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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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MES buffer molecule bound to the CHAPK enzyme putative substrate binding site. The CHAPK protein is shown in transparent surface and secondary structure cartoon representation; the calcium ion is also shown.
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Figure 2: MES buffer molecule bound to the CHAPK enzyme putative substrate binding site. The CHAPK protein is shown in transparent surface and secondary structure cartoon representation; the calcium ion is also shown.

Mentions: The globular CHAPK protein has a relatively long and deep hydrophobic groove. When sequence conservation is mapped onto the surface, one notices that several residues lining the groove are highly conserved (Figure 1D; the sequence alignment underlying this figure is in Additional file1: Table S1). In the native structure, a MES molecule is located in this groove (Figure 2, PDB entry 4CSH), while in the derivative structure a HEPES molecule is present (PDB entry 4CT3). These molecules may well be mimicking the natural peptidoglycan substrate of the protein. Residues in the groove that might contact the peptidoglycan substrate are: Phe36, Asp47, Tyr49, Tyr50, Gln53 and Cys54 from the loop between helices 1 and 2; Asp56 and Thr59 from helix 2; Arg71, Trp73 and Asn75 from the loop between helix 2 and beta-strand A; Trp115 and His117 from the BC-loop and Asn136 and Trp137 from the DE-loop.


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)

MES buffer molecule bound to the CHAPK enzyme putative substrate binding site. The CHAPK protein is shown in transparent surface and secondary structure cartoon representation; the calcium ion is also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: MES buffer molecule bound to the CHAPK enzyme putative substrate binding site. The CHAPK protein is shown in transparent surface and secondary structure cartoon representation; the calcium ion is also shown.
Mentions: The globular CHAPK protein has a relatively long and deep hydrophobic groove. When sequence conservation is mapped onto the surface, one notices that several residues lining the groove are highly conserved (Figure 1D; the sequence alignment underlying this figure is in Additional file1: Table S1). In the native structure, a MES molecule is located in this groove (Figure 2, PDB entry 4CSH), while in the derivative structure a HEPES molecule is present (PDB entry 4CT3). These molecules may well be mimicking the natural peptidoglycan substrate of the protein. Residues in the groove that might contact the peptidoglycan substrate are: Phe36, Asp47, Tyr49, Tyr50, Gln53 and Cys54 from the loop between helices 1 and 2; Asp56 and Thr59 from helix 2; Arg71, Trp73 and Asn75 from the loop between helix 2 and beta-strand A; Trp115 and His117 from the BC-loop and Asn136 and Trp137 from the DE-loop.

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