English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/54059
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL | DATACITE
Exportar a otros formatos:


Insights into the structure-function relationships of pneumococcal cell wall lysozymes: LytC and Cpl-1

AuthorsMonterroso, Begoña ; Saiz, José L.; García, Pedro ; García, José Luis ; Menéndez, Margarita
Issue Date17-Oct-2008
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry, 283 : 28618-28628 (2008)
AbstractThe 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 (Kd = 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.
The integrity of the cell wall is essential for bacterial survival, and peptidoglycan-metabolizing enzymes have to work closely together to prevent the rupture of the cell wall and the cell lysis during cell growth and division (1). In particular, the potentially suicidal activity of autolytic enzymes should be strictly controlled (2), and tight binding to the cell envelope (3) seems to work as a mechanism for the regulation and control of many murein hydrolases from different bacterial species (4). In Streptococcus pneumoniae, the presence of phosphorylcholine residues on lipoteichoic and teichoic acids (5), a characteristic shared by other related microorganisms (6), determines the activity of murein hydrolases. In addition to the catalytic module (CM),3 these enzymes comprise a choline-binding module (CBM) that specifically recognizes and binds the choline residues on the cell wall (4). Moreover, it has been suggested that phosphorylcholine residues would be acting as a selective pressure factor to preserve the cell wall recognition module in all of the murolytic enzymes encoded by pneumococcus so far characterized. The CBM, usually located at the C terminus of choline-binding proteins, is made up of tandemly arranged homologous repeats of about 20 amino acids (Pfam accession code PF01473) (7). Each repeat forms a β-hairpin followed by a loop and a coiled region that tend to fold into a left-handed β-solenoid (8-10). Choline binds at the interface of two consecutive β-hairpins, in a cavity lined by three aromatic side chains (8-10), and the multivalent interacting surface presented by the CBM can provide a strong binding to the pneumococcal envelope. The same type of modular organization is found in the endolysins produced by most pneumococcal bacteriophages and suggests a genetic interchange during evolution between the host and the parasites (7).
Only two autolytic enzymes (autolysins) have been unequivocally identified so far in S. pneumoniae: the well known LytA amidase and the LytC lysozyme (11). LytC is an autolysin designed to remodel the cell wall, with maximum activity at 30 °C. This feature suggests that LytC could be more active in habitats like the upper, well ventilated respiratory tract (11). In fact, LytC plays a role in the colonization of the rat nasopharynx (12), where it could also contribute to DNA release in competent cells (13). LytC is directed to the outer surface by a leader peptide (33 residues), and it remains tightly bound to the cell wall in a mature, active form that comprises a CBM made of 11 (p1-p11) repeating units (264 residues) and a CM belonging to the GH-25 family of glycosyl hydrolases (204 residues). The unprocessed form is also detected in the cytoplasm (11). The slow hydrolysis of pneumococcal cultures carried out by LytC contrasts with the fast, uncontrolled lysis of the host cell performed by Cpl-1, the lysozyme encoded by the pneumococcal phage Cp-1, in order to release the infective particles after the virion replication (11). Cpl-1 and LytC are built of homologous modules (Fig. 1) although assembled in opposite locations (7). In Cpl-1, whose three-dimensional structure has been solved (9), the catalytic module is localized at the N terminus (188 residues), and the CBM (139 residues) is made up of six repeating units (p1-p6) and a short terminal tail (7). However, only two choline-binding sites, located at the interfaces of the three first repeats (p1-p2 and p2-p3), seem to be functional, according to the crystal structure of Cpl-1 (9). In contrast, molecular modeling of LytC reveals a higher number of potential sites for choline. In addition, the alternation of shorter (17 residues) and longer (21-23 residues) repeats (see Fig. 1) in LytC leads to an uneven distribution of choline-binding sites along the CBM surface (14), previously unseen in other CBMs of known structure (8-10). We have performed a comparative study of LytC and Cpl-1 lysozymes using different approaches. The differences found in structure, stability, conformational plasticity, choline-binding avidity, and complex dynamics contribute to determine the lytic profiles of these two lysozymes. Overall, LytC and Cpl-1 represent a good illustrative example of how natural selection can tailor at the genetic level different solutions using homologous building blocks and module shuffling
Description11 páginas. 7 figuras, 4 tablas, 1 esquema --pags NROS. 28618-28628
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M802808200
Appears in Collections:(CIB) Artículos
Files in This Item:
File Description SizeFormat 
J. Biol. Chem.-2008-Monterroso-28618-28.pdf1,09 MBAdobe PDFThumbnail
Show full item record
Review this work

Related articles:

WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.