Helix-helix interactions between homo- and heterodimeric γ-carboxyglutamate-containing conantokin peptides and their derivatives
Helix-helix interactions between homo- and heterodimeric γ-carboxyglutamate-containing conantokin peptides and their derivatives
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DOI：
10.1074/jbc.m609087200
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发表时间：
2007-04-27
影响因子：
4.8
通讯作者：
Prorok, Mary
中科院分区：
文献类型：
--
作者：
Dai, Qiuyun;Sheng, Zhenyu;Prorok, Mary
圆锥角蛋白是天然存在的富含 γ-羧基谷氨酸 (Gla) 的小肽家族，可特异性拮抗离子型谷氨酸受体的 N-甲基-D-天冬氨酸 (NMDA) 亚型。该家族的一个成员 conantokin-G (con-G) 经过 Ca2+ 介导的自组装形成反向平行螺旋二聚体。该复合物中的亚基相互作用取决于单体内间隔 i、i + 4、i + 7、i + 11 间隔的 Gla 残基的分子间 Ca2+ 桥接。在这里，我们进一步探讨了控制这种螺旋-螺旋相互作用的分子决定因素。合成了选定的变体以评估非 Gla 残基对圆锥豆蛋白自缔合的贡献。 Con-G 二聚化被证明是放热的并伴随着正热容变化。使用 conantokin-R (con-R) 的位置 Gla 变体，非二聚化 conantokin，i、i + 4、i + 7、i + 11 Gla 间距单独显示不足以进行自组装。建立了 con-G 和 con-T（K7 γ）这两种具有最佳 Gla 间距的肽的 Ca2+ 依赖性反平行异二聚化。最后，研究了共价约束的 con-G 二肽对表达 NMDA 受体 NR1a、NR1b、NR2A 和 NR2B 亚基组合的 HEK293 细胞中 NMDA 诱发电流的影响。反平行二肽的独特之处在于其增强 NR1a/2A 受体电流的能力，并且与单体 con-G 一样，对 NR1a/2B 和 NR1b/2B 组合具有抑制作用。相比之下，平行物种在所有测试的亚基组合中完全不活跃。这些结果表明，在生理Ca2+浓度下，con-G二聚体的平衡水平很可能以反平行方向存在，并对NMDA受体活性产生不同于单体的影响。
The conantokins are a family of small, naturally occurring gamma-carboxyglutamate ( Gla)-rich peptides that specifically antagonize the N-methyl-D-aspartate ( NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G ( con-G), undergoes Ca2+-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca2+ bridging of Gla residues spaced at i, i + 4, i + 7, i + 11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R ( con-R), a non-dimerizing conantokin, i, i + 4, i + 7, i + 11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca2+-dependent antiparallel heterodimerization of con-G and con-T( K7 gamma), two peptides that harbor optimal Gla spacing, was established. Last, the effects of covalently constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca2+ concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.