8.A.1 The Membrane Fusion Protein (MFP) Family
Proteins of the MFP family function as auxiliary proteins or 'adaptors', connecting a primary porter in the cytoplasmic membrane of a Gram-negative bacterium with an outer membrane factor (OMF; TC #1.B.17) protein that serves a porin or channel function in the outer membrane (Touzé et al., 2004). Thus, in conjunction with an MFP and an OMF, the primary porter in the cytoplasmic membrane pumps molecules out of the cytoplasm, across both membranes of the cell envelope into the external milieu without equilibration with solutes in the periplasm. Crosslinking studies of the AcrA (TC #8.A.1.6)-AcrB (TC #2.A.6.2.2)-TolC (TC #1.B.17.1.1) system revealed that AcrA could be crosslinked to both AcrB (via the C-terminal β-barrel domain of AcrA) and TolC (via the central coiled-coil of AcrA) (Touzé et al., 2004). Mutations in MFPs allow cross activity with different RND-type transporters (Krishnamoorthy et al., 2008).
Most MFPs are about 350-500 residues and probably either span the cytoplasmic membrane once at their N-termini or are anchored to the cytoplasmic membrane via a lipoyl moiety. These proteins cluster in the phylogenetic tree into subfamilies in accordance with the type of cytoplasmic membrane transport system [MFS (TC #2.A.1); RND (TC #2.A.6) or ABC (TC #3.A.1)], with which they interact. At least one MFP, MexA of Pseudomonas aeruginosa, appears to function normally when its N-terminal transmembrane helix is artificially removed (Yoneyama et al., 2000). Evidence that the E. coli MFP, EmrA, which functions with a drug efflux MFS permease, is trimeric and can bind drugs to its periplasmic domain (Borges-Walmsley et al., 2003).
The structure of MexA of P. aeruginosa was solved (Higgins et al., 2004). The protein is elongated with three linearly arranged subdomains as suggested based on secondary structural predictions (Dinh et al., 1994). The molecule consists of an N-terminal lipoyl domain, a central 47 Å long α-helical hairpin domain, and a C-terminal six-stranded β-barrel. In the crystal, hairpins of neighboring MexA monomers pack side by side to form twisted arcs. These MFPs are not true membrane fusion proteins, but serve as 'adaptors' that assemble and control conformational channel opening in the complex (Higgins et al., 2004; Touzé et al., 2004). The crystal structures of other MFPs have been solved (e.g., see Yum et al., 2009). Many transport operons contain two- or three genes encoding distinct MFPs. Zgurskaya et al., 2009 discuss the diversity of MFPs in the context of current views on the mechanism and structure of MFP-dependent transporters.
Gram-positive bacteria have MFP homologues that function as essential accessory factors for the export of bacteriocins and competence peptides via ABC type transporters (Harley et al., 2000). They exist in two sizes, full length proteins (i.e., TC# 8.A.1.4.1) and internally truncated proteins with shortened central α-helical coiled-coil domains (TC# 8.A.1.5.1). The 'adaptor' function proposed above for Gram-negative bacterial MFPs does not explain the requirement of Gram-positive bacterial transporters for these auxiliary proteins.
HlyD (8.A.1.3.1), an MFP that functions with an ABC exporter, was subjected to random point mutation. The different mutants were blocked at different stages of HlyA translocation. Some proved to be defective in HlyA folding. These mutants mapped to the C-terminal lipoyl repeat motif involved in switching from the helical hairpin to the extended form of HlyD during assembly of the functional channel. It was concluded that HlyD is an integral component of the transport pathway, but that it also functions in the final folding of HlyA to its active form (Pimenta et al., 2005).
Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. In the E. coli AcrAB-TolC pump, the inner membrane transporter, AcrB, requires the outer membrane factor, TolC, and the periplasmic adapter protein, AcrA. Xu et al. (2011) proposed a hexameric model of the adapter protein, a trimer of dimers. Its channel-forming property determines the substrate specificity. The hexameric adapter protein binds to the outer membrane factor in an intermeshing cogwheel manner and to the periplasmic region of the inner membrane transporter. An adapter-bridging model for the assembly of the tripartite pump was proposed (Xu et al., 2011).