Stable calcium-free myocilin olfactomedin domain variants reveal challenges in differentiating between benign and glaucoma-causing mutations
Stable calcium-free myocilin olfactomedin domain variants reveal challenges in differentiating between benign and glaucoma-causing mutations
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DOI：
10.1074/jbc.ra119.009419
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发表时间：
2019-08-23
影响因子：
4.8
通讯作者：
Lieberman, Raquel L.
中科院分区：
文献类型：
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作者：
Hill, Shannon E.;Kwon, Michelle S.;Lieberman, Raquel L.
非同义基因突变可能对编码蛋白质的稳定性、结构和生物功能有益、中性或有害，但这些突变的影响通常不易预测。例如，肌纤蛋白 (mOLF) 的 β-螺旋桨嗅觉素结构域在结构、稳定性和聚集之间表现出复杂的相互关系。 mOLF 内的许多突变与青光眼有关；由此产生的变体稳定性较差，易于聚集，并且隔离在细胞内，从而导致细胞毒性。在这里，我们报告了第一个在钙结合位点携带取代的稳定 mOLF 变体，其表现出与青光眼变体无法区分的溶液特性。这些稳定变体在 1.8-2.0 埃分辨率下的晶体结构揭示了我们无法通过分子动力学模拟预测的特征，包括环结构的丢失、螺旋解旋和叶片移位。结合稳定取代和选定的引起青光眼的单点突变体的双突变体挽救了体外折叠和稳定性缺陷。在全长肌纤蛋白的背景下，稳定单变体的分泌与WT蛋白的分泌无法区分，并且双突变体的分泌程度不同。总之，我们的发现表明，尽管存在构象转换，mOLF 仍可以耐受特定的取代，从而使蛋白质保持稳定，这强调了区分良性变异和引起青光眼的变异的复杂性，并为 myocilin 可能的生物学功能提供了新的见解。
Nonsynonymous gene mutations can be beneficial, neutral, or detrimental to the stability, structure, and biological function of the encoded protein, but the effects of these mutations are often not readily predictable. For example, the beta-propeller olfactomedin domain of myocilin (mOLF) exhibits a complex interrelationship among structure(s), stability, and aggregation. Numerous mutations within mOLF are linked to glaucoma; the resulting variants are less stable, aggregation-prone, and sequestered intracellularly, causing cytotoxicity. Here, we report the first stable mOLF variants carrying substitutions in the calcium-binding site that exhibit solution characteristics indistinguishable from those of glaucoma variants. Crystal structures of these stable variants at 1.8-2.0-angstrom resolution revealed features that we could not predict by molecular dynamics simulations, including loss of loop structure, helix unwinding, and a blade shift. Double mutants that combined a stabilizing substitution and a selected glaucoma-causing single-point mutant rescued in vitro folding and stability defects. In the context of full-length myocilin, secretion of stable single variants was indistinguishable from that of the WT protein, and the double mutants were secreted to varying extents. In summary, our finding that mOLF can tolerate particular substitutions that render the protein stable despite a conformational switch emphasizes the complexities in differentiating between benign and glaucoma-causing variants and provides new insight into the possible biological function of myocilin.