Solution Conformation of αA-conotoxin EIVA, a Potent Neuromuscular Nicotinic Acetylcholine Receptor Antagonist from Conus ermineus

Seung-Wook Chi,Kyu-Hwan Park,Jae-Eun Suk,Baldomero M Olivera,J.Michael Mcintosh,Kyou-Hoon Han

doi:10.1074/jbc.m303342200

Abstract

We report the solution three-dimensional structure of an alphaA-conotoxin EIVA determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The alphaA-conotoxin EIVA consists of 30 amino acids representing the largest peptide among the alpha/alphaA-family conotoxins discovered so far and targets the neuromuscular nicotinic acetylcholine receptor with high affinity. alphaA-Conotoxin EIVA consists of three distinct structural domains. The first domain is mainly composed of the Cys3-Cys11-disulfide loop and is structurally ill-defined with a large backbone root mean square deviation of 1.91 A. The second domain formed by residues His12-Hyp21 is extremely well defined with a backbone root mean square deviation of 0.52 A, thus forming a sturdy stem for the entire molecule. The third C-terminal domain formed by residues Hyp22-Gly29 shows an intermediate structural order having a backbone root mean square deviation of 1.04 A. A structurally ill-defined N-terminal first loop domain connected to a rigid central molecular stem seems to be the general structural feature of the alphaA-conotoxin subfamily. A detailed structural comparison between alphaA-conotoxin EIVA and alphaA-conotoxin PIVA suggests that the higher receptor affinity of alphaA-conotoxin EIVA than alphaA-conotoxin PIVA might originate from different steric disposition and charge distribution in the second loop "handle" motif.

Highlights

We report the solution three-dimensional structure of an ␣A-conotoxin EIVA determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics
The ␣A-conotoxin EIVA consists of 30 amino acids representing the largest peptide among the ␣/␣Afamily conotoxins discovered so far and targets the neuromuscular nicotinic acetylcholine receptor with high affinity. ␣A-Conotoxin EIVA consists of three distinct structural domains
A detailed structural comparison between ␣A-conotoxin EIVA and ␣A-conotoxin PIVA suggests that the higher receptor affinity of ␣A-conotoxin EIVA than ␣A-conotoxin PIVA might originate from different steric disposition and charge distribution in the second loop “handle” motif

Summary

EXPERIMENTAL PROCEDURES

Peptide Preparation—␣A-Conotoxin EIVA, originally purified from the venom of C. ermineus, was synthesized and purified as described previously [5]. Disulfide-bonding patterns were assigned by mass spectrometry as well as by NMR. When ␣A-conotoxin EIVA was analyzed by liquid-secondary ionization mass spectrometry, a monoisotopic mass of 3095.2 was obtained [5], suggesting the presence of three disulfide bonds. The formation of three specific disulfide bonds in ␣A-conotoxin EIVA was unambiguously determined by using a well established NMR protocol that relies on the fact that NOEs involving. Structure Calculations—Interproton distance restraints used for computation of structures were derived primarily from the NOESY spectrum recorded with a mixing time of 200 ms obtained at 15 °C. The FELIX program in the NMR Refine module of Biosym 95.0 software (Molecular Simulations Inc., San Diego, CA) was used for quantification of NOE volumes and for converting them into interproton distance restraints. The second step of structure calculation was refinement of the DGII-generated structures

RESULTS

DISCUSSION

Ramachandran plot statistics