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Role of Asn 2 and Glu 7 residues in the oxidative folding and on the conformation of the N ‐terminal loop of apamin
Author(s) -
LeNguyen Dung,
Chiche Laurent,
Hoh François,
MartinEauclaire Marie France,
Dumas Christian,
Nishi Yoshinori,
Kobayashi Yuji,
Aumelas André
Publication year - 2007
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.20755
Subject(s) - chemistry , isomerization , carboxylate , amide , stereochemistry , apamin , diketopiperazines , peptide bond , folding (dsp implementation) , crystallography , peptide , biochemistry , organic chemistry , catalysis , electrical engineering , calcium , engineering
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 Asn 2 and Glu 7 amide signals upon Glu 7 carboxylic group ionization. Thus, apamin and its [ N ‐acetyl], [Glu 7 Gln], [Glu 7 Asp], and [Asn 2 Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu 7 carboxylate group is responsible for the large downfield shifts of the Asn 2 and Glu 7 amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn 2 and Glu 7 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 [Asn 2 Abu] analogue, revealed an essential role of Asn 2 in the β‐turn stability and the cis/trans isomerization of the Ala 5 ‐Pro 6 amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu 7 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 Asn 2 and Glu 7 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