subsequently extended this premise to investigate binding of anti-influenza drugs in the M2 tetramer 9 and uncovered that the water in the channel becomes more liquidlike upon drug binding, thereby contributing a favorable entropic factor to the drug-binding thermodynamics. This experimental result was in close agreement with predications from MD simulations 147 and served as the first experimental verification of drug docking mechanisms in the channel that had previously been suggested from theoretical investigations.…”

Site-Specific Infrared Probes of Proteins

“…Furthermore, they speculated that the tolerance of rilpivirine to mutations in RT is due to the stabilization effect of this H-bonding network. Since many drug molecules contain the C≡N group and water molecules are frequently found in protein binding pockets, we believe that the methods discussed above can be extended to study the mechanism of ligand or drug binding of other biological systems, such as the Influenza A M2 proton channel (28, 91). …”