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Journal of Bacteriology, April 2006, p. 2400-2410, Vol. 188, No. 7
0021-9193/06/$08.00+0     doi:10.1128/JB.188.7.2400-2410.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site

Denise M. Tremblay,^2,3, Mariella Tegoni,^1, Silvia Spinelli,¹ Valérie Campanacci,¹ Stéphanie Blangy,¹ Céline Huyghe,¹ Aline Desmyter,¹ Steve Labrie,^2,4 Sylvain Moineau,^2,3,4* and Christian Cambillau^1*

Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités d'Aix-Marseille I & II, Campus de Luminy, case 932, 13288 Marseille CEDEX 09, France,¹ Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire,² Félix d'Hérelle Reference Center for Bacterial Viruses,³ Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Québec City, Québec, Canada, G1K 7P4⁴

Received 15 December 2005/ Accepted 18 January 2006

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-Å resolution structure of RBP, was found to bind tightly (K_d = 0.26 µM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.

These authors contributed equally to the work.

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