Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

Hdl Handle:
http://hdl.handle.net/10147/324895
Title:
Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K
Authors:
Sanz-Gaitero, Marta; Keary, Ruth; Garcia-Doval, Carmela; Coffey, Aidan; van Raaij, Mark J
Citation:
Sanz-Gaitero M et al. Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K. Virology Journal. 2014 Jul 26;11(1):133
Issue Date:
26-Jul-2014
URI:
http://dx.doi.org/10.1186/1743-422X-11-133; http://hdl.handle.net/10147/324895
Abstract:
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 (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Methods The CHAPK 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 CHAPK 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, CHAPK 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 CHAPK 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 CHAPK 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.
Item Type:
Article
Language:
en
Keywords:
INFECTION CONTROL
Local subject classification:
CELL BIOLOGY; METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

Full metadata record

DC FieldValue Language
dc.contributor.authorSanz-Gaitero, Martaen_GB
dc.contributor.authorKeary, Ruthen_GB
dc.contributor.authorGarcia-Doval, Carmelaen_GB
dc.contributor.authorCoffey, Aidanen_GB
dc.contributor.authorvan Raaij, Mark Jen_GB
dc.date.accessioned2014-08-18T08:57:06Z-
dc.date.available2014-08-18T08:57:06Z-
dc.date.issued2014-07-26-
dc.identifier.citationSanz-Gaitero M et al. Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K. Virology Journal. 2014 Jul 26;11(1):133en_GB
dc.identifier.urihttp://dx.doi.org/10.1186/1743-422X-11-133-
dc.identifier.urihttp://hdl.handle.net/10147/324895-
dc.description.abstractAbstract 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 (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. Methods The CHAPK 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 CHAPK 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, CHAPK 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 CHAPK 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 CHAPK 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.-
dc.language.isoenen
dc.subjectINFECTION CONTROLen_GB
dc.subject.otherCELL BIOLOGYen_GB
dc.subject.otherMETHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUSen_GB
dc.titleCrystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage Ken_GB
dc.typeArticleen
dc.language.rfc3066en-
dc.rights.holderMarta Sanz-Gaitero et al.; licensee BioMed Central Ltd.-
dc.description.statusPeer Reviewed-
dc.date.updated2014-08-08T15:14:20Z-
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