The cryo-EM structure of the T4S system core complex revealed a double-walled structure in the cap of the O-layer (Supplementary Fig. 10a
. The crystal structure of the cap is not double-walled. This is because the lipid moiety of TraN/VirB7 was not built (the SAD-derived map showed electron density connecting to Cys15 of TraN/VirB7 (the lipidation site) but the density was not sufficiently well-defined for unambiguous fitting). However, when the crystal structure of the outer membrane complex is superimposed on the cryo-EM structure of the entire complex (), Cys15 of TraN aligns perfectly at the base of the outer wall of the cap, suggesting that this part of the cap might indeed contain the lipidated part of TraN (indicated in in black dots).
The superposition of the structure of the outer membrane complex and that of the core complex (; such a superposition is valid as one complex is derived from the other by proteolysis) also reveals that the trans-membrane helices overlap partially with the first half of the inner wall of the cap, suggesting that they might form this region of the cap. However, the crystal structure captures them in a different conformation than the one observed in the cryo-EM structure: while in the cryo-EM structure, they form a narrow vertical constriction, in the crystal structure, they lie in a “relaxed” state at a 45° angle. Thus, the two structures may represent different states. Presumably, by removing the entire N-terminal half of TraF, the constraints that this half places on the N-terminal lever arm of TraFCT
might have been removed, releasing the lever arms and leading to relaxation of the trans-membrane helices. Indeed, as illustrated in , sequences N-terminal to the αn
1 helix would directly connect to the I-layer (schematically shown in by dashed red lines) and are likely to bring the N-terminal arm of each TraF/VirB10 subunit down. This is consistent with the fact that the shelf formed by the N-terminal lever arms of TraF/VirB10 subunits has shifted up in the crystal structure compared to its position in the cryo-EM structure of the full-length core complex (, Supplementary Figs. 10a and 10b
in orange and red, respectively). Thus, we propose that the N-terminal arms of the TraF/VirB10 subunits might act in concert to exert conformational changes in the T4S system channel upon signals sensed by the N-terminal domain of the protein (see below).
In A. tumefaciens
, VirB10 is known to undergo a conformational change induced by the energizing T4S system components18
. The structure of the T4S system outer membrane complex reveals that VirB10 forms the outer membrane channel. But VirB10 is also known to insert in the inner membrane, making contact not only with the inner membrane channel component VirB8, but also with the ATPases13,19,20
. Thus, VirB10 is in a unique position to relay conformational changes taking place in the ATPases and to effect pore opening and closure at the outer membrane. Also, in A. tumefaciens
, VirB10 does not directly contact the T-DNA (the T4S substrate), but regulates its handover from the VirB6/VirB8 complex in the inner membrane to VirB9 and VirB2, the major pilin21
. As VirB10 lines the interior of the outer membrane complex, it is difficult to envisage how the substrate could not interact with it, unless it is insulated from the substrate by another layer of protein, presumably made of the VirB2 pilin. We thus propose that the VirB2 pilin forms a cylindrical conduit encased within the VirB10 ring. This hypothesis is also consistent with the observation that, in the state captured in the crystal structure (where VirB2 is absent), the TraF/VirB10 trans-membrane helices have somewhat caved in.
The crystal structure of the T4S system outer membrane complex reveals an outer membrane structure of unprecedented size and complexity. This structure is held together by a dense network of protein-protein interactions, which provides a rich targeting ground for inhibitor design. Most striking among them is the extensive interaction that the N-terminal lever arms of the TraFCT subunits make with numerous subunits along the tetradecameric structure. We hypothesized that these sequences are at the heart of a nano-device regulating T4S. If confirmed, this mechanism could become key to the design of inhibitor compounds specifically targeting the T4S machinery.