Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein.

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein.

[1]  W. DeGrado,et al.  Determining the orientation of uniaxially rotating membrane proteins using unoriented samples: a 2H, 13C, AND 15N solid-state NMR investigation of the dynamics and orientation of a transmembrane helical bundle. , 2007, Journal of the American Chemical Society.

[2]  Vikas Nanda,et al.  Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function. , 2005, Journal of molecular biology.

[3]  Vikas Nanda,et al.  The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment , 2005, Protein science : a publication of the Protein Society.

[4]  Wenbin Luo,et al.  Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness. , 2011, Biochimica et biophysica acta.

[5]  R. Griffin,et al.  RESONANCE ASSIGNMENTS FOR SOLID PEPTIDES BY DIPOLAR-MEDIATED 13C/15N CORRELATION SOLID-STATE NMR , 1998 .

[6]  R. Griffin,et al.  Magic angle spinning NMR investigation of influenza A M2(18-60): support for an allosteric mechanism of inhibition. , 2010, Journal of the American Chemical Society.

[7]  Cinque S. Soto,et al.  pH-induced conformational change of the influenza M2 protein C-terminal domain. , 2008, Biochemistry.

[8]  Huan‐Xiang Zhou,et al.  Insight into the Mechanism of the Influenza A Proton Channel from a Structure in a Lipid Bilayer , 2010, Science.

[9]  S. Cady,et al.  Amantadine-induced conformational and dynamical changes of the influenza M2 transmembrane proton channel , 2008, Proceedings of the National Academy of Sciences.

[10]  H. A. Blough.  Fatty acid composition of individual phospholipids of influenza virus. , 1971, The Journal of general virology.

[11]  M. R. Rosenberg,et al.  Coexistence of two adamantane binding sites in the influenza A M2 ion channel , 2010, Proceedings of the National Academy of Sciences.

[12]  R. Lamb,et al.  Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel , 2009, Proceedings of the National Academy of Sciences.

[13]  R. Lamb,et al.  An amantadine-sensitive chimeric BM2 ion channel of influenza B virus has implications for the mechanism of drug inhibition , 2009, Proceedings of the National Academy of Sciences.

[14]  Jun Wang,et al.  Specific binding of adamantane drugs and direction of their polar amines in the pore of the influenza M2 transmembrane domain in lipid bilayers and dodecylphosphocholine micelles determined by NMR spectroscopy. , 2011, Journal of the American Chemical Society.

[15]  Wenbin Luo,et al.  Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation. , 2009, Biochemistry.

[16]  J. Chou,et al.  Mechanism of drug inhibition and drug resistance of influenza A M2 channel , 2009, Proceedings of the National Academy of Sciences.

[17]  E. Möncke-Buchner,et al.  The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein , 2005, European Biophysics Journal.

[18]  Xianghong Jing,et al.  Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel , 2008, Proceedings of the National Academy of Sciences.

[19]  R. Lamb,et al.  The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging. , 2000, Virology.

[20]  A. Pekosz,et al.  Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production , 2006, Journal of Virology.

[21]  J. Wang,et al.  Transmembrane domain of M2 protein from influenza A virus studied by solid-state (15)N polarization inversion spin exchange at magic angle NMR. , 2000, Biophysical journal.

[22]  Conggang Li,et al.  Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel. , 2007, Biochimica et biophysica acta.

[23]  Kiyonori Takegoshi,et al.  13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR , 2001 .

[24]  S. Cady,et al.  Structure of amantadine-bound M2 transmembrane peptide of influenza A in lipid bilayers from magic-angle-spinning solid-state NMR: the role of Ser31 in amantadine binding. , 2009, Journal of molecular biology.

[25]  Cinque S. Soto,et al.  Structure of the Amantadine Binding Site of Influenza M2 Proton Channels In Lipid Bilayers , 2010, Nature.

[26]  R. Bertram,et al.  Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus. , 2007, Biophysical journal.

[27]  Mei Hong,et al.  Mechanisms of Proton Conduction and Gating in Influenza M2 Proton Channels from Solid-State NMR , 2010, Science.

[28]  Huan-Xiang Zhou,et al.  Conformational heterogeneity of the M2 proton channel and a structural model for channel activation , 2009, Proceedings of the National Academy of Sciences.

[29]  R. Lamb,et al.  Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue. , 1995, Biophysical journal.

[30]  C. Jaroniec,et al.  Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides. , 2001, Journal of the American Chemical Society.

[31]  P. Saffman,et al.  Brownian motion in biological membranes. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Xianghong Jing,et al.  Influenza Virus M2 Protein Mediates ESCRT-Independent Membrane Scission , 2010, Cell.

[33]  T. Gullion,et al.  Rotational-Echo, Double-Resonance NMR , 1989 .

[34]  R. Lamb,et al.  The M2 Proton Channels of Influenza A and B Viruses* , 2006, Journal of Biological Chemistry.

[35]  H. Klenk,et al.  On the structure of the influenza virus envelope. , 1972, Virology.

[36]  S. Cady,et al.  Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation , 2009, Journal of biomolecular NMR.

[37]  R. Lamb,et al.  The interplay of functional tuning, drug resistance, and thermodynamic stability in the evolution of the M2 proton channel from the influenza A virus. , 2008, Structure.

[38]  J. Chou,et al.  Structure and mechanism of the M2 proton channel of influenza A virus , 2008, Nature.

[39]  R. Lamb,et al.  Influenza Virus M2 Ion Channel Protein Is Necessary for Filamentous Virion Formation , 2010, Journal of Virology.

[40]  R. Lamb,et al.  The Gate of the Influenza Virus M2 Proton Channel Is Formed by a Single Tryptophan Residue* , 2002, The Journal of Biological Chemistry.

[41]  Cinque S. Soto,et al.  Structural basis for the function and inhibition of an influenza virus proton channel , 2008, Nature.

[42]  Wenbin Luo,et al.  Structure and function of the influenza A M2 proton channel. , 2009, Biochemistry.