Structure–function relationships of the NMDA receptor antagonist peptide, conantokin‐R

FEBS Letters

Warder, Scott E¹  Prorok, Mary¹  Blandl, Tamas¹  Castellino, Francis J¹ 

[1] Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA

关键词: Conantokin-R;    N-Methyl-D-aspartate receptor;    Peptide conformation;    Cation binding;    γ-Carboxyglutamate;    con-G;    conantokin-G;    con-R;    conantokin-R;    con-T;    conantokin-T;    Gla;    γ-carboxyglutamic acid;    NMDA;    N-methyl-D-aspartate;    DE-MALDI-TOF;    delayed extraction-matrix assisted laser desorption ionization-time of flight mass spectrometry;    HPLC;    high performance liquid chromatography;    CD;    circular dichroism;    ITC;    isothermal titration calorimetry;    NOE;    nuclear Overhauser effect;    NOESY;    nuclear Overhauser effect spectroscopy;    TOCSY;    total correlation spectroscopy;   

DOI  :  10.1016/S0014-5793(00)01309-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Conantokin-R (con-R) is a γ-carboxyglutamate-containing 27-residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con-R was determined through an assay involving inhibition of the spermine-enhanced binding of the NMDA receptor channel blocker, [³H]MK-801, to rat brain membranes, and yielded an IC₅₀ of 93 nM. This value represents a 2–5 times better potency than con-G or con-T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal-free form of con-R is indicative of a low α-helical content. There is an increase in α-helicity upon the addition of divalent cations, such as Ca²⁺, Mg²⁺, or Zn²⁺. Isothermal titration calorimetry experiments showed one detectable Mg²⁺ binding site with a K _d of 6.5 μM, and two binding sites for Zn²⁺, with K _d values of 150 nM and 170 μM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional ¹H-NMR. Analyses of the α-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an α-helical conformation for residues 1–19. Synthetic con-R-derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy-terminus of the wild-type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The α-helical secondary structures of the two truncation peptides were more stable than full-length con-R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability.

【 授权许可】

Unknown