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J. Biol. Chem., Vol. 276, Issue 10, 7391-7396, March 9, 2001

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Sequence Requirements for the N-Methyl-D-aspartate Receptor Antagonist Activity of Conantokin-R^*

Tamas Blandl, Jaroslav Zajicek, Mary Prorok, and Francis J. Castellino

From the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

Conantokin-R (con-R), a -carboxyglutamate-containing 27-residue peptide, is a natural peptide inhibitor of the N-methyl-D-aspartate (NMDA) subtype glutamate receptor. Synthetic analogs of con-R were generated to evaluate the importance of the individual structural elements of this peptide in its NMDA receptor antagonist activity, measured by inhibition of the spermine-enhanced binding of the NMDA receptor-specific channel blocker, [³H]MK-801, to rat brain membranes. Progressive C-terminal truncations of the 27-residue peptide revealed stages of severe activity loss. These occurred at con-R[1-11] and con-R[1-7], corresponding to the deletions of Leu¹²-Pro²⁷ and Met⁸-Pro²⁷ respectively. A second set of analogs featured single Ala substitutions in the fully active con-R[1-17] fragment. The replacement of Met⁸ and Leu¹² by Ala resulted in approximate 20- and 55-fold decreases of inhibitor potency, respectively. In addition to these two residues, the only other positions where a single Ala substitution led to substantial losses (from 11-fold to >1000-fold) of activity were those of the first five N-terminal amino acids. Based on the above findings, the binding epitope of con-R was localized to the N-terminal turn of the helix and other residues on one face along two subsequent turns. This contribution pattern of the side chains in activity closely resembles the results obtained with another member of this peptide family, conantokin-T. The secondary structure and metal ion binding properties of the con-R variants were also evaluated using circular dichroism spectroscopy. Divalent cation-dependent increases of -helix content were observed in most analogs. However, analogs with replacement of Gla¹¹ and Gla¹⁵, as well as truncation fragments shorter than 15 residues, lost the ability to be stabilized by metal ions. These results confirmed the location of the primary divalent cation binding locus at Gla¹¹ and Gla¹⁵. Additional interactions were indicated by the reduced -helix stability in the Ala analogs of Gla⁴, Lys⁷, and Arg¹⁴.

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