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PDBsum entry 2kih
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Transport protein
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PDB id
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2kih
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References listed in PDB file
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Key reference
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Title
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Mechanism of drug inhibition and drug resistance of influenza a m2 channel.
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Authors
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R.M.Pielak,
J.R.Schnell,
J.J.Chou.
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Ref.
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Proc Natl Acad Sci U S A, 2009,
106,
7379-7384.
[DOI no: ]
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PubMed id
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Abstract
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The influenza A virus M2 proton channel equilibrates pH across the viral
membrane during entry and across the trans-Golgi membrane of infected cells
during viral maturation. It is an important target of adamantane-family
antiviral drugs, but drug resistance has become a critical problem. Two
different sites for drug interaction have been proposed. One is a lipid-facing
pocket between 2 adjacent transmembrane helices (around Asp-44), at which the
drug binds and inhibits proton conductance allosterically. The other is inside
the pore (around Ser-31), at which the drug directly blocks proton passage.
Here, we describe structural and functional experiments on the mechanism of drug
inhibition and resistance. The solution structure of the S31N drug-resistant
mutant of M2, a mutant of the highly pathogenic avian influenza subtype H5N1,
shows that replacing Ser-31 with Asn has little effect on the structure of the
channel pore, but dramatically reduces drug binding to the allosteric site.
Mutagenesis and liposomal proton flux assays show that replacing the key residue
(Asp-44) in the lipid-facing binding pocket with Ala has a dramatic effect on
drug sensitivity, but that the channel remains fully drug sensitive when
replacing Ser-31 with Ala. Chemical cross-linking studies indicate an inverse
correlation between channel stability and drug resistance. The lipid-facing
pocket contains residues from 2 adjacent channel-forming helices. Therefore, it
is present only when the helices are tightly packed in the closed conformation.
Thus, drug-resistant mutants impair drug binding by destabilizing helix-helix
assembly.
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Figure 1.
Proposed adamantane binding sites of the M2 channel. (A) The
lipid-facing adamantane binding pocket composed of critical
channel gating elements from 2 adjacent TM helices (2RLF). (B)
The proposed drug-binding site inside the channel pore (3C9J).
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Figure 5.
Structural analysis of the S31N drug resistant mutant. (A)
Ribbon representation of a structural model of the S31N mutant,
illustrating the 2 lysines used in cross-linking. (B) Results of
structural refinement of the helix–helix interface of
S31N(18–60) based on experimental NOE data. The dashed lines
represent intersubunit NOEs involving the side-chain NH[2] of
Asn-31. For comparison, a similar view of WT(18–60) (PDB,
2RLF) is shown. The side chains of both Ser-31 in the WT and
Asn-31 in the mutant are positioned at the helix–helix
interface. (C) SDS/PAGE of M2(18–60) variants after being
treated with 75 μM DSP for 15 min (for details, see Methods).
(D) Same as in C except 2.5 mM DSP and 60 min of reaction time
were applied.
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