Acid activation mechanism of the influenza A M2 proton channel

Contributed by William F. DeGrado, September 23, 2016 (sent for review July 13, 2016; reviewed by José D. Faraldo-Gómez and Wei Yang)
October 24, 2016
113 (45) E6955-E6964

Significance

The influenza A M2 channel (AM2) transports protons into the influenza virus upon acid activation. It is an important pharmacological target as well as a prototypical case to study proton conduction through biological channels. The current work provides the most complete computational characterization to date of the physical basis for the acid activation mechanism of the AM2 proton channel. Our results show that lowering the pH value gradually opens the Trp41 gate and decreases the deprotonation barrier of the His37 tetrad, leading to channel activation. Our result also demonstrates that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain.

Abstract

The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.

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Acknowledgments

This research was supported by National Institutes of Health Grants R01-GM053148 (to G.A.V., J.M.J.S., and R.L.), R01-GM056432 (to W.F.D.), and R01-GM088204 (to M.H.) and a Carlsberg Foundation International Fellowship (J.J.M.). The researchers used computing facilities provided by the Extreme Science and Engineering Discovery Environment, which is supported by National Science Foundation Grant OCI-1053575, as well as by the University of Chicago Research Computing Center and the US Department of Defense High Performance Computing Modernization Program.

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References

1
LH Pinto, LJ Holsinger, RA Lamb, Influenza virus M2 protein has ion channel activity. Cell 69, 517–528 (1992).
2
RA Lamb, LJ Holsinger, LH Pinto, The influenza A virus M2 ion channel protein and its role in the influenza virus life cycle. Cellular Receptors for Animal Viruses (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY), pp. 303–321 (1994).
3
LH Pinto, RA Lamb, The M2 proton channels of influenza A and B viruses. J Biol Chem 281, 8997–9000 (2006).
4
AL Polishchuk, et al., A pH-dependent conformational ensemble mediates proton transport through the influenza A/M2 protein. Biochemistry 49, 10061–10071 (2010).
5
C Wang, RA Lamb, LH Pinto, Activation of the M2 ion channel of influenza virus: A role for the transmembrane domain histidine residue. Biophys J 69, 1363–1371 (1995).
6
IV Chizhmakov, et al., Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells. J Physiol 494, 329–336 (1996).
7
J Hu, et al., Histidines, heart of the hydrogen ion channel from influenza A virus: Toward an understanding of conductance and proton selectivity. Proc Natl Acad Sci USA 103, 6865–6870 (2006).
8
Y Tang, F Zaitseva, RA Lamb, LH Pinto, The gate of the influenza virus M2 proton channel is formed by a single tryptophan residue. J Biol Chem 277, 39880–39886 (2002).
9
R Acharya, et al., Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus. Proc Natl Acad Sci USA 107, 15075–15080 (2010).
10
M Sharma, et al., Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer. Science 330, 509–512 (2010).
11
F Hu, W Luo, M Hong, Mechanisms of proton conduction and gating in influenza M2 proton channels from solid-state NMR. Science 330, 505–508 (2010).
12
JK Williams, Y Zhang, K Schmidt-Rohr, M Hong, pH-dependent conformation, dynamics, and aromatic interaction of the gating tryptophan residue of the influenza M2 proton channel from solid-state NMR. Biophys J 104, 1698–1708 (2013).
13
LH Pinto, et al., A functionally defined model for the M2 proton channel of influenza A virus suggests a mechanism for its ion selectivity. Proc Natl Acad Sci USA 94, 11301–11306 (1997).
14
D Salom, BR Hill, JD Lear, WF DeGrado, pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus. Biochemistry 39, 14160–14170 (2000).
15
JD Lear, Proton conduction through the M2 protein of the influenza A virus; a quantitative, mechanistic analysis of experimental data. FEBS Lett 552, 17–22 (2003).
16
R Liang, H Li, JMJ Swanson, GA Voth, Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel. Proc Natl Acad Sci USA 111, 9396–9401 (2014).
