Nakai Tadashi, Nakai Yota, Takami Naoki, Nakai Emi, Okajima Toshihide
Graduate School of Science and Technology, Hiroshima Institute of Technology, Hiroshima 731-5193, Japan.
Faculty of Life Sciences, Hiroshima Institute of Technology, Hiroshima 731-5193, Japan.
ACS Omega. 2025 Jul 25;10(30):33862-33871. doi: 10.1021/acsomega.5c05386. eCollection 2025 Aug 5.
Cyclization is a promising strategy to enhance protein stability, but its applicability is often limited by structural constraints such as the distance between the N- and C-terminal regions. Here, we report the rational design and characterization of a cyclized Tobacco Etch Virus protease (cTEVp) using the SpyRing system, which enables covalent cyclization through SpyTag/SpyCatcher-mediated isopeptide bond formation. We applied this approach to a widely used engineered TEVp variant (L56V, S135G, S219V, Δ238-242) and employed AlphaFold structure prediction to optimize linker length and domain positioning. Despite the ∼40 Å separation between the N- and C-termini of native TEVp, AlphaFold modeling suggested that the fused SpyTag and SpyCatcher domains can adopt a favorable configuration for intramolecular cyclization. The resulting cTEVp exhibited proteolytic activity comparable to the noncyclized TEVp, indicating that structural constraint via SpyRing-mediated cyclization did not impair enzymatic function. Importantly, cTEVp displayed significantly improved thermostability relative to its noncyclized counterpart, as demonstrated by higher retention of soluble enzyme and residual activity following heat treatment at 50 °C. Our findings validate the effectiveness of SpyRing-mediated cyclization in improving TEVp stability and highlight the utility of computationally guided cyclization as a generalizable strategy for engineering thermally resilient proteins. This study establishes a framework that integrates structure prediction and rational protein engineering, contributing to the development of robust biocatalysts for synthetic biology and industrial biotechnology applications.
环化是一种增强蛋白质稳定性的有前景的策略，但其适用性常常受到诸如N端和C端区域之间距离等结构限制的制约。在此，我们报道了一种使用SpyRing系统对烟草蚀纹病毒蛋白酶（cTEVp）进行理性设计和表征的方法，该系统能够通过SpyTag/SpyCatcher介导的异肽键形成实现共价环化。我们将此方法应用于一种广泛使用的工程化TEVp变体（L56V、S135G、S219V、Δ238 - 242），并采用AlphaFold结构预测来优化连接子长度和结构域定位。尽管天然TEVp的N端和C端之间相隔约40 Å，但AlphaFold建模表明，融合的SpyTag和SpyCatcher结构域可以采用有利于分子内环化的构象。所得的cTEVp表现出与未环化的TEVp相当的蛋白水解活性，这表明通过SpyRing介导的环化所产生的结构限制并未损害酶的功能。重要的是，与未环化的对应物相比，cTEVp显示出显著提高的热稳定性，如在50°C热处理后可溶性酶和残余活性的更高保留率所证明的那样。我们的研究结果验证了SpyRing介导的环化在提高TEVp稳定性方面的有效性，并突出了计算指导的环化作为一种用于工程化热弹性蛋白的可推广策略的实用性。这项研究建立了一个整合结构预测和理性蛋白质工程的框架，有助于开发用于合成生物学和工业生物技术应用的强大生物催化剂。
