Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal “molecular thread” stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.

羧基体是自然界中发现的自组装蛋白质细胞器的范例，提供酶的区室化和增强碳固定的途径。在 α-羧基体中，无序连接蛋白 CsoS2 在羧基体组装和 Rubisco 封装中发挥重要作用。然而，其作用机制尚不完全清楚。在这里，我们使用最少的壳成分合成设计 α-羧基体壳，并确定它们的冷冻电镜结构，以破译壳组装和封装的原理。这些结构表明，本质上无序的 CsoS2 C 末端结构良好，可作为通过多个壳蛋白界面缝合的通用“分子线”。我们进一步在 CsoS2 中发现了一个高度保守的重复关键相互作用基序 [IV]TG，它对壳组装和结构至关重要。我们的研究为 CsoS2 控制的羧基体壳组装和货物封装提供了通用机制，并进一步推进了羧基体的合成工程，用于多种生物技术应用。