Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling
Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling
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DOI：
10.1093/protein/gzv004
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发表时间：
2015-04-01
影响因子：
2.4
通讯作者：
Nelson, Daniel C.
中科院分区：
文献类型：
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作者：
Heselpoth, Ryan D.;Yin, Yizhou;Nelson, Daniel C.
内溶素是噬菌体衍生的肽聚糖水解酶，是一类新兴的蛋白质类治疗药物。虽然链球菌内溶素PlyC已在体外和体内验证了其治疗功效，但该酶固有的热敏感结构与瞬时长期稳定性相关，从而阻碍了将该酶开发为抗菌剂的可行性。在此，我们使用FoldX驱动的计算蛋白质工程方法对PlyC的PlyCA催化亚基的半胱氨酸、组氨酸依赖性酰胺水解酶/肽酶（CHAP）结构域进行了热稳定化。通过结合使用FoldX和Rosetta算法以及目视检查，组装了具有预测稳定化ΔΔG值的PlyC点突变候选物的最终列表，并进行了热特性分析。实验发现八个点突变中的五个是不稳定的，这一结果很可能是由于对具有相应3.3埃X射线晶体结构的复杂且动态的九亚基全酶进行计算建模所致。然而，其中一个突变体PlyC（PlyCA）T406R经实验表明，其热变性温度比野生型提高了约2.2℃，动力学稳定性是野生型的16倍。预计该突变会在N端糖基水解酶结构域的Q106侧链和C端CHAP结构域的R406侧链之间引入一个对热有利的氢键。
Endolysins are bacteriophage-derived peptidoglycan hydrolases that represent an emerging class of proteinaceous therapeutics. While the streptococcal endolysin PlyC has been validated in vitro and in vivo for its therapeutic efficacy, the inherent thermosusceptible structure of the enzyme correlates to transient long-term stability, thereby hindering the feasibility of developing the enzyme as an antimicrobial. Here, we thermostabilized the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain of the PlyCA catalytic subunit of PlyC using a FoldX-driven computational protein engineering approach. Using a combination of FoldX and Rosetta algorithms, as well as visual inspection, a final list of PlyC point mutant candidates with predicted stabilizing Delta Delta G values was assembled and thermally characterized. Five of the eight point mutations were found experimentally to be destabilizing, a result most likely attributable to computationally modeling a complex and dynamic nine-subunit holoenzyme with a corresponding 3.3-angstrom X-ray crystal structure. However, one of the mutants, PlyC (PlyCA) T406R, was shown experimentally to increase the thermal denaturation temperature by similar to 2.2 degrees C and kinetic stability 16-fold over wild type. This mutation is expected to introduce a thermally advantageous hydrogen bond between the Q106 side chain of the N-terminal glycosyl hydrolase domain and the R406 side chain of the C-terminal CHAP domain.