Imidazole–Imidazole Hydrogen Bonding in the pH-Sensing Histidine Side Chains of Influenza A M2

Abstract

The arrangement of histidine side chains in influenza A M2 tetramer determines their pK a values, which define pH-controlled proton conduction critical to the virus lifecycle. Both water-associated and hydrogen-bonded imidazole–imidazolium histidine quaternary structures have been proposed, based on crystal structures and NMR chemical shifts, respectively. Here we show, using the conduction domain construct of M2 in lipid bilayers, that the imidazole rings are hydrogen bonded even at a pH of 7.8 in the neutral charge state. An intermolecular 8.9 ± 0.3 Hz 2h J NN hydrogen bond is observed between H37 Nε and Nδ recorded in a fully protonated sample with 100 kHz magic-angle spinning. This interaction could not be detected in the drug-bound sample.

Virus Structures and Dynamics by Magic-Angle Spinning NMR

“…At high pH, increased dynamics of the histidine residues in the pore and breaking of the hydrogen bond were observed. Both also play a role in the RMT inhibitory mechanism, as discussed below (23).…”

“…The two models can be differentiated by MAS NMR through histidine chemical shifts, as well as by probing whether a hydrogen bond exists between two adjacent histidine sidechains. The imidazole-imidazolium and imidazole-imidazole hydrogen bonds of AM2 His-37 were probed at low and high pH, respectively (23,24). At low pH, the existence of a hydrogen bond was detected, suggesting a low-barrier hydrogen bond mechanism is operational (24).…”

“…Because the NMR active isotopes of carbon and nitrogen, 13 C and 15 N, are only 1.11% and 0.37% naturally abundant, respectively, isotopic enrichment of one or both in the sample under investigation is required for in-depth structural and dynamic characterization of any biological samples. These isotopes can be introduced into viruses (7-11) or recombinantly generated viral proteins and/or nucleic acids (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) by adding them to the growth medium or synthetically (32,33). Labels can be introduced either uniformly (i.e., every carbon and/or nitrogen atom will be enriched in the respective isotope) or into judiciously chosen sites (e.g., into selected amino acid types or following various sparse labeling schemes).…”

“…Viral exterior structural isomer to another), and hydrogen bonding (22)(23)(24)(44)(45)(46)(47). Two possible conduction models involve different histidine tautomers and charges and different hydrogen bonding partners (Figure 2c).…”

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR

“…Solid-state NMR, however, can provide unique information about solvent accessibility and membrane association [88][89][90] . Recent examples at fast MAS using proton detection include the human voltage dependent anion channel (VDAC), the alkane transporter AIkL from Pseudomonas putida 67 and influenza A M2 91 . Transfer mechanisms in solidstate include the Nuclear Overhauser Effect (NOE) and spin diffusion, however, in the fast spinning regime, especially when using deuterated samples, spin diffusion is largely suppressed and magnetization transfer occurs mainly through NOE 67,92 .…”