Gu Jingmin, Feng Yingang, Feng Xin, Sun Changjiang, Lei Liancheng, Ding Wei, Niu Fengfeng, Jiao Lianying, Yang Mei, Li Yue, Liu Xiaohe, Song Jun, Cui Ziyin, Han Dong, Du Chongtao, Yang Yongjun, Ouyang Songying, Liu Zhi-Jie, Han Wenyu
Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.
Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
PLoS Pathog. 2014 May 15;10(5):e1004109. doi: 10.1371/journal.ppat.1004109. eCollection 2014 May.
The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.
来源于葡萄球菌噬菌体GH15的溶菌酶LysGH15,对耐甲氧西林金黄色葡萄球菌(MRSA)表现出广泛的裂解谱和强大的裂解活性。在此,我们发现全长LysGH15及其CHAP结构域的裂解活性依赖于钙离子。为阐明其分子机制,我们测定了LysGH15三个独立结构域的结构。出乎意料的是,LysGH15 CHAP结构域的晶体结构在半胱氨酸-组氨酸-谷氨酸-天冬酰胺四重活性位点凹槽附近揭示了一个“类EF手型”钙离子结合位点。迄今为止,LysGH15 CHAP结构域中的钙离子结合位点在同源蛋白中是独特的,它代表了CHAP家族中首次报道的钙离子结合位点。更重要的是,钙离子作为一个开关,在调节CHAP结构域的活性和非活性状态之间起着重要作用。对酰胺酶-2结构域进行基于结构的诱变表明,催化过程中锌离子和E282都是必需的,这使我们能够提出一种催化机制。核磁共振(NMR)光谱和滴定引导诱变确定了SH3b结构域中参与与底物相互作用的残基(如N404、Y406、G407和T408)。据我们所知,我们的结果构成了关于葡萄球菌噬菌体溶菌酶生化特性的首个结构信息,代表了在理解这类溶菌酶方面向前迈出的关键一步。