Blandl T, Zajicek J, Prorok M, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
J Biol Chem. 2001 Mar 9;276(10):7391-6. doi: 10.1074/jbc.M006648200. Epub 2000 Nov 28.
Conantokin-R (con-R), a gamma-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, [(3)H]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 Leu(12)-Pro(27) and Met(8)-Pro(27) respectively. A second set of analogs featured single Ala substitutions in the fully active con-R[1-17] fragment. The replacement of Met(8) and Leu(12) 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 alpha-helix content were observed in most analogs. However, analogs with replacement of Gla(11) and Gla(15), 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 Gla(11) and Gla(15). Additional interactions were indicated by the reduced alpha-helix stability in the Ala analogs of Gla(4), Lys(7), and Arg(14).
芋螺毒素Conantokin-R（Con-R）是一种含27个氨基酸残基的γ-羧基谷氨酸肽，是N-甲基-D-天冬氨酸（NMDA）亚型谷氨酸受体的天然肽抑制剂。通过抑制精胺增强的NMDA受体特异性通道阻滞剂[³H]MK-801与大鼠脑膜的结合来测定Con-R的NMDA受体拮抗剂活性，进而生成Con-R的合成类似物，以评估该肽的各个结构元件在其活性中的重要性。对27个氨基酸残基的肽进行逐步C末端截短，揭示了严重活性丧失的阶段。这些阶段出现在Con-R[1-11]和Con-R[1-7]，分别对应于Leu(12)-Pro(27)和Met(8)-Pro(27)的缺失。第二组类似物的特点是在完全活性的Con-R[1-17]片段中进行单个丙氨酸取代。用丙氨酸取代Met(8)和Leu(12)分别导致抑制剂效力下降约20倍和55倍。除了这两个残基外，单个丙氨酸取代导致活性大幅丧失（从11倍到>1000倍）的唯一其他位置是前五个N末端氨基酸的位置。基于上述发现，Con-R的结合表位定位于螺旋的N末端转角以及沿随后两个转角的一侧的其他残基。侧链在活性中的这种贡献模式与该肽家族的另一个成员芋螺毒素Conantokin-T的结果非常相似。还使用圆二色光谱法评估了Con-R变体的二级结构和金属离子结合特性。在大多数类似物中观察到二价阳离子依赖性的α-螺旋含量增加。然而，用丙氨酸取代Gla(11)和Gla(15)的类似物以及短于15个残基的截短片段失去了被金属离子稳定的能力。这些结果证实了主要二价阳离子结合位点位于Gla(11)和Gla(15)。Gla(4)、Lys(7)和Arg(14)的丙氨酸类似物中α-螺旋稳定性的降低表明存在其他相互作用。
