Crystal Structure of pb9, the Distal Tail Protein of Bacteriophage T5: a Conserved Structural Motif among All Siphophages

Abstract

fThe tail of Caudovirales bacteriophages serves as an adsorption device, a host cell wall-perforating machine, and a genome delivery pathway. In Siphoviridae, the assembly of the long and flexible tail is a highly cooperative and regulated process that is initiated from the proteins forming the distal tail tip complex. In Gram-positive-bacterium-infecting siphophages, the distal tail (Dit) protein has been structurally characterized and is proposed to represent a baseplate hub docking structure. It is organized as a hexameric ring that connects the tail tube and the adsorption device. In this study, we report the characterization of pb9, a tail tip protein of Escherichia coli bacteriophage T5. By immunolocalization, we show that pb9 is located in the upper part of the cone of the T5 tail tip, at the end of the tail tube. The crystal structure of pb9 reveals a two-domain protein. Domain A exhibits remarkable structural similarity with the N-terminal domain of known Dit proteins, while domain B adopts an oligosaccharide/ oligonucleotide-binding fold (OB-fold) that is not shared by these proteins. We thus propose that pb9 is the Dit protein of T5, making it the first Dit protein described for a Gram-negative-bacterium-infecting siphophage. Multiple sequence alignments suggest that pb9 is a paradigm for a large family of Dit proteins of siphophages infecting mostly Gram-negative hosts. The modular structure of the Dit protein maintains the basic building block that would be conserved among all siphophages, combining it with a more divergent domain that might serve specific host adhesion properties.

Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components

“…Together with the localization of pb9, this strongly suggests that pb9 is the Dit protein of phage T5. This was recently confirmed by the resolution of the crystal structure of pb9, which reveals a two-domain protein, one domain of which shows structural similarity with the N-terminal domain of the Dit proteins of phages SPP1, p2, and TP901-1, infecting Gram-positive bacteria (56).…”

“…We used antibodies directed against the pb9 and pb3 proteins to localize these proteins within the tail tip. We visualized the sites of cross-links and gold labeling with anti-pb9 in the upper part of the cone, right under the collar onto which are attached the L-fibers (see accompanying paper by Flayhan et al [56]) and in the lower part of the cone with anti-pb3 IgGs (Fig. 3C).…”

“…While the proteins forming the top of the tail-tail terminator T5p142 and tail completion T5p143-are likely organized like their homologous proteins in most other siphophages, the proteins forming the tail tip architecture show an unusual topology. The conical structure of the tail tip is formed by two proteins: pb9, the Dit protein located in the upper part (56), and pb3, located in the lower part in contact with the central straight fiber. The Dit protein pb9 would serve as the docking site for the long L-shaped side fibers and their collar base (formed by pb1 and the newly identified T5p132 protein).…”

Molecular architecture of tailed double-stranded DNA phages

“…Proteins analogous to Dit were also found in the Gram-positive infecting phage SPP1 155 and in the Gram-negative infecting phage T5, 143,156 which do not possess baseplates, but have tapered tail tip complexes. Phage T5 encodes a separate protein analogous to p2_Tal and T4_gp27, 143,156 which binds to Dit and attaches a long central tail spike, whereas phage SPP1 has a Tal/gp27-like region at the N-terminus of its central fiber protein.…”

“…Phage T5 encodes a separate protein analogous to p2_Tal and T4_gp27, 143,156 which binds to Dit and attaches a long central tail spike, whereas phage SPP1 has a Tal/gp27-like region at the N-terminus of its central fiber protein. 157 The central part of the tail tip complex has a common architecture in the long-tailed phages.…”