Insights into the Structure-Function Relationships of Pneumococcal Cell Wall Lysozymes, LytC and Cpl-1

Abstract

The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30°C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4°C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 ؎ 0.5 sites/monomer) behave as equivalent (K d ‫؍‬ 2.7 ؎ 0.3 mM). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37°C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37°C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.

Improving the Lethal Effect of Cpl-7, a Pneumococcal Phage Lysozyme with Broad Bactericidal Activity, by Inverting the Net Charge of Its Cell Wall-Binding Module

“…The purity of the isolated protein was checked by SDS-PAGE (12% acrylamide-bisacrylamide) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry before storage at Ϫ20°C in 20 mM phosphate buffer (pH 6.0). Purification of the other enzybiotics was performed as previously described (17,(25)(26)(27), and protein concentrations were determined spectrophotometrically using the …”

“…Viability was determined after 1 h of incubation. b Ϫ, no effect; ϩ, decrease of 1 log unit in viable cells; ϩϩ, decrease of 2 log units in viable cells; ϩϩϩ, decrease of 3 log units in viable cells; ϩϩϩϩ, decrease of Ն4 log units in viable cells; *, Ͻ10 CFU · ml respective molar absorption coefficients at 280 nm (17,(25)(26)(27). Before use, all proteins were equilibrated in 20 mM sodium phosphate buffer, pH 6.0 (P i buffer).…”

Cpl-7, a Lysozyme Encoded by a Pneumococcal Bacteriophage with a Novel Cell Wall-binding Motif*

“…The crystallographic structure of Cpl-1, both in its free-state and bound to muropeptide analogues, has been reported (9,10) and its solution behavior characterized both in the absence and in the presence of choline (11,12). In contrast, although the CM of Cpl-7 has been recently crystallized (13), there is no available information about the overall structure of the enzyme or its CWBR.…”

“…Major changes would arise from substitutions at positions 12 and 96. The change of Ser 12 by alanine would prevent the H-bond formed between the serine oxygen of Cpl-1 and the lactil group of MurNAc at position Ϫ1 (10). Moreover, the substitution of Asp 96 by histidine also implies the loss of the H-bonds discerned in Cpl-1 between the carboxylate oxygens of Asp 96 and the Lys moiety of the peptide bound to the same MurNAc unit.…”

Insights into the regulation of bacteriophage endolysin: multiple means to the same end

“…Dimerization-based endolysin activity regulation has been reported in the case of Cpl-1 endolysin produced by Cp-1 bacteriophage that infects S. pneumoniae. Cpl-1 endolysin shows enhanced activity upon dimerization that is a result of the interaction between its CBD and the bacterial cell wall component choline (Sanz et al, 1992;Buey et al, 2007;Monterroso et al, 2008). It is worth adding here that introduction of a disulphide bond leading to the stabilization of the dimeric form of Cpl-1 resulted in higher activity of the endolysin and reduced plasma clearance (Resch et al, 2011).…”