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Cpl-1 overall fold The Catalytic Module Choline Binding Modular Organization Concluding Remarks References |
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Streptococcus pneumoniae is a common and important human pathogen associated with pneumonia, septicemia, meningitis and otitis media. The high morbidity and mortality caused by pneumococcal diseases, particularly in infants, elderly and immunocompromised patients, is exacerbated by the increasing prevalence of antibiotic-resistant strains and the suboptimal efficacy of available vaccines. Since the integrity of the peptidoglycan is essential for bacterial survival, bacteriophage coded cell wall lysins may constitute effective antibacterial agents against their host. All the pneumococcal endolysins so far described, display a modular structure such that, in addition to the catalytic module, most of them posses a choline-binding module to facilitate their anchoring to the choline-containing teichoic acid of the pneumococcal cell wall.
Very recently the crystal structures of the free and choline bound states of the Cpl-1 lysin (339 aa), encoded by the pneumococcal phage Cp-1 have been determined by using SAD technique (1,2) (see Figure 1). This is the first complete structure of a modular Choline-Binding Protein up to now repported. Cpl-1 cleaves the glycosidic N-acetylmuramoyl-(b1,4)-N-acetylglucosamine bonds of the pneumococcal glycan chain. The 3D structure presents a catalytic module (green) and a choline-binding module (cyan and magenta) joined by an acidic linker (orange).
The catalytic module displays an irregular (a/b)5b3 barrel. Among the acidic residues lining the substrate-binding site (Figure 2a), two pairs of negatively charged residues (Asp10-Asp182 and Asp92-Glu94) are disposed facing each other in the central hole (Figure 2b). Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25, in which hydrolysis occurs via a net inversion of the anomeric configuration with Asp10 acting as the general base, helping to activate the nucleophilic water molecule, and Glu94 acting as the general acid, protonating the departing oxygen atom in a concerted fashion as the bond cleaves.
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The cell wall anchoring module is formed by six similar choline-binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain (cyan in Figure 3) configuring two choline-binding sites (blue squares), and a ?-sheet domain that contributes to bring together the whole structure (magenta in Figure 3). The crystal structure of Cpl-1 complexed with choline showed that choline is located at the interface of two consecutive repeats in such a way that three structurally conserved aromatic residues (two Trp and one Tyr) form a cavity in which choline methyl groups are placed (Figure 4). In addition, a Lys residue caps the site and could stabilise the phosphate group of phosphoryl-choline through one H-bond.
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