17
ML DiFrancesco, U-P Hansen, G Thiel, A Moroni, I Schroeder, Effect of cytosolic pH on inward currents reveals structural characteristics of the proton transport cycle in the influenza A protein M2 in cell-free membrane patches of Xenopus oocytes. PLoS One 9, e107406 (2014).
18
C Ma, et al., Asp44 stabilizes the Trp41 gate of the M2 proton channel of influenza A virus. Structure 21, 2033–2041 (2013).
19
SD Cady, et al., Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463, 689–692 (2010).
20
J Wang, et al., Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus. Proc Natl Acad Sci USA 110, 1315–1320 (2013).
21
Y Wu, et al., Flipping in the pore: Discovery of dual inhibitors that bind in different orientations to the wild-type versus the amantadine-resistant S31N mutant of the influenza A virus M2 proton channel. J Am Chem Soc 136, 17987–17995 (2014).
22
M Hong, WF DeGrado, Structural basis for proton conduction and inhibition by the influenza M2 protein. Protein Sci 21, 1620–1633 (2012).
23
AL Stouffer, 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. Structure 16, 1067–1076 (2008).
24
JL Thomaston, et al., High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction. Proc Natl Acad Sci USA 112, 14260–14265 (2015).
25
JL Thomaston, WF DeGrado, Crystal structure of the drug-resistant S31N influenza M2 proton channel. Protein Sci 25, 1551–1554 (2016).
26
JR Schnell, JJ Chou, Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591–595 (2008).
27
Y Miao, R Fu, HX Zhou, TA Cross, Dynamic short hydrogen bonds in histidine tetrad of full-length M2 proton channel reveal tetrameric structural heterogeneity and functional mechanism. Structure 23, 2300–2308 (2015).
28
F Hu, W Luo, SD Cady, M Hong, Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness. Biochim Biophys Acta 1808, 415–423 (2011).
29
C Li, H Qin, FP Gao, TA Cross, Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel. Biochim Biophys Acta 1768, 3162–3170 (2007).
30
F Hu, K Schmidt-Rohr, M Hong, NMR detection of pH-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel. J Am Chem Soc 134, 3703–3713 (2012).
31
Q Zhong, DM Newns, P Pattnaik, JD Lear, ML Klein, Two possible conducting states of the influenza A virus M2 ion channel. FEBS Lett 473, 195–198 (2000).
32
C Wei, A Pohorille, Activation and proton transport mechanism in influenza A M2 channel. Biophys J 105, 2036–2045 (2013).
33
E Khurana, et al., Molecular dynamics calculations suggest a conduction mechanism for the M2 proton channel from influenza A virus. Proc Natl Acad Sci USA 106, 1069–1074 (2009).
34
H Chen, Y Wu, GA Voth, Proton transport behavior through the influenza A M2 channel: Insights from molecular simulation. Biophys J 93, 3470–3479 (2007).
35
M Yi, TA Cross, HX Zhou, A secondary gate as a mechanism for inhibition of the M2 proton channel by amantadine. J Phys Chem B 112, 7977–7979 (2008).
36
M Yi, TA Cross, HX Zhou, Conformational heterogeneity of the M2 proton channel and a structural model for channel activation. Proc Natl Acad Sci USA 106, 13311–13316 (2009).
37
Y Wu, GA Voth, A computational study of the closed and open states of the influenza a M2 proton channel. Biophys J 89, 2402–2411 (2005).
38
H Dong, G Fiorin, WF DeGrado, ML Klein, Proton release from the histidine-tetrad in the M2 channel of the influenza A virus. J Phys Chem B 118, 12644–12651 (2014).
39
HX Zhou, A theory for the proton transport of the influenza virus M2 protein: Extensive test against conductance data. Biophys J 100, 912–921 (2011).
40
KL Roberts, GP Leser, C Ma, RA Lamb, The amphipathic helix of influenza A virus M2 protein is required for filamentous bud formation and scission of filamentous and spherical particles. J Virol 87, 9973–9982 (2013).
41
NW Schmidt, A Mishra, J Wang, WF DeGrado, GCL Wong, Influenza virus A M2 protein generates negative Gaussian membrane curvature necessary for budding and scission. J Am Chem Soc 135, 13710–13719 (2013).
42
C Ma, et al., Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel. Proc Natl Acad Sci USA 106, 12283–12288 (2009).
43
T Wang, M Hong, Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles. Biochemistry 54, 2214–2226 (2015).
