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首页> 外文期刊>Journal of Molecular Biology >Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies.
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Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies.

机译：静电相互作用介导obscurin与小锚蛋白的结合1：生化和分子模型研究。

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摘要

Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last alpha-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation.
机译：小锚蛋白1（sAnk1;也称为Ank1.5）是骨骼肌和心肌细胞中肌浆网（SR）的必需蛋白，被认为与大模块蛋白obscurin的C端结合。围绕收缩装置。体外使用融合蛋白，结合定点诱变和表面等离振子共振测量，我们先前表明sAnk1上的obscurin结合位点部分由六个赖氨酸和精氨酸残基组成。在这里，我们显示在obscurin的sAnk1的高亲和力结合位点（残基6316和6345之间）中的四个带电残基（由三个谷氨酸和赖氨酸组成）是必需的，但不足以使obscurin上的该位点与sAnk1结合具有高亲和力。我们还确定了sAnk1中的特定互补突变，该突变可以部分或完全补偿由obscurin的电荷转换突变引起的结合变化。我们使用分子建模来开发结合到sAnk1的obscurin残基6322-6339的结构模型。这些模型基于布朗和分子动力学模拟的结合，预测sAnk1上对obscurin的结合位点被组织为两个锚蛋白样重复序列，最后一个α-螺旋段与附近的螺旋分子成一定角度，从而允许赖氨酸6338暗素的形成与sAnk1的天冬氨酸111形成离子相互作用。通过双突变周期实验验证了这一预测。我们的结果与一个模型一致，在该模型中，obscurin和sAnk1上特定的侧链对之间的静电相互作用促进结合和复合物形成。

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