title: Dynamic nuclear polarization study of inhibitor binding to the M2(18-60) proton transporter from influenza A.
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(18-60) 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 (15)N-labeled ammonium of rimantadine was observed near A30 (13)Cβ and G34 (13)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.
authors.affiliation: Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Loren B
authors.initials: LB
authors.lastname: Andreas
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Barnes
authors.forename: Björn
authors.initials: B
authors.lastname: Corzilius
authors.forename: James J
authors.initials: JJ
authors.lastname: Chou
authors.forename: Eric A
authors.initials: EA
authors.lastname: Miller
authors.forename: Marc
authors.initials: M
authors.lastname: Caporini
authors.forename: Melanie
authors.initials: M
authors.lastname: Rosay
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D003080:Cold Temperature; D005990:Glycerol / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D012299:Rimantadine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D000998:Antiviral Agents; D005990:Glycerol; D008024:Ligands; D009587:Nitrogen Isotopes; D012299:Rimantadine; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins