
ABSTRACT Antiamoebin I is a membrane-active peptaibol produced by fungi of the species Emericellopsis which is capable of forming ion channels in membranes. Previous structure determinations by x-ray crystallography have shown the molecule is mostly helical, with a deep bend in the center of the polypeptide, and that the backbone structure is independent of the solvent used for crystallization. In this study, the solution structure of antiamoebin was determined by NMR spectroscopy in methanol, a solvent from which one of the crystal structures was determined. The ensemble of structures produced exhibit a right-handed helical C terminus and a left-handed helical conformation toward the N-terminus, in contrast to the completely right-handed helices found in the crystal structures. The NMR results also suggest that a "hinge" region exists, which gives flexibility to the polypeptide in the central region, and which could have functional implications for the membrane insertion process. A model for the membrane insertion and assembly process is proposed based on the antiamoebin solution and crystal structures, and is contrasted with the assembly and insertion mechanism proposed for other ion channel-forming polypeptides.
INTRODUCTION
The antiamoebins are a family of peptides produced by fungi of the species Emericellopsis that have antibiotic properties against the organism responsible for amoebic dysentery (Thirumalachur, 1968). Up to 16 microheterogeneous members of the family have been identified, termed antiamoebin I, II, etc., (Pandey et al., 1977; Pandey et al., 1978; Jaworski and Brukner, 2000). Antiamoebin I has the primary structure:
The peptaibols, including antiamoebin, are considered good models for studying ion channels, as they form stable, well characterized channels and are relatively abundant (Wallace, 2000). They also fall in the intermediate size range where both NMR spectroscopic and x-ray crystallographic techniques can be used to investigate three-dimensional structure.
High-resolution crystal structures of antiamoebin I published by Snook et al. (1998) and Karle et al. (1998) in different environments (methanol and octanol, respectively) and in different crystallographic space groups, show that the molecule has essentially the same backbone conformation in both environments. The polypeptide backbone atoms of the two crystal structures have a positional root mean square deviation (RMSD) of 0.24 Angstrom (Wallace et al., 2000). The structures are mostly helical, with an N-terminal a-helix (residues 1-9) followed by a small segment of 3^sub 10^-helix (residues 10-12) joined to an overlapping series of beta-turns at the C-terminus (residues 12-16), which is sometimes described as a beta-bend-ribbon (Di Blasio et al., 1992). The molecule has a deep bend centered on residue Hyp^sup 11^, forming an angle of 560 between the two helix axes in the methanol crystal form (Snook et al., 1998).
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[Author Affiliation]
T. P. Galbraith,* R. Harris,^ ^^ P. C. Driscoll,^^ (sec) and B. A. Wallace*
*School of Crystallography, Birkbeck College, University of London, London WC1 E 7HX, UK; ^Bloomsbury Centre for Structural Biology, Birkbeck College and University College, University of London, London WC1 E, UK; ^^Department of Biochemistry and Molecular Biology, University College, University of London, London WC1 E 613T, UK; and (sec)Ludwig Institute of Cancer Research, UCL School of Medicine Branch, London WlP 813T, UK
[Author Affiliation]
Submitted June 17, 2002, and accepted for publication August 05, 2002.
R. Harris's present address is The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037 USA.
Address reprint requests to B. A. Wallace. Tel.: (+44) 20 7631 6857; Fax: (+44) 20 7631 6803; E-mail: b.wallace@mail.cryst.bbk.ac.uk.