3D Solution Structure of –Conotoxin MII by NMR
Spectroscopy: Effects of Solution Environment on Helicity
Hill, Oomen, Miranda, Bingham, Alewood, and Craik
Biochemistry 1998, 37, 15621-15630
Presented by Charles Vaske
Conus Magus
Baldomero Olivera, University of Utah
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Nicotinic Acetlycholine Receptor
• Pentameric gated ion channel
• Present in muscle and neuronal tissue
• Diverse neuronal species• Implicated in
schizophrenia, addiciton, AD, Parkinson’s Tourette’s Tsigelny, et al, UCSD
-Conotoxin MII
• Antagonist specific to 32 nAChR• Only known inhibitor specific to a single
nAChR species• Structure previously unknown• Some homologous structures known
-ConotoxinsToxin Sequence Species Specificty
MII GCCSNPVCHLEHSNLC* C. magus neuronalPnIA ** GCCSLPPCAANNPDYC* C. pennaceus neuronalPnIB ** GCCSLPPCALSNPDYC* C. pennaceus neuronalEpI ** GCCSDPRCNMNNPDYC* C. episcopatus neuronalEI RDOCCYHPTCNMSNPQIC* C. ermineus muscle
MI GRCC HPACGKNYS C* C. magus muscleGI ** ECC NPACGRHYS C* C. geographus muscleGIA ECC NPACGRHYS CGK* C. geographus muscleGII ECC HPACGKHFS C* C. geographus muscleSI ICC NPACGPKYS C* C. striatus muscleSIA YCC HPACGKNFD C* C. striatus muscleSII GCCC NPACGPNYG CGTSCS C. striatus muscle
ImI GCCSDPRCAWR C* C. imperialis neuronal
** Structure solved
Structure Determination
3 NMR solutions:– 10% D20 / 90% H20
– 30% Deuterated Acetonitrile (CD3CN)– 30% Trifluoroethanol (TFE)
NOESY, TOCSY, DQF-COSYOne proline residue
Figure 2 - Fingerprint region of NOESY
Figure 3 - 2ndary Chemical Shift
Figure 4 - NOE peak summaries
H20
CD
3CN
TFE
Figure 5 - Circular Dichroism
Figure 6 - 3D Structure
RMSD from experimental restraintsInterproton distances (Å) (376) 0.045 ± 0.001
Dihedral angles (deg) (12) 1.01 ± 0.01
RMSD from idealized geometryBonds (Å) 0.012 ± 0.01
Angles (deg) 3.04 ± 0.01
Impropers (deg) 0.39 ± 0.01
Energies (kcal mol-1)Etotal 32.5 ± 0.2
ENOE 22.2 ± 0.2
Ecdih 0.74 ± 0.01
EL-J -59.1 ± 0.5
Ebond + Eangle + Eimproper 61.3 ± 0.2
Pairwise RMSD for backbone atoms (N, C, C) (Å)
1-16 0.07 ± 0.06
20 Final Structures
Amphipathic helix
Solvent Exposed
Buried
Figure 7
Figure 8 - Comparison to PnIb
MII GCCSNPVCHLEHSNLC*PnIB GCCSLPPCALSNPDYC*
RMSD 0.94Å
Conclusions
• Single main conformation• Hydrophobic environment tightens
secondary structure• Can form hypotheses about binding to
nAChR