Membrane Fusion Proteins of Type I Secretion System and Tripartite Efflux Pumps Share a Binding Motif for TolC in Gram-Negative Bacteria

Abstract

The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli . We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.

Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model

“…Since both the tip regions of the AcrA hexamer and the TolC trimer display six-bladed cogwheel structures, the two proteins could achieve tight packing without a gap, inducing opening of the TolC channel. This model appears to better fit the EM map, including the dilated feature at the junction of AcrA and TolC, and results from previous biochemical and genetic studies (Xu et al, 2009a(Xu et al, , 2009b(Xu et al, , 2011a(Xu et al, , 2011b(Xu et al, , 2012Yum et al, 2009;Kim et al, 2010Kim et al, , 2015Lee et al, 2012Lee et al, , 2013Lee et al, , 2014. Taken together, these data suggest the 'adapter bridging model' can be considered as a general assembly mechanism for RNDtype tripartite drug efflux pumps in Gram-negative bacteria.…”

“…Results from in vivo crosslinking experiments have greatly impacted subsequent research, including work on the E. coli heavy-metal efflux pump CusABC that appeared to oppose the adaptor wrapping model (Su et al, 2011). Functional and structural data favoring a 3:6:3 ratio and denying direct contact between AcrB and TolC have recently accumulated (Narita et al, 2003;Stegmeier et al, 2006;Mima et al, 2007;Xu et al, 2009aXu et al, , 2009bXu et al, , 2010Xu et al, , 2011aXu et al, , 2011bXu et al, , 2012Yum et al, 2009;Kim et al, 2010Kim et al, , 2015Janganan et al, 2011;Su et al, 2011;Lee et al, 2012Lee et al, , 2013Lee et al, , 2014Du et al, 2014). Thus, the plausibility of the adapter wrapping model is now weakened (Du et al, 2015), although it is still supported by some researchers (Yamaguchi et al, 2015).…”

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB

“…The procedure for MIC measurement has been previously described (Lee et al, 2012. Briefly, overnight cultures grown in LBS medium were diluted 1:100 in the same medium.…”

“…However, the mutants that were used for in vivo cross-linking appeared to be nonfunctional (Kim et al, 2010;Xu et al, 2011b), and therefore, these studies need to be reevaluated. In contrast, a recent assembly model for tripartite efflux pumps proposes a direct interaction between the α-barrel of MFPs with the α-barrel of OMFs in a tip-to-tip manner, like intermeshing cogwheels (Kim et al, 2010;Xu et al, 2011aXu et al, , 2011bLee et al, 2012Lee et al, , 2013Lee et al, , 2014Song et al, 2014). A conserved structural motif at the α-barrel of MFPs called the RLS motif has been shown to be important for this interaction.…”

News and views on protein secretion systems

“…This initial recognition of the secretion machine by the substrate triggers the recruitment of the OM TolC protein, thus completing the channel through which the substrate is transported [25]. During this process, one suggestion is that HlyD may, via its interaction with the periplasmic TolC helical tube, trigger a conformational change, which can enlarge the internal diameter of the pore [26]. The structural analysis of the T1SS actually relies mostly on the understanding of efflux pumps system [27].…”