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- PDB-2kqt: Solid-state NMR structure of the M2 transmembrane peptide of the ... -

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Basic information

Entry
Database: PDB / ID: 2kqt
TitleSolid-state NMR structure of the M2 transmembrane peptide of the influenza A virus in DMPC lipid bilayers bound to deuterated amantadine
ComponentsM2 protein
KeywordsTRANSPORT PROTEIN / influenza / transmembrane / amantadine / REDOR
Function / homology
Function and homology information


symbiont-mediated suppression of host autophagy / proton transmembrane transporter activity / protein complex oligomerization / monoatomic ion channel activity / symbiont genome entry into host cell via pore formation in plasma membrane / host cell plasma membrane / virion membrane / identical protein binding / membrane
Similarity search - Function
Influenza virus matrix protein 2 / Influenza Matrix protein (M2)
Similarity search - Domain/homology
(3S,5S,7S)-tricyclo[3.3.1.1~3,7~]decan-1-amine / Matrix protein 2 / Matrix protein 2
Similarity search - Component
MethodSOLID-STATE NMR / simulated annealing, Monte Carlo
Model detailsfewest violations, model 1
AuthorsCady, S.D. / Schmidt-Rohr, K. / Wang, J. / Soto, C.S. / DeGrado, W.F. / Hong, M.
CitationJournal: Biophys.J. / Year: 2007
Title: Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus.
Authors: Hu, J. / Asbury, T. / Achuthan, S. / Li, C. / Bertram, R. / Quine, J.R. / Fu, R. / Cross, T.A.
History
DepositionNov 18, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0Feb 9, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 26, 2011Group: Other
Revision 1.3Feb 26, 2020Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_database_status.status_code_cs / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.4Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data
Revision 1.5May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI
Remark 0THIS ENTRY 2KQT REFLECTS AN ALTERNATIVE MODELING OF THE ORIGINAL STRUCTURAL DATA 2H95 and 2KAD

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: M2 protein
B: M2 protein
C: M2 protein
D: M2 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)11,0725
Polymers10,9214
Non-polymers1511
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)17 / 24structures with the least restraint violations
RepresentativeModel #1fewest violations

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Components

#1: Protein/peptide
M2 protein


Mass: 2730.295 Da / Num. of mol.: 4 / Fragment: residues 22-46 / Source method: obtained synthetically / Details: Solid-phase synthesis / References: UniProt: Q9YP62, UniProt: O70632*PLUS
#2: Chemical ChemComp-308 / (3S,5S,7S)-tricyclo[3.3.1.1~3,7~]decan-1-amine / Amantadine


Mass: 151.249 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N

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Experimental details

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Experiment

ExperimentMethod: SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 13C-13C Spin Diffusion
1222D 13C-13C Spin Diffusion
1332D 13C-13C Spin Diffusion
1412D 13C-15N HETCOR
1522D 13C-15N HETCOR
1632D 13C-15N HETCOR
1711D 2H-13C REDOR, single 13C pulse
1821D 2H-13C REDOR, single 13C pulse
1931D 2H-13C REDOR, single 13C pulse
11011D 2H-13C REDOR, single 13C pulse
11111D 2H-13C REDOR, single 2H pulse
11221D 2H-13C REDOR, single 2H pulse
11331D 2H-13C REDOR, single 2H pulse
11442H Static Quadrupolar Echo
11552H Static Quadrupolar Echo
11632H Static Quadrupolar Echo

