In recent years, new protein engineering methods have produced more than a dozen symmetric, self-assembling protein cages whose structures have been validated to match their design models with near-atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X-ray crystallography of designed protein oligomers that form two-component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen-bonding networks over exclusively hydrophobic interactions to stabilize the designed protein-protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen-bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I21 3 is generated by an off-target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.

中文翻译：

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两个新蛋白质笼的设计和结构说明了蛋白质工程的成功和持续的挑战。

近年来，新的蛋白质工程方法已经产生了十几种对称的自组装蛋白质笼，其结构已经过验证，可以以接近原子的精度匹配其设计模型。然而，在实验室中测试的许多蛋白质笼设计并没有形成所需的组件，提高设计的成功率一直是最近的重点。在这里，我们展示了通过 X 射线晶体学设计的蛋白质寡聚物解决的两种蛋白质结构，这些寡聚物形成具有四面体对称性的双组分笼。为了改善过去对难溶蛋白质的趋势，我们使用了一种计算协议，该协议有利于氢键网络的形成，而不是完全疏水的相互作用，以稳定设计的蛋白质-蛋白质界面。初步表征显示高度可溶的表达，和溶液研究表明两种设计的蛋白质都成功形成了笼子。对于其中一个设计，晶体结构以高分辨率确认形成了预期的四面体笼，尽管几个翻转的氨基酸侧链旋转异构体导致界面偏离了预期的精确氢键模式。另一个设计的笼状结构表明，在获得晶体的条件下，形成了非笼状结构，其中空间群 I21 3 中的多孔 3D 蛋白质网络由脱靶双同聚界面产生。这些结果说明了开发用于极性界面设计的计算方法的一些持续挑战，并为越来越多的用于下游应用的工程蛋白质材料列表添加了两个潜在有价值的新条目。