Partitioning and Localization of Spin-Labeled Amantadine in Lipid Bilayers: An Epr Study

Subczynski, Witold K.; Wojas, J.; Pezeshk, Vida; Pezeshk, Abbas

doi:10.1021/js970381n

Cited by 30 publications

(31 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is well in excess of the expected stoichiometric M2 : amantadine ratio, which is either 4 : 1 based on the crystal structure or 1: 1 based on the solution NMR structure. The use of excess amantadine is largely due to the concern that the lipophilic drug, which readily partitions into the lipid membrane from the aqueous solution 29; 30; 31 , may not all end up in the channel. Nevertheless, the question arises as to whether the observed M2 chemical shift changes result from non-specific effects of amantadine on the lipid bilayer that then indirectly change the peptide structure, or whether it is due to direct amantadine-peptide interactions.…”

Section: Resultsmentioning

confidence: 99%

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

Cady

Mishanina

Hong

2009

Journal of Molecular Biology

137

186

View full text Add to dashboard Cite

The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the sidechain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.

show abstract

Section: Resultsmentioning

confidence: 99%

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

Cady

Mishanina

Hong

2009

Journal of Molecular Biology

137

186

View full text Add to dashboard Cite

show abstract

“…The amantadine-bound samples in our experiments contain 8-fold molar excess of amantadine to the peptide, or 32-fold excess amantadine to the channel. The excess amantadine partitions into the bilayer (Subczynski et al, 1998, Wang et al, 2004). Paramagnetic relaxation enhancement NMR data indicate that amantadine is located at the interfacial region of the DMPC bilayer with the amine group pointing to the surface (Li et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

Cady

Hong

2009

J Biomol NMR

View full text Add to dashboard Cite

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the 1H rotating-frame spin-lattice relaxation (T1ρ) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by 2H, 15N and 13C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent 1H T1ρ relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 ± 0.9 μs for the apo peptide to 0.89 ± 0.41 μs for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of ln(T1ρ−1) indicates that the activation energy of motion increased from 14.0 ± 4.0 kJ/mol for the apo peptide to 23.3 ± 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.

show abstract

“…However, neither of these second sites appears to be required for virus replication. Amantadine partitions into lipid bilayers (41)(42)(43) giving the drug access to the cytoplasmic side of the four-helix bundle. Whether the external rimantadine binding site, that we consider to be a second drug-binding site, has another role in properties of A/M2 function that are not directly associated with inhibition of channel activity remains to be determined.…”

Section: An A/m2 and Bm2 Chimeric Tm Domain Renders The Bm2 Ion Channelmentioning

confidence: 99%

Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel

Jing

Ohigashi

et al. 2008

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

166

184

View full text Add to dashboard Cite

with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24 -36 into BM2, yielded a pHactivated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.amantadine inhibition of influenza virus ͉ amantadine resistance ͉ drug-binding site ͉ influenza reverse genetics

show abstract

Partitioning and Localization of Spin-Labeled Amantadine in Lipid Bilayers: An Epr Study

Cited by 30 publications

References 40 publications

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

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

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel

Contact Info

Product

Resources

About