Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Abstract

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

From conjugation to T4S systems in Gram‐negative bacteria: a mechanistic biology perspective

“…At this point in time, two architectures of the T4S systems have been proposed. One was observed by negativestain EM on purified materials and also observed in situ by cryo-ET [14,16,56, preprint: 69,73] and consisted of multiple hexameric ATPase barrels, some laterally located [14, preprint: 69,73], and some both laterally and centrally located [16,56] ( Figs 3B and C, and 4). The other was observed in situ by cryo-ET [15] and consisted of stacked ATPases, one of them a hexamer of dimers ( Fig 3C).…”

“…Indeed, VirD4 and VirB4 have strikingly similar structures . Moreover, in both the VirB3‐10 structure by Low et al and that of VirB3‐10/VirD4 by Redzej et al , the VirB4 barrels are distinctly formed of trimers of dimers with the dimers appearing to interact loosely with one another. Thus, it is not far‐fetched to hypothesize that VirD4 might form mixed hexamers with VirB4, a transition that would facilitate the handover and transition of the T4S system from a protein transporter mediated by VirB4 to a DNA transporter mediated by VirD4.…”

“…A similar secretion chamber was described for the Legionella dot/icm system [preprint : 69]. In the latest Legionella tomography work, a stalk is also visible, made by DotG/VirB10, and forms an uninterrupted funnel/channel [56]. Right panels: Schematics of IMC ATPases.…”

“…The white arrows indicate the transfer route for each option. The T4S system and the relaxosome are as in Fig 4. ª 2019 The Author EMBO reports 20: e47012 | 2019 a central channel, and some other times the presence of one [14][15][16]56,73]. Because of the multiple processes sometimes taking place simultaneously during conjugation, the mechanism of conjugation is by and large still unclear, although recent breakthroughs have shed light on some of its aspects.…”

Two pKM101‐encoded proteins, the pilus‐tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer

“…Mobile genetic elements are composed of genes encoding three functional modules: (i) the translocation channel, (ii) mobilization (mob) proteins that assemble as a catalytically active relaxosome to initiate DNA substrate nicking at cognate origin-of-transfer (oriT) sequences and (iii) the T4CP that physically links the relaxosome with the translocation channel (de la Cruz et al, 2010). To facilitate genetic manipulations of large MGEs, these functional modules can be expressed from separate plasmids, as shown recently for the pKM101 and R388 transfer systems (Gordon et al, 2017;Redzej et al, 2017). In this study, we expressed tra genes encoding the Tra pKM101 channel/ pilus from the nontransmissible plasmid pCGR125 ( Fig.…”