PDBsum entry 2kih

Transport protein

PDB id: 2kih

Contents

Protein chains

38 a.a.

PDB id:

2kih

Name:

Transport protein

Title:

S31n mutant of m2 proton channel

Structure:

Matrix protein 2. Chain: a, b, c, d. Synonym: proton channel protein m2. Engineered: yes. Mutation: yes

Source:

Influenza a virus. Organism_taxid: 381517. Strain: a/udorn/307/1972 h3n2. Gene: m. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

10 models

Authors:

R.M.Pielak

Key ref:

R.M.Pielak et al. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel. Proc Natl Acad Sci U S A, 106, 7379-7384. PubMed id: 19383794 DOI: 10.1073/pnas.0902548106

Date:

05-May-09 Release date: 19-May-09

Protein chains

P0DOF5  (M2_I72A2) -  Matrix protein 2 from Influenza A virus (strain A/Udorn/307/1972 H3N2)

Seq: 97 a.a.

Struc: 38 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1073/pnas.0902548106

Proc Natl Acad Sci U S A 106:7379-7384 (2009)

PubMed id: 19383794

Mechanism of drug inhibition and drug resistance of influenza A M2 channel.

R.M.Pielak, J.R.Schnell, J.J.Chou.

ABSTRACT

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.

Selected figure(s)

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).

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.

Figures were selected by an automated process.