44
J Wang, et al., Molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza A virus M2. J Am Chem Soc 133, 12834–12841 (2011).
45
H Dong, G Fiorin, WF Degrado, ML Klein, Exploring histidine conformations in the M2 channel lumen of the influenza A virus at neutral pH via molecular simulations. J Phys Chem Lett 4, 3067–3071 (2013).
46
V Carnevale, G Fiorin, BG Levine, WF Degrado, ML Klein, Multiple proton confinement in the M2 channel from the influenza A virus. J Phys Chem C Nanomater Interfaces 114, 20856–20863 (2010).
47
TW Allen, OS Andersen, B Roux, Energetics of ion conduction through the gramicidin channel. Proc Natl Acad Sci USA 101, 117–122 (2004).
48
MT Colvin, LB Andreas, JJ Chou, RG Griffin, Proton association constants of His 37 in the Influenza-A M218-60 dimer-of-dimers. Biochemistry 53, 5987–5994 (2014).
49
SY Liao, Y Yang, D Tietze, M Hong, The influenza M2 cytoplasmic tail changes the proton-exchange equilibria and the backbone conformation of the transmembrane histidine residue to facilitate proton conduction. J Am Chem Soc 137, 6067–6077 (2015).
50
IV Chizhmakov, et al., Differences in conductance of M2 proton channels of two influenza viruses at low and high pH. J Physiol 546, 427–438 (2003).
51
JK Williams, et al., Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR. J Am Chem Soc 135, 9885–9897 (2013).
52
T Leiding, J Wang, J Martinsson, WF DeGrado, SP Arsköld, Proton and cation transport activity of the M2 proton channel from influenza A virus. Proc Natl Acad Sci USA 107, 15409–15414 (2010).
53
JA Mould, et al., Mechanism for proton conduction of the M(2) ion channel of influenza A virus. J Biol Chem 275, 8592–8599 (2000).
54
E Khurana, RH Devane, M Dal Peraro, ML Klein, Computational study of drug binding to the membrane-bound tetrameric M2 peptide bundle from influenza A virus. Biochim Biophys Acta 1808, 530–537 (2011).
55
AL Stouffer, et al., Structural basis for the function and inhibition of an influenza virus proton channel. Nature 451, 596–599 (2008).
56
W Luo, R Mani, M Hong, Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR. J Phys Chem B 111, 10825–10832 (2007).
57
M Hong, KJ Fritzsching, JK Williams, Hydrogen-bonding partner of the proton-conducting histidine in the influenza M2 proton channel revealed from 1H chemical shifts. J Am Chem Soc 134, 14753–14755 (2012).
58
AD MacKerell, et al., All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102, 3586–3616 (1998).
59
Jr AD MacKerell, M Feig, 3rd CL Brooks, Improved treatment of the protein backbone in empirical force fields. J Am Chem Soc 126, 698–699 (2004).
60
RB Best, et al., Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. J Chem Theory Comput 8, 3257–3273 (2012).
61
JB Klauda, et al., Update of the CHARMM all-atom additive force field for lipids: Validation on six lipid types. J Phys Chem B 114, 7830–7843 (2010).
62
WL Jorgensen, J Chandrasekhar, JD Madura, RW Impey, ML Klein, Comparison of simple potential functions for simulating liquid water. J Chem Phys 79, 926–935 (1983).
63
T Darden, D York, L Pedersen, Particle mesh Ewald: An N-log(N) method for Ewald sums in large systems. J Chem Phys 98, 10089–10092 (1993).
64
JC Phillips, et al., Scalable molecular dynamics with NAMD. J Comput Chem 26, 1781–1802 (2005).
65
SE Feller, Y Zhang, RW Pastor, BR Brooks, Constant pressure molecular dynamics simulation: The Langevin piston method. J Chem Phys 103, 4613–4621 (1995).
66
L Verlet, Computer “experiments” on classical fluids. I. Thermodynamical properties of Lennard-Jones molecules. Phys Rev 159, 98–103 (1967).
67
HJC Berendsen, D Vanderspoel, R Vandrunen, GROMACS: A message-passing parallel molecular dynamics implementation. Comput Phys Commun 91, 43–56 (1995).
68
MJ Abraham, et al., GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).
69
G Bussi, D Donadio, M Parrinello, Canonical sampling through velocity rescaling. J Chem Phys 126, 014101 (2007).
70
HJC Berendsen, JPM Postma, WF Vangunsteren, A Dinola, JR Haak, Molecular dynamics with coupling to an external bath. J Chem Phys 81, 3684–3690 (1984).
71
PH König, et al., Toward theoretical analysis of long-range proton transfer kinetics in biomolecular pumps. J Phys Chem A 110, 548–563 (2006).
