Zouharova Monika, Herman Petr, Bednarova Lucie, Vetyskova Veronika, Hadravova Romana, Postulkova Klara, Zemanova Lucie, Vondrasek Jiri, Vydra Bousova Kristyna
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo namesti 5/542, 16000 Prague, Czech Republic.
Second Faculty of Medicine, Charles University, V Úvalu 84, 15006 Prague, Czech Republic.
ACS Omega. 2025 Feb 20;10(8):7741-7751. doi: 10.1021/acsomega.4c07953. eCollection 2025 Mar 4.
The evolution of proteins is primarily driven by the combinatorial assembly of a limited set of pre-existing modules known as protein domains. This modular architecture not only supports the diversity of natural proteins but also provides a robust strategy for protein engineering, enabling the design of artificial proteins with enhanced or novel functions for various industrial applications. Among these functions, oligomerization plays a crucial role in enhancing protein activity, such as by increasing the binding capacity of antibodies. To investigate the potential of engineering oligomerization, we examined the transferability of the sequence domain encoded by exon 5 (Ex5), which was originally responsible for the oligomerization of ameloblastin (AMBN). We designed a two-domain protein composed of Ex5 in combination with a monomeric, globular, and highly stable protein, specifically calmodulin (CaM). CaM represents the opposite protein character to AMBN, which is highly disordered and has a dynamic character. This engineered protein, termed eCaM, successfully acquired an oligomeric function, inducing self-assembly under specific conditions. Biochemical and biophysical analyses revealed that the oligomerization of eCaM is both concentration- and time-dependent, with the process being reversible upon dilution. Furthermore, mutating a key oligomerization residue within Ex5 abolished the self-assembly of eCaM, confirming the essential role of the Ex5 motif in driving oligomerization. Our findings demonstrate that the oligomerization properties encoded by Ex5 can be effectively transferred to a new protein context, though the positioning of Ex5 within the protein structure is critical. This work highlights the potential of enhancing monomeric proteins with oligomeric functions, paving the way for industrial applications and the development of proteins with tailored properties.
蛋白质的进化主要由一组有限的、预先存在的称为蛋白质结构域的模块的组合组装驱动。这种模块化结构不仅支持天然蛋白质的多样性，还为蛋白质工程提供了一种强大的策略，能够设计出具有增强或新功能的人工蛋白质，用于各种工业应用。在这些功能中，寡聚化在增强蛋白质活性方面起着关键作用，例如通过增加抗体的结合能力。为了研究工程化寡聚化的潜力，我们研究了外显子5（Ex5）编码的序列结构域的可转移性，该结构域最初负责成釉蛋白（AMBN）的寡聚化。我们设计了一种由Ex5与单体、球状且高度稳定的蛋白质（具体为钙调蛋白（CaM））组合而成的双结构域蛋白质。CaM代表了与高度无序且具有动态特性的AMBN相反的蛋白质特性。这种工程化蛋白质称为eCaM，成功获得了寡聚功能，在特定条件下诱导自组装。生化和生物物理分析表明，eCaM的寡聚化既依赖于浓度也依赖于时间，稀释后该过程是可逆的。此外，突变Ex5内的一个关键寡聚化残基消除了eCaM的自组装，证实了Ex5基序在驱动寡聚化中的重要作用。我们的研究结果表明，Ex5编码的寡聚化特性可以有效地转移到新的蛋白质环境中，尽管Ex5在蛋白质结构中的定位至关重要。这项工作突出了增强具有寡聚功能的单体蛋白质的潜力，为工业应用和定制特性蛋白质的开发铺平了道路。
