Fine mapping of the α-actinin binding site within cysteine-rich protein
Fine mapping of the α-actinin binding site within cysteine-rich protein
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DOI：
10.1042/bj3500269
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发表时间：
2000-08
影响因子：
4.1
通讯作者：
B. D. Harper;M. Beckerle;P. Pomiès
中科院分区：
文献类型：
--
作者：
B. D. Harper;M. Beckerle;P. Pomiès
富含半胱氨酸的蛋白 (CRP) 是高度保守的 LIM（源自三种基因产物 lin-11、isl-1 和 mec-3 的缩写）结构域蛋白家族，与肌肉分化有关。迄今为止，所有 CRP 家族成员均已被证明与丝状肌动蛋白交联剂 α-辅肌动蛋白相互作用。这种相互作用所需的 CRP 区域先前已被广泛定位到该分子的 N 末端一半。在这里，我们报道了 CRP 的 α-辅肌动蛋白结合区域，我们通过结合印迹叠加和蛋白质免疫印迹技术绘制了图谱，该区域仅限于蛋白质 N 末端富含甘氨酸重复序列内的 18 个残基序列。结合区域的定点诱变分析揭示了单个赖氨酸残基（人 CRP1 中的第 65 位赖氨酸）的至关重要性。与野生型 CRP 相比，该位点的改变导致 α-辅肌动蛋白结合减少 10 倍。关键的赖氨酸残基位于短的α螺旋内，这增加了诱变诱导的α-辅肌动蛋白结合能力的改变可能归因于关键结构元件的破坏的可能性。
The cysteine-rich proteins (CRPs) are a family of highly conserved LIM (an acronym derived from the three gene products lin-11, isl-1 and mec-3) domain proteins that have been implicated in muscle differentiation. All CRP family members characterized so far have been shown to interact with the filamentous actin cross-linker alpha-actinin. The region of CRP required for this interaction has previously been broadly mapped to the molecule's N-terminal half. Here we report that the alpha-actinin-binding region of CRP, which we have mapped by using a combination of blot overlay and Western immunoblot techniques, is confined to an 18-residue sequence occurring within the protein's N-terminal glycine-rich repeat. A site-directed mutagenesis analysis of the binding region has revealed the critical importance of a single lysine residue (lysine 65 in human CRP1). Alterations at this site lead to a 10-fold decrease in alpha-actinin binding in comparison with wild-type CRP. The critical lysine residue localizes within a short alpha-helix, raising the possibility that mutagenesis-induced alterations in alpha-actinin-binding capacity might be attributed to the disruption of a key structural element.