Department of Biochemistry, University of Washington, Seattle, WA, USA.
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Nature. 2021 Jan;589(7842):468-473. doi: 10.1038/s41586-020-03120-8. Epub 2021 Jan 6.
Ordered two-dimensional arrays such as S-layers and designed analogues have intrigued bioengineers, but with the exception of a single lattice formed with flexible linkers, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.
我们描述了一种通过在双螺旋蛋白构建模块之间设计刚性界面来生成共组装双层的计算方法，并利用它设计了 p6m 晶格。设计的阵列组件在毫摩尔浓度下是可溶的，但当在纳摩尔浓度下组合时，它们会迅速组装成几乎类似于体外和细胞中计算设计模型的近晶态微米级阵列，而无需二维支架。由于材料是从头开始设计的，因此可以很容易地对组件进行功能化和重新配置其对称性，从而形成具有可区分表面的配体阵列，我们证明了这些配体阵列可以驱动广泛的受体聚集、下游蛋白募集和信号转导。我们使用原子力显微镜在支持双层上进行研究，并在活细胞上进行定量显微镜检查，结果表明，在膜上组装的阵列具有类似于体外形成的阵列的组件化学计量和结构，并且我们的材料因此可以将有序结构强加于细胞膜等基本无序的基底上。与以前表征的细胞表面受体结合组装体（如抗体和纳米笼）不同，这些组装体很快被内吞，我们发现，在细胞表面组装的大阵列可以以可调节的方式抑制内吞作用，这对于延长受体结合和免疫逃逸具有潜在的治疗意义。我们的工作为合成细胞生物学提供了基础，在该生物学中，多蛋白宏观材料被设计用于调节细胞反应并重塑合成和生命系统。
