Hundt Nikolas, Preller Matthias, Swolski Olga, Ang Angella M, Mannherz Hans G, Manstein Dietmar J, Müller Mirco
Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
FEBS J. 2014 Dec;281(23):5279-91. doi: 10.1111/febs.13068. Epub 2014 Oct 13.
Cytoplasmic β-actin supports fundamental cellular processes in healthy and diseased cells including cell adhesion, migration, cytokinesis and maintenance of cell polarity. Mutations in ACTB, the gene encoding cytoplasmic β-actin, lead to severe disorders with a broad range of symptoms. The two dominant heterozygous gain-of-function β-actin mutations p.R183W and p.E364K were identified in patients with developmental malformations, deafness and juvenile-onset dystonia (p.R183W) and neutrophil dysfunction (p.E364K). Here, we report the recombinant production and functional characterization of the two mutant proteins. Arg183 is located near the nucleotide-binding pocket of actin. Our results from biochemical studies and molecular dynamics simulations show that replacement by a tryptophan residue at position 183 establishes an unusual stacking interaction with Tyr69 that perturbs nucleotide release from actin monomers and polymerization behavior by inducing a closed state conformation. The replacement of Glu364 by a lysine residue appears to act as an allosteric trigger event leading to the preferred formation of the closed state. Thus, our approach indicates that both mutations affect interdomain mobility and nucleotide interactions as a basis for the formation of disease phenotypes in patients.
细胞质β-肌动蛋白在健康细胞和患病细胞中支持基本的细胞过程，包括细胞黏附、迁移、胞质分裂和细胞极性维持。编码细胞质β-肌动蛋白的基因ACTB发生突变会导致出现具有广泛症状的严重疾病。在患有发育畸形、耳聋和青少年型肌张力障碍（p.R183W）以及中性粒细胞功能障碍（p.E364K）的患者中鉴定出了两种主要的杂合功能获得性β-肌动蛋白突变p.R183W和p.E364K。在此，我们报告了这两种突变蛋白的重组生产和功能表征。精氨酸183位于肌动蛋白的核苷酸结合口袋附近。我们的生化研究和分子动力学模拟结果表明，183位的精氨酸被色氨酸残基取代后，与酪氨酸69形成了异常的堆积相互作用，通过诱导闭合状态构象干扰了肌动蛋白单体的核苷酸释放和聚合行为。谷氨酸364被赖氨酸残基取代似乎起到了变构触发事件的作用，导致优先形成闭合状态。因此，我们的方法表明，这两种突变都会影响结构域间的流动性和核苷酸相互作用，这是患者疾病表型形成的基础。
