新加坡南洋理工大学的Bin Wu研究小组近日取得一项新成果。他们研究了高精度蛋白连接的结构基础及应用。这一研究成果于2025年1月2日发表在国际顶尖学术期刊《美国化学会杂志》上。

研究人员展示了载子和底物结合形式的MmConnectase（Methanococcthem maripaludis, MmCET）的X射线晶体结构。通过与其非活性对偶体MjCET（Methanococcthem janaschi）进行比较分析，揭示了MmCET具有高精度结联活性的结构基础。研究人员提出修改N端底物识别基元来抑制MmCET的可逆蛋白酶活性，从而在复杂的生物环境中实现高精度的蛋白质连接，例如在含血清的细胞培养中。为了进一步证明其提高的加工效率和精度，单分子蛋白质展开实验表明，他们优化的Connectase与OaAEP1（C247A）结合，可以进行蛋白质的逐步串联连接，从而形成明确的蛋白质聚合物。

研究人员表示，酶催化的蛋白质修饰在各种应用中变得非常宝贵，在精度，偶联效率和生物相容性方面优于化学方法。尽管连接酶（如分选酶A和OaAEP1）取得了重大进展，但它们在异质生物环境中的作用仍然受到有限的靶序列特异性的限制。2021年，Lupas的团队引入了Connectase，这是一种用于蛋白质连接的改造古细菌蛋白酶家族，但其低处理能力和缺乏结构信息，阻碍了实际生物和生物物理应用的进一步工程设计。

附：英文原文

Title: Structural Basis of High-Precision Protein Ligation and Its Application

Author: Kelvin Han Chung Chong, Lichao Liu, Rae Chua, Yoke Tin Chai, Zhuojian Lu, Renming Liu, Eddie Yong Jun Tan, Jinxi Dong, Yek How Khoh, Jianqing Lin, Franklin L. Zhong, Julien Lescar, Peng Zheng, Bin Wu

Issue&Volume: January 2, 2025

Abstract: Enzyme-catalyzed protein modifications have become invaluable in diverse applications, outperforming chemical methods in terms of precision, conjugation efficiency, and biological compatibility. Despite significant advances in ligases, such as sortase A and OaAEP1, their use in heterogeneous biological environments remains constrained by limited target sequence specificity. In 2021, Lupas’ group introduced Connectase, a family of repurposed archaeal proteases for protein ligations, but its low processivity and lack of structural information have impeded further engineering for practical biological and biophysical applications. Here, we present the X-ray crystallographic structures of MmConnectase (Methanococcus maripaludis, MmCET) in both apo and substrate-bound forms. Comparative analysis with its inactive paralogue, MjCET (Methanococcus janaschi), reveals the structural basis of MmCET’s high-precision ligation activity. We propose modifications to the N-terminal substrate recognition motifs to suppress MmCET’s reversible protease activity, enabling high-precision protein ligations in complex biological environments, such as serum-containing cell cultures. To further demonstrate the enhanced processivity and precision, single-molecule protein unfolding experiments showed that our optimized Connectase, in conjunction with OaAEP1(C247A), can perform stepwise tandem ligations of protein leading to a well-defined protein polymer.

DOI: 10.1021/jacs.4c10689

Source: https://pubs.acs.org/doi/abs/10.1021/jacs.4c10689