Limits...
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.

Show MeSH

Related in: MedlinePlus

Presence of metal ions in the CHAPK crystal structure. A. X-ray fluorescence emission spectrum collected from a CHAPK crystal irradiated with monochromatic synchrotron radiation (12.7 KeV). B. Detail of the calcium ion coordination. Coordinating atoms are one Oδ atom of each of Asp45 and Asp47 residues, both Oδ atoms of Asp56, the main chain oxygen atoms of Tyr49 and His51 and an ordered water molecule (behind the calcium ion in this view). C. Detail of the zinc coordination. The zinc ion is sandwiched between Cys54 and the sulphate group of the MES ion, about 10 Å away from the calcium ion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Presence of metal ions in the CHAPK crystal structure. A. X-ray fluorescence emission spectrum collected from a CHAPK crystal irradiated with monochromatic synchrotron radiation (12.7 KeV). B. Detail of the calcium ion coordination. Coordinating atoms are one Oδ atom of each of Asp45 and Asp47 residues, both Oδ atoms of Asp56, the main chain oxygen atoms of Tyr49 and His51 and an ordered water molecule (behind the calcium ion in this view). C. Detail of the zinc coordination. The zinc ion is sandwiched between Cys54 and the sulphate group of the MES ion, about 10 Å away from the calcium ion.

Mentions: While building and refining the protein model, relatively strong density peaks were observed near the terminal atoms of the side-chains of Cys54 and Asp56 in each of the four protein chains in the asymmetric unit, suggesting the presence of metal ions. X-ray fluorescence spectroscopy is a powerful method to identify trace elements in biological samples[18]. Therefore, we recorded an X-ray fluorescence spectrum from a frozen native CHAPK protein crystal, which revealed significant amounts of zinc and calcium (Figure 3A). Sulphur (from methionine, cysteine residues and buffer molecules) and chlorine (from the crystallization buffer) were also detected. The presence of trace amounts of titanium and copper is likely the result of interaction of the beam with certain beamline or sample holder components not related to the sample.


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)

Presence of metal ions in the CHAPK crystal structure. A. X-ray fluorescence emission spectrum collected from a CHAPK crystal irradiated with monochromatic synchrotron radiation (12.7 KeV). B. Detail of the calcium ion coordination. Coordinating atoms are one Oδ atom of each of Asp45 and Asp47 residues, both Oδ atoms of Asp56, the main chain oxygen atoms of Tyr49 and His51 and an ordered water molecule (behind the calcium ion in this view). C. Detail of the zinc coordination. The zinc ion is sandwiched between Cys54 and the sulphate group of the MES ion, about 10 Å away from the calcium ion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Presence of metal ions in the CHAPK crystal structure. A. X-ray fluorescence emission spectrum collected from a CHAPK crystal irradiated with monochromatic synchrotron radiation (12.7 KeV). B. Detail of the calcium ion coordination. Coordinating atoms are one Oδ atom of each of Asp45 and Asp47 residues, both Oδ atoms of Asp56, the main chain oxygen atoms of Tyr49 and His51 and an ordered water molecule (behind the calcium ion in this view). C. Detail of the zinc coordination. The zinc ion is sandwiched between Cys54 and the sulphate group of the MES ion, about 10 Å away from the calcium ion.
Mentions: While building and refining the protein model, relatively strong density peaks were observed near the terminal atoms of the side-chains of Cys54 and Asp56 in each of the four protein chains in the asymmetric unit, suggesting the presence of metal ions. X-ray fluorescence spectroscopy is a powerful method to identify trace elements in biological samples[18]. Therefore, we recorded an X-ray fluorescence spectrum from a frozen native CHAPK protein crystal, which revealed significant amounts of zinc and calcium (Figure 3A). Sulphur (from methionine, cysteine residues and buffer molecules) and chlorine (from the crystallization buffer) were also detected. The presence of trace amounts of titanium and copper is likely the result of interaction of the beam with certain beamline or sample holder components not related to the sample.

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