Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin.
The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-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 alpha-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 beta-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 beta-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.
Le-Nguyen
Dung
D
Centre de Pharmacologie et Biotechnologie pour la Santé-CNRS FRE 3009, Faculté de Pharmacie, Montpellier, France.
Chiche
Laurent
L
Hoh
François
F
Martin-Eauclaire
Marie France
MF
Dumas
Christian
C
Nishi
Yoshinori
Y
Kobayashi
Yuji
Y
Aumelas
André
A
D000595:Amino Acid Sequence; D000818:Animals; D001030:Apamin / Q000031:analogs & derivatives / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry; D018360:Crystallography, X-Ray; D019281:Dimerization; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D012996:Solutions; D013816:Thermodynamics
D012996:Solutions; D001030:Apamin; D018698:Glutamic Acid; D001216:Asparagine; D011392:Proline