Ahn Minkoo, Hagan Christine L, Bernardo-Gancedo Ana, De Genst Erwin, Newby Francisco N, Christodoulou John, Dhulesia Anne, Dumoulin Mireille, Robinson Carol V, Dobson Christopher M, Kumita Janet R
Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
Institute of Structural and Molecular Biology, University College London and Birkbeck College, London, United Kingdom.
Biophys J. 2016 Dec 6;111(11):2358-2367. doi: 10.1016/j.bpj.2016.10.028.
The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.
人溶菌酶转化为淀粉样纤维与一种罕见但致命的非神经性系统性淀粉样变性的遗传形式相关。大量聚集蛋白的积累被认为是由疾病相关溶菌酶变体的瞬时中间物种的形成引发的，这主要是由于在生理相关条件下全局协同性的丧失。有趣的是，所有五个天然存在的、具有淀粉样变性的单点突变都位于溶菌酶的β结构域，该区域在瞬时中间物种形成过程中主要处于未折叠状态。鉴于α结构域缺乏已知的天然存在的、具有淀粉样变性的单点突变，我们选择了三个特定突变来研究位置对天然态动力学的影响，这是通过核磁共振光谱和质谱分析的氢-氘（HD）交换实验来进行研究的。我们将一个使α结构域不稳定的突变（I23A）的影响与特征明确的I59Tβ结构域变体的影响进行了比较。我们还研究了一个在结构域界面处对天然态稳定性影响较小的突变（I56V）的影响，并将其与C螺旋内具有相似稳定性的变体（I89V）的影响进行了比较。我们发现，当变体具有相似的降低的天然态稳定性时，突变的位置（I23A与I59T）对天然态动力学至关重要，α结构域突变在生理相关条件下形成瞬时中间物种的能力明显较低。有趣的是，与含有α结构域突变的变体相比，界面处的突变（I56V）在高温下促进瞬时中间物种形成方面具有更大的影响，尽管这种突变仅导致溶菌酶天然态稳定性的微小变化。这些发现表明，在人溶菌酶形成与致病性淀粉样蛋白形成相关的易于聚集的瞬时中间物种的倾向背景下，特定突变的位置是决定其天然态动力学性质的一个重要因素。
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