Functional Interactions of VirB11 Traffic ATPases with VirB4 and VirD4 Molecular Motors in Type IV Secretion Systems

Abstract

Pilus biogenesis and substrate transport by type IV secretion systems require energy, which is provided by three molecular motors localized at the base of the secretion channel. One of these motors, VirB11, belongs to the superfamily of traffic ATPases, which includes members of the type II secretion system and the type IV pilus and archaeal flagellar assembly apparatus. Here, we report the functional interactions between TrwD, the VirB11 homolog of the conjugative plasmid R388, and TrwK and TrwB, the motors involved in pilus biogenesis and DNA transport, respectively. Although these interactions remained standing upon replacement of the traffic ATPase by a homolog from a phylogenetically related conjugative system, namely, TraG of plasmid pKM101, this homolog could not replace the TrwD function for DNA transfer. This result suggests that VirB11 works as a switch between pilus biogenesis and DNA transport and reinforces a mechanistic model in which VirB11 proteins act as traffic ATPases by regulating both events in type IV secretion systems. Type IV secretion systems (T4SSs) export proteins and virulence effectors to other bacteria and eukaryotic cells (1-3). They are also responsible for genetic exchange between bacteria during conjugation (4, 5). T4SSs are large macromolecular assemblies formed by 12 different protein subunits, named VirB1 to VirB11 and VirD4, following the Agrobacterium tumefaciens T4SS nomenclature. Three of these proteins (VirD4, VirB4, and VirB11) are hexameric ATPases (6-8) that provide the energy for substrate transport and T4SS biogenesis.VirB11 proteins are traffic ATPases that belong to a large AAA ϩ secretion protein superfamily, which also includes proteins of type II secretion systems and the type IV pilus biogenesis and archaeal flagellar assembly machineries (9). All of them are soluble, hexameric proteins located at the cytoplasmic side of the inner membrane. The monomer is characterized by the presence of an N-terminal domain (NTD) and a C-terminal domain (CTD), which are connected by a flexible linker that plays a key role in the catalytic cycle (10-12). Comparison of the crystal structures of VirB11 from Brucella suis (13) and its homolog in Helicobacter pylori, HP0525 (10,14), revealed that this linker is responsible for a large domain swap of the NTD over the CTD without affecting the hexameric assembly (13).VirB11 was reported to assist VirB4 during pilus biogenesis by dislocating pilin subunits from the inner membrane to the periplasmic space, thus promoting pilus polymerization (15). VirB4 proteins are the largest and most conserved components of T4SSs. TrwK, the VirB4 homolog of plasmid R388, consists of a hexameric double ring with a barrel-shaped structure (16). The atomic structure of the C-terminal domain of the VirB4 homolog in Thermoanaerobacter pseudethanolicus was recently obtained (17), revealing a striking structural similarity with TrwB, the VirD4 homolog of the R388 plasmid. TrwB was extensively characterized as a DNA-dependent ATPase (6, 18), playing an ess...

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

“…To fulfill their coupling functions, T4CPs must interact with secretion substrates and cognate T4SS channels. The latter contacts were not a major focus of our study, but prior work has shown that T4CPs interact with VirB10-like subunits, as well as the VirB4-and VirB11-like ATPases (15,18,30,44,47,82). This interaction network is required not only for early-stage reactions mediating substrate transfer across the inner membrane but also for transduction of intracellular signals across the T4SS channel, enabling substrate translocation to the cell exterior.…”

“…The nature of these or other T4SS ATPase interactions is not fully defined but is predicted to involve contacts between their cytoplasmic domains (37,82,87,88). Accordingly, the VirD4 moieties of the chimeric T4CPs must productively engage with the pKM101-encoded VirB4-like TraB and VirB11-like TraG ATPases for protein transfer through the pKM101 channel.…”

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

“…VirD4 and VirB4, though expected to form hexamers, have been identified in monomeric and dimeric forms as well (Gomis‐Ruth et al , 2001; Schroder et al , 2002; Rabel et al , 2003; Arechaga et al , 2008; Mihajlovic et al , 2009; Durand et al , 2011; Pena et al , 2012; Wallden et al , 2012). All three ATPases interact with one another and these interactions likely direct the two processes in which T4S systems are involved: pilus biogenesis and substrate secretion (Atmakuri et al , 2004; Ripoll‐Rozada et al , 2013). VirD4 also interacts with the N‐terminally located transmembrane helices of VirB10 (Llosa et al , 2003; Ripoll‐Rozada et al , 2013; Segura et al , 2013).…”

“…All three ATPases interact with one another and these interactions likely direct the two processes in which T4S systems are involved: pilus biogenesis and substrate secretion (Atmakuri et al , 2004; Ripoll‐Rozada et al , 2013). VirD4 also interacts with the N‐terminally located transmembrane helices of VirB10 (Llosa et al , 2003; Ripoll‐Rozada et al , 2013; Segura et al , 2013). Although the general organization of the conjugative T4S system is well known, the details of the substrate secretion mechanism are still unclear.…”

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

“…A second ATPase, VirB11, was shown to be important for pilus assembly 112 , but no VirB11 homologue has been identified for the F-plasmid-encoded T4SS so far 113 . Therefore, the assembly of the F pilus might be powered solely by the activity of the F system's VirB4 homologue, with the help of some other T4SS IMC components.…”