Role of Asn2 and Glu7 residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

• Published in: Biopolymers Vol. 86; no. 5-6; pp. 447 - 462
• Main Authors: Le-Nguyen, Dung, Chiche, Laurent, Hoh, François, Martin-Eauclaire, Marie France, Dumas, Christian, Nishi, Yoshinori, Kobayashi, Yuji, Aumelas, André
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05.08.2007
• Subjects: apamin; carboxyl group ionization; disulfide bond; NMR; proline isomerization; structure; X-ray
• ISSN: 0006-3525; 1097-0282
• DOI: 10.1002/bip.20755

The X‐ray structure of [N‐acetyl]‐apamin has been solved at 0.95 Å resolution. It consists of an 1‐7 N‐terminal loop stabilized by an Asn‐β‐turn motif (2–5 residues) and a helical structure spanning the 9–18 residues tightly linked together by two disulfide bonds. However, neither this accurate X‐ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn2 and Glu7 amide signals upon Glu7 carboxylic group ionization. Thus, apamin and its [N‐acetyl], [Glu7Gln], [Glu7Asp], and [Asn2Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu7 carboxylate group is responsible for the large downfield shifts of the Asn2 and Glu7 amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn2 and Glu7 amide protons as well as the N‐terminal α‐amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn2Abu] analogue, revealed an essential role of Asn2 in the β‐turn stability and the cis/trans isomerization of the Ala5‐Pro6 amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu7 carboxyl group. However, neither destabilization of the β‐turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn2 and Glu7 residues appeared essential for the N‐terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 447–462, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com