Structural and biochemical characterization reveals LysGH15 as an unprecedented "EF-hand-like" calcium-binding phage lysin.

Jingmin Gu,Mei Yang,Ziyin Cui,Jun Song,Liancheng Lei,Xiaohe Liu,Yongjun Yang,Yingang Feng,Songying Ouyang,Wei Ding,Yue Li,Changjiang Sun,Xin Feng,Cheng Du ,Dong Wook Han ,Fujun Niu ,Zhijie Liu ,Libin Jiao ,Han Wang

doi:10.1371/journal.ppat.1004109

Abstract

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.

Highlights

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, in immunocompromised patients [1]
We report that the lytic activity of LysGH15 and its CHAP domain is dependent on calcium ions
To elucidate the molecular mechanism, we determined the structures of three individual LysGH15 domains using X-ray crystallography or nuclear magnetic resonance (NMR)

Summary

Introduction

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, in immunocompromised patients [1]. Over the past three decades, the incidence of methicillin-resistant S. aureus (MRSA) infection, as caused by communityassociated MRSA (CA-MRSA) isolates, has dramatically increased worldwide, which raises serious concerns within the medical community [2]. Lysin can rapidly and lyse Gram-positive bacteria when exogenously applied [8]. Because the bacterial cell wall is conserved and is necessary for the life cycle, the current lack of reports on the development of bacterial resistance against lysin is not surprising [9]. The species- or type-specificity guarantees that the lysin will not affect the normal microflora [10]. Lysin is thought to be a promising potential antibacterial agent

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Conclusion