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Sample preparation

Details
Solution-IDContentsSolvent system
15.8 mg [U-99% 13C; U-99% 15N] at S31, I32 and D44 M2-TM, 21.6 mg DMPC, 10 mM NaH2PO4, 10 mM Na2HPO4, 1 mM EDTA, 0.1 mM NaN3, .42 mg [U-2H] d15-1-aminoadamantane*HCl, 10mM pH 7.5 phosphate buffer, 50% hydration10mM pH 7.5 phosphate buffer, 50% hydration
25.0 mg [U-99% 13C; U-99% 15N] at S31, I32 and D44 M2-TM, 10.4 mg DMPC, 10 mM NaH2PO4, 10 mM Na2HPO4, 1 mM EDTA, 0.1 mM NaN3, 0.09 mg [U-2H] d15-1-aminoadamantane*HCl, 10mM pH 7.5 phosphate buffer, 50% hydration10mM pH 7.5 phosphate buffer, 50% hydration
35.2 mg [U-99% 13C; U-99% 15N] at L26, V27, A29 and G34 M2-TM, 19.9 mg DMPC, 10 mM NaH2PO4, 10 mM Na2HPO4, 1 mM EDTA, 0.1 mM NaN3, 0.38 mg [U-2H] d15-1-aminoadamantane*HCl, 10mM pH 7.5 phosphate buffer, 50% hydration10mM pH 7.5 phosphate buffer, 50% hydration
47 mg [U-99% 13C; U-99% 15N] at L26, V27, A29 and G34 M2-TM, 27 mg DMPC, 10 mM NaH2PO4, 10 mM Na2HPO4, 1 mM EDTA, 0.1 mM NaN3, 2.0 mg [U-2H] d15-1-aminoadamantane*HCl, 10mM pH 7.5 phosphate buffer, 50% hydration10mM pH 7.5 phosphate buffer, 50% hydration
57 mg [U-99% 13C; U-99% 15N] at L26, V27, A29 and G34 M2-TM, 27 mg DMPC, 10 mM NaH2PO4, 10 mM Na2HPO, 1 mM EDTA, 0.1 mM NaN3, 0.5 mg [U-2H] d15-1-aminoadamantane*HCl, 10mM pH 7.5 phosphate buffer, 50% hydration10mM pH 7.5 phosphate buffer, 50% hydration
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
5.8 mg/mLM2-TM-1[U-99% 13C; U-99% 15N] at S31, I32 and D441
21.6 mg/mLDMPC-21
10 mMNaH2PO4-31
10 mMNa2HPO4-41
1 mMEDTA-51
.1 mMNaN3-61
.42 mg/mLd15-1-aminoadamantane*HCl-7[U-2H]1
5.0 mg/mLM2-TM-8[U-99% 13C; U-99% 15N] at S31, I32 and D442
10.4 mg/mLDMPC-92
10 mMNaH2PO4-102
10 mMNa2HPO4-112
1 mMEDTA-122
0.1 mMNaN3-132
0.09 mg/mLd15-1-aminoadamantane*HCl-14[U-2H]2
5.2 mg/mLM2-TM-15[U-99% 13C; U-99% 15N] at L26, V27, A29 and G343
19.9 mg/mLDMPC-163
10 mMNaH2PO4-173
10 mMNa2HPO4-183
1 mMEDTA-193
.1 mMNaN3-203
0.38 mg/mLd15-1-aminoadamantane*HCl-21[U-2H]3
7 mg/mLM2-TM-22[U-99% 13C; U-99% 15N] at L26, V27, A29 and G344
27 mg/mLDMPC-234
10 mMNaH2PO4-244
10 mMNa2HPO4-254
1 mMEDTA-264
.1 mMNaN3-274
2.0 mg/mLd15-1-aminoadamantane*HCl-28[U-2H]4
7 mg/mLM2-TM-29[U-99% 13C; U-99% 15N] at L26, V27, A29 and G345
27 mg/mLDMPC-305
10 mMNaH2PO4-315
10 mMNa2HPO4-325
1 mMEDTA-335
.1 mMNaN3-345
0.5 mg/mLd15-1-aminoadamantane*HCl-35[U-2H]5
Sample conditionspH: 7.5 / Pressure: ambient / Temperature: 243 K

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NMR measurement

Radiation wavelengthRelative weight: 1
NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE4001
Bruker AvanceBrukerAVANCE6002

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin1.3Bruker Biospincollection
TopSpin1.3Bruker Biospinchemical shift assignment
TopSpin1.3Bruker Biospinprocessing
TopSpin1.3Bruker Biospindata analysis
TopSpin1.3Bruker Biospinpeak picking
X-PLOR_NIH, IN-HOUSE METHODSchwieters, Kuszewski, Tjandra and Clorerefinement
XwinNMRBruker Biospincollection
XwinNMRBruker Biospinchemical shift assignment
RefinementMethod: simulated annealing, Monte Carlo / Software ordinal: 1
Details: The temperature was initially set to 1,000,000K and decreased by 10% every 100 steps until a temperature of 25 K was reached. The constants (CI-CIV) were obtained through a trial-and-error ...Details: The temperature was initially set to 1,000,000K and decreased by 10% every 100 steps until a temperature of 25 K was reached. The constants (CI-CIV) were obtained through a trial-and-error process. Some side chain rotamers were changed to maximize agreement with the radial distances., An ensemble of models was obtained by selecting the top scoring model after one round of Monte Carlo/Simulated Annealing minimization and refining again with the inverse kinematics algorithm. The distance potential constant CIII was set to 50 kcal/mol-radians^2. Since the radial distance provided excellent restraints between the drug and M2, we were able to position the amantidine molecule near with S31 without the need for further minimization. THE POSITION OF THE AMANTADINE LIGAND (PDB CODE 308) IS IDENTICAL IN ALL OF THE MINIMIZED STRUCTURE SINCE IT IS THE AVERAGE LIGAND POSITION RELATIVE TO THE BACKBONE. WE HAVE MEASURED A 13C-2H DIPOLAR COUPLING FROM SEVERAL PEPTIDE BACKBONE AND SIDECHAIN CARBONS TO THE DEUTERONS ON THE 308 LIGAND. WE SIMULATED THE THE RADIUS OF THE M2 CHANNEL PORE FOR THE SITES WHERE WE OBSERVED 13C-2H DIPOLAR COUPLING, AND FOUND PORE RADII FOR THESE SITES THAT CORRESPONDED WITH THE MEASURED 13C-2H DIPOLAR COUPLING AT THAT SITE. THUS, IN THE FINAL STRUCTURE MINIMIZATION, WE USED THE PORE RADII AS A CONSTRAINT RATHER THAN PEPTIDE-DRUG DISTANCES SINCE THE LIGAND IS ROTATING IN THE CHANNEL.
NMR constraintsProtein chi angle constraints total count: 8
NMR representativeSelection criteria: fewest violations
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 24 / Conformers submitted total number: 17

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