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