Dynamic Nuclear Polarization Study of Inhibitor Binding
to the M2<sub>18–60</sub> Proton Transporter from Influenza
A
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Abstract
We demonstrate the use of dynamic
nuclear polarization (DNP) to
elucidate ligand binding to a membrane protein using dipolar recoupling
magic angle spinning (MAS) NMR. In particular, we detect drug binding
in the proton transporter M2<sub>18–60</sub> from influenza
A using recoupling experiments at room temperature and with cryogenic
DNP. The results indicate that the pore binding site of rimantadine
is correlated with previously reported widespread chemical shift changes,
suggesting functional binding in the pore. Futhermore, the <sup>15</sup>N-labeled ammonium of rimantadine was observed near A30 <sup>13</sup>Cβ and G34 <sup>13</sup>Cα, suggesting a possible hydrogen
bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker
external binding site(s) in a ZF-TEDOR spectrum. This approach is
generally applicable, particularly for weakly bound ligands, in which
case the application of MAS NMR dipolar recoupling requires the low
temperatures to quench dynamic exchange processes. For the fully protonated
samples investigated, we observed DNP signal enhancements of ∼10
at 400 MHz using only 4–6 mM of the polarizing agent TOTAPOL.
At 600 MHz and with DNP, we measured a distance between the drug and
the protein to a precision of 0.2 Å