72
D Riccardi, et al., “Proton holes” in long-range proton transfer reactions in solution and enzymes: A theoretical analysis. J Am Chem Soc 128, 16302–16311 (2006).
73
R Liang, JMJ Swanson, GA Voth, Benchmark study of the SCC-DFTB approach for a biomolecular proton channel. J Chem Theory Comput 10, 451–462 (2014).
74
AD Becke, Density-functional exchange-energy approximation with correct asymptotic behavior. Phys Rev A Gen Phys 38, 3098–3100 (1988).
75
C Lee, W Yang, RG Parr, Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys Rev B Condens Matter 37, 785–789 (1988).
76
S Grimme, J Antony, S Ehrlich, H Krieg, A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J Chem Phys 132, 154104 (2010).
77
G Lippert, J Hutter, M Parrinello, A hybrid Gaussian and plane wave density functional scheme. Mol Phys 92, 477–487 (1997).
78
C Hartwigsen, S Goedecker, J Hutter, Relativistic separable dual-space Gaussian pseudopotentials from H to Rn. Phys Rev B 58, 3641–3662 (1998).
79
T Laino, F Mohamed, A Laio, M Parrinello, An efficient linear-scaling electrostatic coupling for treating periodic boundary conditions in QM/MM simulations. J Chem Theory Comput 2, 1370–1378 (2006).
80
T Laino, F Mohamed, A Laio, M Parrinello, An efficient real space multigrid QM/MM electrostatic coupling. J Chem Theory Comput 1, 1176–1184 (2005).
81
PE Blöchl, Electrostatic decoupling of periodic images of plane-wave-expanded densities and derived atomic point charges. J Chem Phys 103, 7422–7428 (1995).
82
J VandeVondele, J Hutter, An efficient orbital transformation method for electronic structure calculations. J Chem Phys 118, 4365–4369 (2003).
83
J VandeVondele, et al., QUICKSTEP: Fast and accurate density functional calculations using a mixed Gaussian and plane waves approach. Comput Phys Commun 167, 103–128 (2005).
84
C Knight, GE Lindberg, GA Voth, Multiscale reactive molecular dynamics. J Chem Phys 137, 22A525 (2012).
85
JG Nelson, Y Peng, DW Silverstein, JMJ Swanson, Multiscale reactive molecular dynamics for absolute pK a predictions and amino acid deprotonation. J Chem Theory Comput 10, 2729–2737 (2014).
86
S Lee, R Liang, GA Voth, JMJ Swanson, Computationally efficient multiscale reactive molecular dynamics to describe amino acid deprotonation in proteins. J Chem Theory Comput 12, 879–891 (2016).
87
T Yamashita, Y Peng, C Knight, GA Voth, Computationally Efficient Multiconfigurational Reactive Molecular Dynamics. J Chem Theory Comput 8, 4863–4875 (2012).
88
S Plimpton, Fast parallel algorithms for short-range molecular dynamics. J Comput Phys 117, 1–19 (1995).
89
TJF Day, AV Soudackov, M Cuma, UW Schmitt, GA Voth, A second generation multistate empirical valence bond model for proton transport in aqueous systems. J Chem Phys 117, 5839–5849 (2002).
90
Y Wu, HL Tepper, GA Voth, Flexible simple point-charge water model with improved liquid-state properties. J Chem Phys 124, 024503 (2006).
91
RW Hockney, JW Eastwood Computer Simulation Using Particles (McGraw-Hill, New York), pp. 540 (1981).
92
Y Sugita, A Kitao, Y Okamoto, Multidimensional replica-exchange method for free-energy calculations. J Chem Phys 113, 6042–6051 (2000).
93
B Roux, The calculation of the potential of mean force using computer simulations. Comput Phys Commun 91, 275–282 (1995).
94
MP Allen, DJ Tildesley Computer Simulation of Liquids (Oxford Univ Press, New York, 1990).
95
S Kumar, D Bouzida, RH Swendsen, PA Kollman, JM Rosenberg, The weighted histogram analysis method for free-energy calculations on biomolecules. 1. The method. J Comput Chem 13, 1011–1021 (1992).
96
DG Levitt, Interpretation of biological ion channel flux data--reaction-rate versus continuum theory. Annu Rev Biophys Biophys Chem 15, 29–57 (1986).
97
B Roux, M Karplus, Ion-transport in a gramicidin-like channel: Dynamics and mobility. J Phys Chem 95, 4856–4868 (1991).
98
DG Levitt, General continuum theory for multiion channel. I. Theory. Biophys J 59, 271–277 (1991).
99
TB Woolf, B Roux, Conformational flexibility of o-phosphorylcholine and o-phosphorylethanolamine: A molecular dynamics study of solvation effects. J Am Chem Soc 116, 5916–5926 (1994).

