Research Article: Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin

Date Published: May 15, 2014

Publisher: Public Library of Science

Author(s): Jingmin Gu, Yingang Feng, Xin Feng, Changjiang Sun, Liancheng Lei, Wei Ding, Fengfeng Niu, Lianying Jiao, Mei Yang, Yue Li, Xiaohe Liu, Jun Song, Ziyin Cui, Dong Han, Chongtao Du, Yongjun Yang, Songying Ouyang, Zhi-Jie Liu, Wenyu Han, Gongyi Zhang.


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.

Partial Text

Although Staphylococcus aureus is a common habitant of the human skin and respiratory tract, several highly pathogenic strains are major causes of hospital-associated infections and can be life threatening, particularly in immunocompromised patients [1]. Over the past three decades, the incidence of methicillin-resistant S. aureus (MRSA) infection, particularly as caused by community-associated MRSA (CA-MRSA) isolates, has dramatically increased worldwide, which raises serious concerns within the medical community [2]. USA300 is the most prevalent CA-MRSA strain and accounts for up to 97% of all CA-MRSA infections [3]. The treatment of infections caused by CA-MRSA has become increasingly difficult due to the emergence of multidrug resistance [4]. Therefore, an urgent need exists for novel therapeutic agents directed against this formidable pathogen [5], [6].

In this study, we report that the lytic activity of the LysGH15 and its CHAP domain is critically dependent on a calcium ion. To confirm these findings, the structures of the individual CHAP, amidase-2, and SH3b domains of LysGH15 were determined. This study represents the first report of the structure of a staphylococcal phage lysin.




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