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Crystal structure of the lytic CHAP(K) domain of the endolysin LysK from Staphylococcus aureus bacteriophage K.

Sanz-Gaitero M, Keary R, Garcia-Doval C, Coffey A, van Raaij MJ - Virol. J. (2014)

Bottom Line: The resulting structures were completed, refined and analyzed.When compared to previously solved CHAP domains, CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56.A zinc ion was found more loosely bound.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Estructura de Macromoleculas, Centro Nacional de Biotecnologia (CNB-CSIC), Calle Darwin 3, E-28049 Madrid, Spain. mjvanraaij@cnb.csic.es.

ABSTRACT

Background: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAP(K)), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAP(K) cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.

Methods: The CHAP(K) domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.

Results: The structure of CHAP(K) revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.

Conclusions: The high-resolution structure of the CHAP(K) domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAP(K) with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.

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Crystal structure of the N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK) of the endolysin LysK from staphylococcal bacteriophage K. (A) Overall structure. Beta-strands are shown in green, alpha-helices in blue and 310-helices in red. The calcium ion is shown in grey, the zinc ion in white. The N-terminal end (Nt), residue 165, the alpha-helices and the beta-strands are labelled. (B). Topology diagram. The same labelling is used as in panel A. (C). Superposition of CHAPK (magenta) onto structure onto the CHAP domain of the streptococcal phage endolysin PlyC (PDB entry 4 F88; cyan). (D). Space-filling representation with conserved residues in almost the same orientation as panel A, but slightly tilted forward to better illustrate the hydrophobic groove, which is indicated with an arrow. The colour coding goes from blue for less conserved residues, via white, to purple for the most conserved residues.
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Figure 1: Crystal structure of the N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK) of the endolysin LysK from staphylococcal bacteriophage K. (A) Overall structure. Beta-strands are shown in green, alpha-helices in blue and 310-helices in red. The calcium ion is shown in grey, the zinc ion in white. The N-terminal end (Nt), residue 165, the alpha-helices and the beta-strands are labelled. (B). Topology diagram. The same labelling is used as in panel A. (C). Superposition of CHAPK (magenta) onto structure onto the CHAP domain of the streptococcal phage endolysin PlyC (PDB entry 4 F88; cyan). (D). Space-filling representation with conserved residues in almost the same orientation as panel A, but slightly tilted forward to better illustrate the hydrophobic groove, which is indicated with an arrow. The colour coding goes from blue for less conserved residues, via white, to purple for the most conserved residues.

Mentions: The CHAPK protein consists of a single globular domain that contains two alpha-helices, two 310-helices and six beta-strands (Figure 1A and B). The amino-terminal part of the protein consists of the two alpha-helices (I and II) interconnected by a long loop. This long loop borders a groove in the protein, at the bottom of which the catalytic site is located (see below). Another loop, containing a 310-helix, connects this amino-terminal part of the protein to a six-stranded beta-sheet that forms the carboxy-terminal part. The six beta-strands are arranged in an anti-parallel beta-sheet in the topology AFBCDE (Figure 1B). The structure of CHAPK had previously been predicted by in silico modelling[13]. The six-stranded beta-sheet was predicted well, but the amino-terminal alpha-helices were incorrectly placed and the calcium-binding loop between them was not present in the model. The main chain atoms of the catalytic site residues were within 2 Å of their predicted positions.


Crystal structure of the lytic CHAP(K) domain of the endolysin LysK from Staphylococcus aureus bacteriophage K.

Sanz-Gaitero M, Keary R, Garcia-Doval C, Coffey A, van Raaij MJ - Virol. J. (2014)

Crystal structure of the N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK) of the endolysin LysK from staphylococcal bacteriophage K. (A) Overall structure. Beta-strands are shown in green, alpha-helices in blue and 310-helices in red. The calcium ion is shown in grey, the zinc ion in white. The N-terminal end (Nt), residue 165, the alpha-helices and the beta-strands are labelled. (B). Topology diagram. The same labelling is used as in panel A. (C). Superposition of CHAPK (magenta) onto structure onto the CHAP domain of the streptococcal phage endolysin PlyC (PDB entry 4 F88; cyan). (D). Space-filling representation with conserved residues in almost the same orientation as panel A, but slightly tilted forward to better illustrate the hydrophobic groove, which is indicated with an arrow. The colour coding goes from blue for less conserved residues, via white, to purple for the most conserved residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126393&req=5

Figure 1: Crystal structure of the N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK) of the endolysin LysK from staphylococcal bacteriophage K. (A) Overall structure. Beta-strands are shown in green, alpha-helices in blue and 310-helices in red. The calcium ion is shown in grey, the zinc ion in white. The N-terminal end (Nt), residue 165, the alpha-helices and the beta-strands are labelled. (B). Topology diagram. The same labelling is used as in panel A. (C). Superposition of CHAPK (magenta) onto structure onto the CHAP domain of the streptococcal phage endolysin PlyC (PDB entry 4 F88; cyan). (D). Space-filling representation with conserved residues in almost the same orientation as panel A, but slightly tilted forward to better illustrate the hydrophobic groove, which is indicated with an arrow. The colour coding goes from blue for less conserved residues, via white, to purple for the most conserved residues.
Mentions: The CHAPK protein consists of a single globular domain that contains two alpha-helices, two 310-helices and six beta-strands (Figure 1A and B). The amino-terminal part of the protein consists of the two alpha-helices (I and II) interconnected by a long loop. This long loop borders a groove in the protein, at the bottom of which the catalytic site is located (see below). Another loop, containing a 310-helix, connects this amino-terminal part of the protein to a six-stranded beta-sheet that forms the carboxy-terminal part. The six beta-strands are arranged in an anti-parallel beta-sheet in the topology AFBCDE (Figure 1B). The structure of CHAPK had previously been predicted by in silico modelling[13]. The six-stranded beta-sheet was predicted well, but the amino-terminal alpha-helices were incorrectly placed and the calcium-binding loop between them was not present in the model. The main chain atoms of the catalytic site residues were within 2 Å of their predicted positions.

Bottom Line: The resulting structures were completed, refined and analyzed.When compared to previously solved CHAP domains, CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56.A zinc ion was found more loosely bound.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Estructura de Macromoleculas, Centro Nacional de Biotecnologia (CNB-CSIC), Calle Darwin 3, E-28049 Madrid, Spain. mjvanraaij@cnb.csic.es.

ABSTRACT

Background: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAP(K)), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAP(K) cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.

Methods: The CHAP(K) domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.

Results: The structure of CHAP(K) revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.

Conclusions: The high-resolution structure of the CHAP(K) domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAP(K) with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.

Show MeSH
Related in: MedlinePlus