Magic-Angle-Spinning NMR of the Drug Resistant S31N M2 Proton Transporter from Influenza A

Andreas, Loren B.; Eddy, Matthew T.; Chou, James J.; Griffin, Robert G.

doi:10.1021/ja3003606

Cited by 56 publications

(112 citation statements)

References 40 publications

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“…S4C). Interestingly, the backbone 13 C chemical shifts for the form studied here show a good correlation with those of only one of two distinct conformers seen by SSNMR of S31N (18-60) in phospholipid bilayers (R 2 = 0.88), indicating that the drug-bound conformation seen here is similar to one of the two conformational forms seen in phospholipid bilayers in the absence of an inhibitor (34). It would be interesting to determine whether the population of the two conformational forms seen by SSNMR is also pH-dependent.…”

Section: M2-s31n Inhibitorsupporting

confidence: 63%

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Wang

Ma³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

204

350

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The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered smallmolecule drugs that inhibit S31N with potencies greater than amantadine's potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.M2-S31N mutant structure | membrane protein structure | M2-S31N inhibitorT he influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine (1-3), which bind directly to the pore of the channel (2-4). Although amantadine has been widely used for several decades, drug resistance has curtailed the use of this family of drugs. Many amantadineresistant influenza viruses can be selected in cell culture (5, 6). A subset of these mutations is found in infected patients undergoing treatment with amantadine (7), and reverse-engineered viruses harboring various pore-lining mutations are competent to replicate in the mouse (8). However, many of these mutations give rise to somewhat attenuated viruses that are less transmissible than WT virus, and they tend to revert in the absence of drug pressure (6, 9). Indeed, large-scale sequencing of transmissible viruses isolated as early as 1918 showed that mutations to pore-lining residues are allowed only within the first turn of the transmembrane (TM) helix at positions 26, 27, and 31 (10). S31N has long been the predominant amantadine-resistant mutation in M2 (11)(12)(13)(14). It predominated in 98-100% of the transmissible amantadineresistant H1N1, H5N1, and H3N2 strains isolated from humans, birds, and swine in the past decade. V27A and L26F are less frequent mutations (10,11,15). Extensive studies of point mutations to the pore-lining residues of M2 have been conducted to understand the paucity of natural variants (16,17). Numerous mutants in the N-terminal aqueous pore retained the ability to conduct protons selectively over other ions, although the magnitude and pH dependence of their conduction varied. However, only a few mutations at the most distal sites, V27A,...

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Section: M2-s31n Inhibitorsupporting

confidence: 63%

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Wang

Ma³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

204

350

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“…It is therefore well suited to the study of biological samples that cannot be dissolved and easily crystallized, or for which crystallization is suspected to significantly alter the samples' function and biological significance. Examples of classes of molecules in this category include membrane proteins that function properly only when embedded in a bilayer membrane (21)(22)(23)(24)(25) and protein fibrils (19,(26)(27)(28) associated with neurodegenerative or other diseases. Figure 1 depicts some of the architectural motifs of membrane proteins and amyloid fibrils.…”

Section: Magic Angle Spinning Nmr Of Membrane Proteins and Amyloid Fimentioning

confidence: 99%

“…For example, membrane-bilayer-bound proteins such as bacteriorhodopsin (bR) (10,32,33), sensory rhodopsin (sR) (22,34), voltage-gated potassium channels (Kv) (35,36), voltagedependent anion channels (VDAC) (37), G protein-coupled receptors (38), integral membrane proteins such as DsbA and DsbB (39), proton channels such as influenza M2 (2,21,40,41), viral fusion proteins (2), and microbial retinal-binding photoreceptor (22) NMR. Figure 1f depicts a two-dimensional (2D) 13 C-13 C radio-frequency-driven recoupling (RFDR) spectrum of VDAC, a 283-amino-acid protein from the mitochondrial outer membrane.…”

Section: Magic Angle Spinning Nmr Of Membrane Proteins and Amyloid Fimentioning

confidence: 99%

Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and ¹H Detection

Andreas²,

Griffin

2015

Annu. Rev. Biochem.

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138

100

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Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.

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“…Several studies of M2CD and M2FL involving the His37 chemical shifts have been recently reported 7,26–30 . Interestingly, these chemical shifts show non-negligible differences even at similar pH.…”

Section: Introductionmentioning

confidence: 99%

“…The origin for these differences is not yet understood: they could result from lipid membrane differences, sample preparation differences, or real structural differences caused by the extra-membrane domains. Extensive peak doubling was detected for one of the M2CD constructs 26 , indicating breaking of the tetramer’s C 4 symmetry and a dimer-of-dimer topology. This peak doubling was observed at pH 7.8, thus it cannot be interpreted by an imidazole-imidazolium dimer model proposed for His37 at acidic pH 29 .…”

Section: Introductionmentioning

confidence: 99%

The Influenza M2 Cytoplasmic Tail Changes the Proton-Exchange Equilibria and the Backbone Conformation of the Transmembrane Histidine Residue to Facilitate Proton Conduction

et al. 2015

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The influenza M2 protein forms an acid-activated tetrameric proton channel important for the virus lifecycle. Residue His37 in the transmembrane domain is responsible for channel activation and proton selectivity. While the structure and dynamics of His37 have been well studied in TM peptide constructs, it has not been investigated in the presence of the full cytoplasmic domain, which increases the proton conductivity by 2-fold compared to the TM peptide. We report here 13C and 15N chemical shifts of His37 in the cytoplasmic-containing M2(21-97), and show that cationic histidines are already present at neutral pH, in contrast to the TM peptide, indicating that the cytoplasmic domain shifts the protonation equilibria. Quantification of the imidazole 15N intensities yielded two resolved proton dissociation constants (pKa’s) of 7.1 and 5.4, which differ from the TM result but resemble the M2(18–60) result, suggesting cooperative proton binding. The average His37 pKa is higher for M2(21–97) than for the shorter constructs. We attribute this higher pKa to direct and indirect effects of the cytoplasmic domain, which is rich in acidic residues. 2D 13C-13C correlation spectra reveal seven His37 Cα-Cβ cross peaks at different pH, some of which are unique to the cytoplasmic-containing M2 and correspond to more ideal α-helical conformations. Based on the pH at which these chemical shifts appear and their sidechain structures, we assign these conformations to His37 in differently charged tetramers. Thus, the cytoplasmic domain facilitates proton conduction through the transmembrane pore by modifying the His37-water proton-exchange equilibria and the His37 backbone conformational distribution.

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Magic-Angle-Spinning NMR of the Drug Resistant S31N M2 Proton Transporter from Influenza A

Cited by 56 publications

References 40 publications

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and ¹H Detection

The Influenza M2 Cytoplasmic Tail Changes the Proton-Exchange Equilibria and the Backbone Conformation of the Transmembrane Histidine Residue to Facilitate Proton Conduction

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Magic-Angle-Spinning NMR of the Drug Resistant S31N M2 Proton Transporter from Influenza A

Cited by 56 publications

References 40 publications

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and 1H Detection

The Influenza M2 Cytoplasmic Tail Changes the Proton-Exchange Equilibria and the Backbone Conformation of the Transmembrane Histidine Residue to Facilitate Proton Conduction

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Magic Angle Spinning NMR of Proteins: High-Frequency Dynamic Nuclear Polarization and ¹H Detection