Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.A broad number of products are manufactured by large-scale bacterial fermentation, including the value-added fermented dairy products. Most bacterial fermentation industries have experienced problems with phage contamination. Phage outbreaks are costly and time-consuming because they can slow or arrest the fermentation process and adversely affect product quality (15). For decades, the dairy industry has relied on an array of strategies to control this natural phenomenon, including rotation of their bacterial cultures (11, 24, 25). However, in spite of these efforts, new virulent lactococcal phages keep emerging. A better understanding of the various mechanisms affecting the genetic diversity of the phage population is necessary for optimal phage control strategies (18).Lactococcal phages are among the most studied bacterial viruses because of the economic importance of their hosts. Hundreds of lactococcal phages have been isolated, and the vast majority of them have a long, contractile tail, thereby belonging to the Siphoviridae family (1). Lactococcus lactis phages are currently classified into 10 genetically distinct groups (10), but only members of 3 of them are highly adapted to multiply in milk, namely, the 936, c2, and P335 groups (11, 24, 25). The first step for such an effective viral infection is host recognition, which necessitates the interaction between the adsorption device located at the distal tail end of the phage and the cell surface receptor (32). Members of the 936 and P335 groups recognize their host through an interaction between their receptor binding protein (RBP) (13) and receptors, probably lipoteichoic acids, at the host cell surface (27, 29-31).We have previously determined the crystal structures of three RBPs, from the virulent lactococcal phages p2 (30, 31) and bIL170 (936 group) (27) and from the temperate phage TP901-1 (P335 group) (29). The RBPs of these phages have a similar architecture of three protomers related by a threefold axis. Each protomer comprises three domains: the N terminus (named shoulders in p2), the interlaced β-prism linker (the “neck” domain), and the jelly-roll domain (2) at the C terminus (the “head” domain). This last domain harbors a saccharide binding site likely involved in host recognition, as it binds with high affinity to phosphoglycerol, a component of teichoic acid (8, 19, 27, 29-31). We have previously shown that the shoulder and neck domains are highly conserved in the RBPs of 936-like phages (8, 19, 27, 29-31). The individuality of the RBP C-terminal domain sequence likely dictates phage specificity for the receptor, which may specifically recognize different substitutions (H, GlcNAc, or d-Ala) of the phosphoglycerol moieties of the L. lactis teichoic acid polymers. Recently, the complete genomic sequence of the reference virulent phage P335 was determined, and comparative analysis revealed that the C terminus of its RBP showed homology to the RBP of the virulent lactococcal phage P475 of the 936 group (17). Such homology between RBP head domains was surprising because the two lactococcal phage groups rarely shared common genes or domains. This observation suggested that modular shuffling of domains can occur between these otherwise genetically distinct phage groups.The overall fold of the N-terminal RBP domain is different in 936- and P335-like phages. In the P335 group, the N-terminal domain comprises a unique helix that fits into the rest of the phage baseplate (28, 29) (Fig. ​(Fig.1A),1A), while in the 936 group, this 140-residue domain is a large β-sandwich with an external α-helix (30) (Fig. ​(Fig.1B).1B). Nonetheless, the N-terminal domains of the two RBPs may still be, related because both appear to be built using a coiled coil, although the 936-like phages have an additional β-sandwich. The β-prism linkers (neck domain) of the two phage groups also differ in sequence and in radius, but they have a similar fold, the latter being also close to that of T4 phage short fiber (33). The linker domain of phage TP901-1 is wider than that of p2 and exhibits a repeated motif (G-X-Y-X-Y, where X is polar and Y nonpolar). Finally, the C-terminal domains of both species share the same fold, a jelly-roll motif (2) also found in adenovirus (5) and reovirus (3, 4, 6).Open in a separate windowFIG. 1.Structures and sequences of RBPs from lactococcal phages. (A) Three-dimensional structure of the RBP from phage TP901-1 (P335 group; blue). (B) Three-dimensional structure of the RBP from phage p2 (936 group; magenta). (C) View of a model associating domains of TP901-1 (N terminus and linker domain, below red line, blue) and p2 (head, above red line, magenta) RBPs. (D) Three-dimensional crystal structure of chimera form 1 (yellow) assembled according to the model in panel C. (E) Sequence alignment of the RBPs of p2 (part) and TP901-1. The secondary structure is described above the alignment. The binding residues are shown with blue dots. The hinge proline (Pro 162/63) is identified by a red arrow. The chimera is composed of the N-terminal domain (residues 17 to 33) and the linker domain residues (residues 34 to 63) from phage TP901-1 RBP and the C-terminal domain (residues 163 to 264) from phage p2 RBP.The question addressed here was whether exchange between the C-terminal domains of two phage groups would lead to a stable protein with conserved binding capacity. To answer this question, we have generated an RBP chimera comprising the N-terminal and linker domains of phage TP901-1 fused to the C-terminal domain of phage p2. We have produced this chimera and determined its crystal structure and its sugar binding capacity. These results indicate that straightforward domain exchange produced a stable chimera with a conserved binding capacity and a structure close to that of each of the parental parts.