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J. Biol. Chem., Vol. 283, Issue 42, 28618-28628, October 17, 2008

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

Begoña Monterroso¹, José Luis Sáiz, Pedro García^¶, José Luis García^¶, and Margarita Menéndez²

From the Instituto de Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain and the CIBER de Enfermedades Respiratorias (CIBERES) and ^¶Centro de Investigaciones Biológicas Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain

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.

Received for publication, April 14, 2008 , and in revised form, July 30, 2008.

^* This work was supported by Dirección General de Investigación Cientifica y Técnica Grants BIO2003-01952, BFU2006-10288, and SAF2006-00390. Additional funding was provided by the COMBACT program (S-BIO-0260/2006) of the Comunidad de Madrid, the CIBER of Respiratory Diseases (CIBERES), an initiative of the ISCIII, and the Glycodynamics Network (FP6-UE and MCTN-CT-2005-019561). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3 and Tables 1 and 2.

¹ Supported by a fellowship from the Spanish Ministerio de Ciencia y Tecnología. Present address: Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, MD 20892.

² To whom correspondence should be addressed. Tel.: 34-915619400; Fax: 34-915642431; E-mail: mmenendez{at}iqfr.csic.es.

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