3D Solution Structure of ?

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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

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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,
  Parkinsons Tourettes

Tsigelny, et al, UCSD
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?-Conotoxin MII

• Antagonist specific to ?3?2 nAChR
• Only known inhibitor specific to a single nAChR
  species
• Structure previously unknown
• Some homologous structures known

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?-Conotoxins
Toxin Sequence Species Specificty
MII GCCSNPVCHLEHSNLC C. magus neuronal
PnIA GCCSLPPCAANNPDYC C. pennaceus neuronal
PnIB GCCSLPPCALSNPDYC C. pennaceus neuronal
EpI GCCSDPRCNMNNPDYC C. episcopatus neuronal
EI RDOCCYHPTCNMSNPQIC C. ermineus muscle

MI GRCC HPACGKNYS C C. magus muscle
GI ECC NPACGRHYS C C. geographus muscle
GIA ECC NPACGRHYS CGK C. geographus muscle
GII ECC HPACGKHFS C C. geographus muscle
SI ICC NPACGPKYS C C. striatus muscle
SIA YCC HPACGKNFD C C. striatus muscle
SII GCCC NPACGPNYG CGTSCS C. striatus muscle

ImI GCCSDPRCAWR C C. imperialis neuronal
Structure solved
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Structure Determination

• 3 NMR solutions
• 10 D20 / 90 H20
• 30 Deuterated Acetonitrile (CD3CN)
• 30 Trifluoroethanol (TFE)
• NOESY, TOCSY, DQF-COSY
• One proline residue

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Figure 2 - Fingerprint region of NOESY
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Figure 3 - 2ndary Chemical Shift
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TFE
H20
CD3CN
Figure 4 - NOE peak summaries
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Figure 5 - Circular Dichroism
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Figure 6 - 3D Structure
20 Final Structures
RMSD from experimental restraints RMSD from experimental restraints
Interproton distances (Å) (376) 0.045 0.001
Dihedral angles (deg) (12) 1.01 0.01
RMSD from idealized geometry RMSD from idealized geometry
Bonds (Å) 0.012 0.01
Angles (deg) 3.04 0.01
Impropers (deg) 0.39 0.01
Energies (kcal mol-1) 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) Pairwise RMSD for backbone atoms (N, C?, C) (Å)
1-16 0.07 0.06
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Amphipathic helix
Solvent Exposed
Buried
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Figure 7
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Figure 8 - Comparison to PnIb
RMSD 0.94Å
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Conclusions

• Single main conformation
• Hydrophobic environment tightens secondary
  structure
• Can form hypotheses about binding to nAChR