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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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Overexpression and activity of CHAPK mutants. A. Sodium dodecyl sulphate polyacryalamide electrophoresis gel of lysates containing over-expressed CHAPK and site-directed mutants. A control not expressing CHAPK is also included. B. Composite zymogram gel of CHAPK, site-directed mutant CHAPK variants and negative control expression lysates.
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Figure 4: Overexpression and activity of CHAPK mutants. A. Sodium dodecyl sulphate polyacryalamide electrophoresis gel of lysates containing over-expressed CHAPK and site-directed mutants. A control not expressing CHAPK is also included. B. Composite zymogram gel of CHAPK, site-directed mutant CHAPK variants and negative control expression lysates.

Mentions: The importance of the calcium ion in relation to the catalytic ability of CHAPK was investigated by creation of mutants containing a single amino acid change to alanine at each of the five residues involved in calcium coordination. Zymogram analysis demonstrated that mutation of residues Asp45, Asp47 and Asp56 resulted in the complete abolishment of the staphylolytic activity of the enzyme (Figure 4). This result indicates that the coordinated calcium ion is essential for the catalytic mechanism of the enzyme and complements a previous study, which showed that the chelator EDTA was able to reduce CHAPK activity by 99%[21]. While mutant His51-Ala retained staphylolytic ability, activity of the enzyme was visibly reduced in comparison with the parental CHAPK. Mutation of Tyr49 to alanine did not appear to affect the staphylolytic ability of the enzyme as the clearing produced on a zymogram gel was comparable to that seen for non-mutated CHAPK (Figure 4). The fact that mutants His51-Ala and Tyr49-Ala retained activity while the other mutants did not may be explained by the fact that main chain oxygen atoms are involved in coordination as opposed to the side chain oxygens. Therefore these residues are more amenable to substitution without eliminating catalytic activity.


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)

Overexpression and activity of CHAPK mutants. A. Sodium dodecyl sulphate polyacryalamide electrophoresis gel of lysates containing over-expressed CHAPK and site-directed mutants. A control not expressing CHAPK is also included. B. Composite zymogram gel of CHAPK, site-directed mutant CHAPK variants and negative control expression lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Overexpression and activity of CHAPK mutants. A. Sodium dodecyl sulphate polyacryalamide electrophoresis gel of lysates containing over-expressed CHAPK and site-directed mutants. A control not expressing CHAPK is also included. B. Composite zymogram gel of CHAPK, site-directed mutant CHAPK variants and negative control expression lysates.
Mentions: The importance of the calcium ion in relation to the catalytic ability of CHAPK was investigated by creation of mutants containing a single amino acid change to alanine at each of the five residues involved in calcium coordination. Zymogram analysis demonstrated that mutation of residues Asp45, Asp47 and Asp56 resulted in the complete abolishment of the staphylolytic activity of the enzyme (Figure 4). This result indicates that the coordinated calcium ion is essential for the catalytic mechanism of the enzyme and complements a previous study, which showed that the chelator EDTA was able to reduce CHAPK activity by 99%[21]. While mutant His51-Ala retained staphylolytic ability, activity of the enzyme was visibly reduced in comparison with the parental CHAPK. Mutation of Tyr49 to alanine did not appear to affect the staphylolytic ability of the enzyme as the clearing produced on a zymogram gel was comparable to that seen for non-mutated CHAPK (Figure 4). The fact that mutants His51-Ala and Tyr49-Ala retained activity while the other mutants did not may be explained by the fact that main chain oxygen atoms are involved in coordination as opposed to the side chain oxygens. Therefore these residues are more amenable to substitution without eliminating catalytic activity.

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