is a common pathogen that plays a major role in various human and animal diseases ranging from skin and soft tissue infections to more serious cases of pneumonia, endocarditis, meningitis and osteomyelitis.1
Treatment of these infections has become increasingly difficult due to the worldwide prevalence of multidrug-resistant strains including methicillin resistant S. aureus
(MRSA) which is a frequent cause of serious nosocomial infections.2
As a consequence it is critical to develop new and effective antibacterials with the potential of eliminating such infections irrespective of antibiotic sensitivity.
Over the past decade numerous studies have focused on developing recombinant bacteriophage (phage)-encoded cell wall hydrolases, termed endolysins (lysins), as novel antibacterial agents as recently reviewed by Loessner (2005),3
and Fenton et al. (2010).5
When applied exogenously as purified recombinant proteins to Gram-positive bacteria, lysins bring about rapid cell lysis and bacterial death.3,6,7
It is this ability to rapidly lyse pathogenic Gram-positive cells upon direct contact with peptidoglycan, also termed “lysis from without,” that has laid the foundation for their exploitation as novel therapeutics.6
The majority of lysins display a modular structure, usually comprising of at least one N-terminal catalytic domain which attacks bacterial cell wall peptidoglycan, combined with a C-terminal cell wall binding domain which directs the lytic domain to its site of action.8,9
In the case of staphylococcal lysins the presence of three domains, comprising an N-terminal cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain and an amidase-2 domain linked to a C-terminal SH3b cell wall binding domain, is a common feature. This organization has been observed in LysK,10
Studies have shown that of the two lytic domains contained within staphylococcal lysins, the CHAP domain confers the principle enzymatic activity of the protein, whereas the amidase domain contributes minimal detectable activity.17-20
Furthermore, a recent study in our laboratory showed that the activity of the single-domain truncated CHAP (later designated CHAPK
), was 2-fold higher than the native three-domain LysK protein.20
Studies have also demonstrated that the CHAP domain of staphylococcal lysins acts as a D
-Ala-Gly endopeptidase, specifically cleaving the peptide bond between D
-alanine and the first glycine in the pentaglycine cross-bridge of staphylococcal cell wall peptidoglycan17,18
Figure 1. CHAPK cleavage site of staphylococcal cell wall peptidoglycan.
CHAP proteins contain three highly conserved amino acid residues, two of which are an invariant cysteine (Cys) and histidine (His) along with a third, polar residue such as asparagine (Asn), aspartic acid (Asp) or glutamic acid (Glu).21
These residues are principally involved in catalytic activity, forming part of the active site of the enzyme as well as being equivalent to the catalytic triad of papain-like thiol proteases.21,22
Site-directed mutagenesis studies on these conserved residues within the CHAP domain of the LysWMY staphylococcal lysin resulted in reduced activity, suggesting that a Cys-His-Asn catalytic triad is crucial for enzymatic function.11
The CHAP domain is a member of the NlpC/P60 family of peptidases and can be found in proteins from bacteria, archaea and eukaryotes of the Trypanosomidae family.22
However, very little structural information is available in relation to the CHAP domains of phage lysins. To date the crystal structure of five lysins has been elucidated, Cpl-1,23
none of which are derived from staphylococcal phage and none of which contain a CHAP domain.
In the current study a comparative modeling approach is employed to predict the three dimensional (3D) structure of the 162-amino-acid CHAP domain protein, CHAPK
, derived from the staphylococcal phage K lysin; LysK.10
The potential of CHAPK
as an alternative antibacterial agent which displays rapid lytic activity against pathogenic staphylococci including MRSA, both in vitro and in vivo, has previously been demonstrated by our group.20,28
Comparative modeling allows accurate prediction of the 3D structure of a target protein (CHAPK
) based on its similarity to a related template protein whose structure has been experimentally resolved by either X-ray crystallography or Nuclear Magnetic Resonance (NMR) spectroscopy.29
The basis of this approach is that evolutionary related proteins share similar 3D structures if they have a statistically significant sequence similarity (generally > 25% identity).30
The model prediction process consists of four consecutive steps starting with template selection, followed by target-template alignment, model building and finally model evaluation.31
Resolution of the 3D structure of CHAPK by comparative modeling can provide a valuable insight into the structural basis of interaction with its cell wall peptidoglycan substrate and mechanism of catalytic activity. This knowledge may subsequently be used in the design of novel lysins with altered and improved catalytic domains, or the creation of designer lysins with unique capabilities (e.g., enhanced turnover rate, altered specificity) furthering the development of these unique enzymes as alternative antibacterial agents in the fight against multi-drug resistant bacterial infections.