Structure and molecular assignment of lactococcal phage TP901-1 baseplate

Abstract

P335 lactococcal phages infect the gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL (-) and bppU(-), lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with light-scattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.

FIGURE 1.

X-ray structure of full-length BppL_TP901-1 trimer. A, two monomers are depicted in surface representation (green and blue), and the third monomer is in ribbon representation (red). Note the position of the N terminus stretch running parallel to the N-terminal helix. B, ribbon comparison of BppL_TP901-1 trimer cleaved form (PDB code 2F0C, gray) and the full-length form (pink, salmon, and yellow). The red arrow (Nt-short) points to the cleavage position in the previous structure, i.e. in the first turn of the shoulder domain α-helix (8).

FIGURE 2.

EM of TP901-1 BppU-BppL complex negatively stained. A, selected class averages observed in various orientations and grouped in function of feet gap size, termed packed and open conformation. White arrows outline a 20-Å feet particle displacement. Each image corresponds to 360 × 360 Å. B, surface representation of the BppU-BppL three-dimensional EM reconstruction at 24.8 Å resolution. Scale bar, 25 Å. C, view of the tripod EM density with three BppL trimers fitted in the three feet. The dome and protuberance regions are attributed to the BppU trimer.

FIGURE 3.

EM of TP901-1 wild-type and mutant BP negatively stained. A, wild-type BP is depicted in light blue, the bppL⁻ mutant in gray, and the bppU⁻ mutant in yellow. The last EM density region contains a dodecamer of Dit and a trimer of Tal. The approximate Dit and Tal positions are indicated by red and blue arrows, respectively. The density corresponding to the wild-type and BppL⁻ mutants has been cut away in the foreground for clarity. B, fitting of one tripod (blue) and three RBPs into their corresponding regions of the wild-type BP EM reconstruction (yellow). The two red arrows indicate the positions of the faint/broken density of the third tripod leg.

FIGURE 4.

Pseudo-atomic model of the TP901-1 BP. A, EM reconstruction of the wild-type TP901-1 BP. From top to bottom, the following molecules, depicted in ribbon rendering, have been fitted into the BP EM density map: Hcp1 hexamers (light blue and yellow); a dodecamer of SPP1 Dit N-terminal moieties (red); the 54 BppL molecules (organized in six tripods of three RBPs each, dark blue); the position and volume of the Tal N-terminal part are tentatively represented by phage p2 ORF16 (pink). B, fitting of the same molecules as in A into the bppU⁻ BP reconstruction (note the absence of BppL and BppU molecules). C, cut away representation of B. D, clipped view of the wild-type TP901-1 BP EM reconstruction with the components assigned highlighted with different colors.

FIGURE 5.

Surface plasmon resonance (BIAcore) experiments with the 3:9 BppU-BppL complex (tripod). A, when the tripods are attached to the CM5 chip and DARPin 20 used as analyte, we measured a K_D of 22 ± 3 nm. B, when DARPin 20 is attached to the chip and the tripod used as analyte, the K_D was 440 times lower than in A, illustrating a strong avidity effect.