[논문]Crystal Structure of LysB4, an Endolysin from Bacillus cereus-Targeting Bacteriophage B4

Hong, Seokho; Son, Bokyung; Ryu, Sangryeol; Ha, Nam-Chul

doi:10.14348/molcells.2018.0379

Crystal Structure of LysB4, an Endolysin from Bacillus cereus-Targeting Bacteriophage B4 원문보기

Molecules and cells, v.42 no.1, 2019년, pp.79 - 86

Hong, Seokho (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University) , Son, Bokyung (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University) , Ryu, Sangryeol (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University) , Ha, Nam-Chul (Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Center for Food Safety and Toxicology, Seoul National University)

Abstract ▼ AI-Helper

Endolysins are bacteriophage-derived enzymes that hydrolyze the peptidoglycan of host bacteria. Endolysins are considered to be promising tools for the control of pathogenic bacteria. LysB4 is an endolysin produced by Bacillus cereus-infecting bacteriophage B4, and consists of an N-terminal enzymatic active domain (EAD) and a C-terminal cell wall binding domain (CBD). LysB4 was discovered for the first time as an L-alanoyl-D-glutamate endopeptidase with the ability to breakdown the peptidoglycan among B. cereus-infecting phages. To understand the activity of LysB4 at the molecular level, this study determined the X-ray crystal structure of the LysB4 EAD, using the full-length LysB4 endolysin. The LysB4 EAD has an active site that is typical of LAS-type enzymes, where $Zn^{2+}$ is tetrahedrally coordinated by three amino acid residues and one water molecule. Mutational studies identified essential residues that are involved in lytic activity. Based on the structural and biochemical information about LysB4, we suggest a ligand-docking model and a putative endopeptidase mechanism for the LysB4 EAD. These suggestions add insight into the molecular mechanism of the endolysin LysB4 in B. cereus-infecting phages.

주제어

표/그림 (6)

$Table 1. X-ray diffraction and refinement statistics$ 표 Table 1. X-ray diffraction and refinement statistics
그림 Fig. 1. The Structure of the LysB4 EAD.
그림 Fig. 2. Structural comparison to Ply500 EAD.
그림 Fig. 3. Ligand docking model with GlcNAc-MurNAc-LAla-D-Glu-meso-DAP-D-Ala.
표 Table 2. Endopeptidase activity assay against several bacteria
그림 Fig. 4. The CD profiles and a putative endopeptidase mechanism of LysB4.

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가설 설정

To gain insight into the molecular basis for the substrate specificity of LysB4 toward the DAP-type peptidoglycan, a ligand docking model was constructed with GlcNAcMurNAc-L-Ala-D-Glu-meso-DAP-D-Ala, which is the stem peptide of the DAP-type peptidoglycan. The sulfate ion is bound adjacent to the active water molecule coordinating Zn²⁺ and is geometrically similar to the tetrahedral intermediate of the sessile peptide bond, which led us to hypothesize that the bound sulfate ion is in the site of the tetrahedral intermediate of the cleaved peptide bond between the Lalanine and the D-glutamic acid in the enzymatic reaction. We also noted that the Trp100 residue at the outer edge of the cleft is within a distance to make an interaction with a pyranose ring of sugar.

제안 방법

, 2013). Combining these findings, we manually docked the ligand peptide into the deep cleft of the LysB4 EAD (Fig. 3A). The aromatic ring of the Trp100 residue made a π-interaction with the pyranose ring of GlcNAc, and the carbonyl group of the peptide bond between L-alanine and D-glutamic acid was located in the position of the sulfate ion (Fig.
Here, we determined the X-ray crystal structure of the LysB4 EAD, using the full-length LysB4 endolysin, to reveal the working mechanism of the endolysin based on the high resolution structure. The ensuing studies give molecular insights into the function of the LysB4 of B.
The K15Q, R50Q, R74T, and D129A mutants of LysB4 were constructed via an overlapping extension PCR protocol (Nelson and Fitch, 2012). To construct pET15b_LysB4 K15Q, the lysB4 gene was amplified from the pET15b_LysB4 vector in two parts, using two sets of primers (BglII-pET15b-F and lysB4-K15Q-R for the front part; lysB4-K15Q-F and lysB4-BamHI-R for the rear part). Two overlapping PCR fragments containing the single mutation were used for the second PCR step generating the lysB4 (K15Q) gene.
To gain insight into the molecular basis for the substrate specificity of LysB4 toward the DAP-type peptidoglycan, a ligand docking model was constructed with GlcNAcMurNAc-L-Ala-D-Glu-meso-DAP-D-Ala, which is the stem peptide of the DAP-type peptidoglycan. The sulfate ion is bound adjacent to the active water molecule coordinating Zn²⁺ and is geometrically similar to the tetrahedral intermediate of the sessile peptide bond, which led us to hypothesize that the bound sulfate ion is in the site of the tetrahedral intermediate of the cleaved peptide bond between the Lalanine and the D-glutamic acid in the enzymatic reaction.

대상 데이터

The resultant PCR product was inserted between the NdeI/BamHI restriction sites of pET15b to construct lysB4 K15Q-containing pET15b (pET15b_LysB4 K15Q). For the construction of pET15b_LysB4 R50Q, pET15b_LysB4 R74T, and pET15b_LysB4 D129A, two corresponding sets of primers were used. The sequences of primers used in this study are listed in the supporting information Supplementary Table S1.

이론/모형

09 Å³/Da Matthews coefficient. The crystal structure of LysB4 was determined at 2.38 Å resolution in the space group of /4₁ via the molecular replacement method using the structure of Ply500 EAD (PDB code: 2VO9) as a search model (Korndorfer et al., 2008)(Table 1).
X-ray diffraction data were processed using HKL2000 software (Otwinowski and Minor, 1997). The structure of LysB4 was determined by the molecular replacement method with MOLREP (Vagin and Teplyakov, 2010) in the CCP4 package (Winn et al., 2011) using the structure of Ply500 EAD (PDB code: 2VO9) as a search model (Korndorfer et al., 2008). The position of Zn²⁺ was determined by zinc single-wavelength anomalous dispersion (Zn-SAD) phasing with Phaser software (McCoy et al.

성능/효과

We suggest that Zn²⁺ or other divalent ions might mediate the ionic interaction to meso-DAP by binding to the negatively charged residues of LysB4, although further study might be needed. In conclusion, our high resolution structure of the LysB4 protein provides insight into the molecular mechanism of how endolysins in bacteriophages recognize and hydrolyze the host pepti-doglycans.

후속연구

, 2018). Thus, further study is required on the endopeptidases from B. cereus phages to understand the breakdown stage for bacteriophages infecting Bacillus.
3C). We suggest that Zn²⁺ or other divalent ions might mediate the ionic interaction to meso-DAP by binding to the negatively charged residues of LysB4, although further study might be needed. In conclusion, our high resolution structure of the LysB4 protein provides insight into the molecular mechanism of how endolysins in bacteriophages recognize and hydrolyze the host pepti-doglycans.

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