Chong Kelvin Han Chung, Liu Lichao, Chua Rae, Chai Yoke Tin, Lu Zhuojian, Liu Renming, Tan Eddie Yong Jun, Dong Jinxi, Khoh Yek How, Lin Jianqing, Zhong Franklin L, Lescar Julien, Zheng Peng, Wu Bin
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 636921, Singapore.
NTU Institute of Structural Biology, Nanyang Technological University, EMB 06-01, 59 Nanyang Drive, Singapore 636921, Singapore.
J Am Chem Soc. 2025 Jan 15;147(2):1604-1611. doi: 10.1021/jacs.4c10689. Epub 2025 Jan 2.
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 (, MmCET) in both apo and substrate-bound forms. Comparative analysis with its inactive paralogue, MjCET (), 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.
酶催化的蛋白质修饰在各种应用中变得非常重要，在精度、偶联效率和生物相容性方面优于化学方法。尽管连接酶取得了重大进展，如分选酶A和OaAEP1，但它们在异质生物环境中的应用仍然受到靶序列特异性有限的限制。2021年，卢帕斯团队引入了连接酶，这是一类用于蛋白质连接的经过重新利用的古细菌蛋白酶，但其低持续性和缺乏结构信息阻碍了其在实际生物学和生物物理应用中的进一步工程改造。在这里，我们展示了Mm连接酶（MmCET）在无apo和底物结合形式下的X射线晶体结构。与其无活性的旁系同源物MjCET的比较分析揭示了MmCET高精度连接活性的结构基础。我们建议对N端底物识别基序进行修饰，以抑制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蛋白质连接，如含血清的细胞培养物。为了进一步证明增强的持续性和精度，单分子蛋白质解折叠实验表明，我们优化后的连接酶与OaAEP1（C247A）结合，可以对蛋白质进行逐步串联连接，从而形成定义明确的蛋白质聚合物。