Information & Authors

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Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 113 | No. 45
November 8, 2016
PubMed: 27791184

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Submission history

Published online: October 24, 2016
Published in issue: November 8, 2016

Keywords

  1. ion channel
  2. proton conduction
  3. multiscale modeling
  4. QM/MM
  5. free-energy sampling

Acknowledgments

This research was supported by National Institutes of Health Grants R01-GM053148 (to G.A.V., J.M.J.S., and R.L.), R01-GM056432 (to W.F.D.), and R01-GM088204 (to M.H.) and a Carlsberg Foundation International Fellowship (J.J.M.). The researchers used computing facilities provided by the Extreme Science and Engineering Discovery Environment, which is supported by National Science Foundation Grant OCI-1053575, as well as by the University of Chicago Research Computing Center and the US Department of Defense High Performance Computing Modernization Program.

Authors

Affiliations

Ruibin Liang
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637;
James Franck Institute, The University of Chicago, Chicago, IL 60637;
Jessica M. J. Swanson
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637;
James Franck Institute, The University of Chicago, Chicago, IL 60637;
Jesper J. Madsen
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637;
James Franck Institute, The University of Chicago, Chicago, IL 60637;
Mei Hong
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;
William F. DeGrado1 [email protected]
Department of Pharmaceutical Chemistry, University of San Francisco, San Francisco, CA 94158
Gregory A. Voth1 [email protected]
Department of Chemistry, The University of Chicago, Chicago, IL 60637;
Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637;
James Franck Institute, The University of Chicago, Chicago, IL 60637;

Notes

1
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: R.L., J.M.J.S., W.F.D., and G.A.V. designed research; R.L. and J.J.M. performed research; R.L., J.M.J.S., J.J.M., M.H., W.F.D., and G.A.V. analyzed data; and R.L., J.M.J.S., M.H., W.F.D., and G.A.V. wrote the paper.
Reviewers: J.D.F.-G., NIH/National Heart, Lung, and Blood Institute; and W.Y., Florida State University.

Competing Interests

The authors declare no conflict of interest.

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    Acid activation mechanism of the influenza A M2 proton channel
    Proceedings of the National Academy of Sciences
    • Vol. 113
    • No. 45
    • pp. 12599-E7139

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