Structure and dynamics of the GABA binding pocket: A narrowing cleft that constricts during activation
Structure and dynamics of the GABA binding pocket: A narrowing cleft that constricts during activation
复制标题
DOI：
10.1523/jneurosci.21-01-00067.2001
复制
发表时间：
2001-01-01
影响因子：
5.3
通讯作者：
Czajkowski, C
中科院分区：
文献类型：
--
作者：
Wagner, DA;Czajkowski, C
光亲和标记和诱变研究已经确定了几个氨基酸，可能有助于配体门控离子通道的配体结合域。然而，这些类型的研究，只产生一个一维的，静态的结合位点结构的描述。在这项研究中，我们使用取代的半胱氨酸可及性方法，不仅要确定结合口袋残基，但也引出有关结合位点的动力学和结构的信息。将GABAA受体（β（2）V199至β（2）S209）的假定环C配体结合域周围的残基单独突变为半胱氨酸，并将突变亚基与野生型α（1）亚基在非洲爪蟾卵母细胞中共表达。甲硫代磺酸N-生物素基氨基乙酯（MTSEA-生物素）与在位置G203、S204、Y205、P206、R207和S209处引入的半胱氨酸反应。这种可及性模式与α-螺旋或β-链都不一致。相反，G203-S209似乎形成了一个水可及的延伸螺旋，而V199-T202似乎埋在蛋白质或膜中。GABA或竞争性拮抗剂SR-95531的共同应用显著减慢了在位置S204、Y205、R207和S209处引入的半胱氨酸的MTSEA-生物素修饰，表明这些残基排列并面向GABA结合口袋。MTSEA-生物素反应速率揭示了从G203-S209的陡峭的可及性梯度，并表明结合口袋是一个深的狭窄裂缝。受体的戊巴比妥活化显著减慢了S204、R207和S209处半胱氨酸的MTSEA-生物素修饰，表明结合位点可能在门控期间收缩。
Photo-affinity labeling and mutagenesis studies have identified several amino acids that may contribute to the ligand binding domains of ligand-gated ion channels. These types of studies, however, only generate a one-dimensional, static description of binding site structure. In this study, we used the substituted cysteine accessibility method not only to identify binding pocket residues but also to elicit information about binding site dynamics and structure. Residues surrounding the putative loop C ligand binding domain of the GABAA receptor (beta (2)V199 to beta (2)S209) were individually mutated to cysteine, and the mutant subunits were coexpressed with wild-type alpha (1) subunits in Xenopus oocytes. N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) reacts with cysteines introduced at positions G203, S204, Y205, P206, R207, and S209. This accessibility pattern is not consistent with either an alpha -helix or beta -strand. Instead, G203-S209 seems to form a water-accessible extended coil, whereas V199-T202 appears to buried in the protein or membrane. Coapplication of either GABA or the competitive antagonist SR-95531 significantly slows MTSEA-biotin modification of cysteines introduced at positions S204, Y205, R207, and S209, demonstrating that these residues line and face into the GABA binding pocket. MTSEA-biotin reaction rates reveal a steep accessibility gradient from G203-S209 and suggests that the binding pocket is a deep narrowing cleft. Pentobarbital activation of the receptor significantly slows MTSEA-biotin modification of cysteines at S204, R207, and S209, suggesting that the binding site may constrict during gating.