Extended Data Figure 6: Identification, oligomerization and interaction analysis of TssA domains.
From: Priming and polymerization of a bacterial contractile tail structure
a, Limited proteolysis of purified TssA. The purified full-length TssA protein (first lane) was submitted to proteinase K limited proteolysis for the time indicated on top of each lane and analysed by SDS–PAGE and Coomassie blue staining. Stable fragments are indicated on the right with their boundaries (numbers identified in the sequence in b) and the corresponding fragment. The uncropped scan of the Coomassie blue stained gel is provided in Supplementary Figure 1. b, TssA protein sequence. The localization of the boundaries of the stable fragments obtained after Proteinase K limited proteolysis and electrospray mass spectrometry analyses are arrowed. The secondary structures observed in the crystal structures (Fig. 2a and Extended Data Fig. 6f, g) are indicated on top of the corresponding sequence. c, Bacterial two-hybrid analysis of TssANt and TssACt interactions (see legend to Extended Data Fig. 1b). d, e, MALS/QELS/UV/RI analysis of the purified TssANt (d) and TssACt (e) fragments. f, g, X-ray structure of the TssANt2 domain (PDB: 4YO3). The rainbow coloured ribbon representation of the TssANt monomer is shown (f, consecutive α-helices numbered α1 to α7) whereas the dimeric structure (g) highlights the helices at the interface (α1, α2 and α6). h, The TssA central core interacts with Hcp and VgrG whereas the TssA arms interact with TssE and TssC. Bacterial two-hybrid analysis of TssANt and TssACt interactions (see legend to Extended Data Fig. 1b). i, j, Surface plasmon resonance interaction study of the purified TssACt (i) or TssANt (j) domains with the Hcp protein (i) or the TssBC complex (j). Sensorgrams (variation of plasmon resonance in arbitrary unit (∆RU) as a function of reaction time (in seconds)) were recorded upon injection of the purified TssA C-terminal (i) or TssANt (j) domains (concentrations of 3.125 (dark grey), 6.25, 12.5, 25, 50 and 100 (light grey) μM) on HC200m chips coated with the purified Hcp protein (i) or the TssBC complex (j) (upper panels). The graph reporting ∆RU as a function of the TssA domain concentration (lower panel) was used to estimate the indicated apparent dissociation constants (Kd).
