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).


 

References:

Baranova, N. and H. Nikaido. (2002). The BaeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate. J. Bacteriol. 184: 4168-4176.

Binet, R., S. Létoffé, J.M. Ghigo, P. Delepelaire, and C. Wandersman. (1997). Protein secretion by Gram-negative bacterial ABC exporters–a review. Gene 192: 7-11.

Borges-Walmsley, M.I., J. Beauchamp, S.M. Kelly, K. Jumel, D. Candlish, S.E. Harding, N.C. Price, and A.R. Walmsley. (2003). Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA. J. Biol. Chem. 278: 12903-12912.

Dinh, T., I.T. Paulsen, and M.H. Saier, Jr. (1994). A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of Gram-negative bacteria. J. Bacteriol. 176: 3825-3831.

Gimmestad M., M. Steigedal, H. Ertesvag, S. Moreno, B.E. Christensen, G. Espin, and S. Valla. (2006). Identification and characterization of an Azotobacter vinelandii Type I secretion system responsible for export of the AlgE-Type Mannuronan C-5-Epimerase. J. of Bacteriol. 188(15):5551-5560.

Glaser, P., H. Sakamoto, J. Bellalou, A. Ullmann, and A. Danchin. (1988). Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO. J. 7: 3997-4004.

Gupta, A., K. Matsui, J.-F. Lo, and S. Silver. (1999). Molecular basis for resistance to silver cations in Salmonella. Nature Med. 5: 183-188.

Guthmiller, J.M., D. Kolodrubetz, and E. Kraig. (1995). Mutational analysis of the putative leukotoxin transport genes in Actinobacillus actinomycetemcomitans. Microb. Pathog. 18: 307-321.

Harley, K.T., G.M. Djordjevic, T.T. Tseng, and M.H. Saier, Jr. (2000). Membrane fusion protein homologues in Gram-positive bacteria. Mol. Microbiol. 36: 516-517.

Higgins, M.K., E. Bokma, E. Koronakis, C. Hughes, and V. Koronakis. (2004). Structure of the periplasmic component of a bacterial drug efflux pump. Proc. Natl. Acad. Sci. USA 101: 9994-9999.

Krishnamoorthy, G., E.B. Tikhonova, and H.I. Zgurskaya. (2008). Fitting periplasmic membrane fusion proteins to inner membrane transporters: mutations that enable Escherichia coli AcrA to function with Pseudomonas aeruginosa MexB. J. Bacteriol. 190: 691-698.

Lee, E.H., S.A. Hill, R. Napier, and W.M. Shafer. (2006). Integration Host Factor is required for FarR repression of the farAB-encoded efflux pump of Neisseria gonorrhoeae. Mol Microbiol. 60: 1381-1400.

Létoffé, S., P. Delepelaire, and C. Wandersman. (1996). Protein secretion in Gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding. EMBO J. 15: 5804-5811.

Nishino, K. and A. Yamaguchi. (2001). Analysis of a complete library of putative drug transporter genes in Escherichia coli. J. Bacteriol. 183: 5803-5812.

Paulsen, I.T., J.H. Park, P.S. Choi, and M.H. Saier, Jr. (1997). A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol. Lett. 156: 1-8.

Pimenta, A.L., K. Racher, L. Jamieson, M.A. Blight, and I.B. Holland. (2005). Mutations in HlyD, part of the Type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin. J. Bacteriol. 187: 7471-7480.

Sulavik, M.C., C. Houseweart, C. Cramer, N. Jiwani, N. Murgolo, J. Greene, B. DiDomenico, K.J. Shaw, G.H. Miller, R. Hare, and G. Shimer. (2001). Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob. Agents Chemother. 45: 1126-1136.

Touzé, T., J. Eswaran, E. Bokma, E. Koronakis, C. Hughes, and V. Koronakis. (2004). Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system. Mol. Microbiol. 53: 697-706.

Van Dyk T.K., L.J. Templeton, K.A. Cantera, P.L. Sharpe, F.S. Sariaslani. (2004). Chacracterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve? Journal of Bacteriol. 186:7196-7204.

Xu, Y., M. Lee, A. Moeller, S. Song, B.Y. Yoon, H.M. Kim, S.Y. Jun, K. Lee, and N.C. Ha. (2011). Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria. J. Biol. Chem. 286: 17910-17920.

Yoneyama, H., H. Maseda, H. Kamiguchi, and T. Nakae. (2000). Function of the membrane fusion protein, MexA, of the MexA, B-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane. J. Biol. Chem. 275: 4628-4634.

Yum, S., Y. Xu, S. Piao, S.H. Sim, H.M. Kim, W.S. Jo, K.J. Kim, H.S. Kweon, M.H. Jeong, H. Jeon, K. Lee, and N.C. Ha. (2009). Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump. J. Mol. Biol. 387: 1286-1297.

Zgurskaya, H.I., Y. Yamada, E.B. Tikhonova, Q. Ge, and G. Krishnamoorthy. (2009). Structural and functional diversity of bacterial membrane fusion proteins. Biochim. Biophys. Acta. 1794: 794-807.

Examples:

TC#NameOrganismal TypeExample
Examples:

TC#NameOrganismal TypeExample
8.A.1.1.1

Membrane Fusion Protein, EmrA, that function with MFS multidrug exporter, EmrB (Nishino and Yamaguchi 2001).

Multiple drugs

EmrA of E. coli

 
8.A.1.1.2The MFP cluster 1 protein, FarA (efflux of antimicrobial long chain fatty acids (Lee et al., 2006); functions with MFS carrier, FarB (TC# 2.A.1.3.20))Bacteria FarA of Neisseria gonorrhoeae (Q9RQ30)
 
8.A.1.1.3

MdtN; acts with MdtO (TC# 2.A.85.6.1) and MdtP (TC# 1.B.17.3.9) (Sulavik et al., 2001).

Bacteria

MdtN of E. coli (B1LPP9)

 
8.A.1.1.4

Membrane fusion protein, YiaV

Proteobacteria

YiaV of E. coli

 
Examples:

TC#NameOrganismal TypeExample
8.A.1.2.1

CzcB of MFP cluster 2 (functions with RND porter CzcA, TC# 1.A.6.1.2 and CzcC, TC# 1.B.17.2.1).

Multiple drugs; heavy metals; oligosaccharides

CzcB of Alcaligenes eutrophus

 
8.A.1.2.2

Uncharacterized MFP of 314aas and 1 TMS.

MFP of Bdellovibrio exovorus

 
Examples:

TC#NameOrganismal TypeExample
8.A.1.3.1MFP cluster 3 (function with ABC porters) (Pimenta et al., 2005)Proteins, peptides HlyD of E. coli
 
8.A.1.3.2MFP (cluster3). CyaD (functions with CyaB (TC# 3.A.1.109.2)) (Glaser et al., 1988)BacteriaCyaD of Bordetella pertussis (P11091)
 
8.A.1.3.3

The MFP, EexE of cluster 3 (functions with the ABC porter EexD, (TC# 3.A.1.110.10) and OMF EexF (TC# 1.b.17.1.3)) (Gimmestad et al., 2006

).

Bacteria

EexE of Azotobacter vinelandii (C1DS85)

 
8.A.1.3.4

Leukotoxin export MFP protein of 457 aas, TdcA (Guthmiller et al. 1995).  Functions with the ABC exporter, LtxB (TC# 3.A.109.8) and the TolC-like OMF protein, TdeA (TC# 1.B.17.3.11).

Leukotoxin export MFP of Aggregatibacter (Actinobacillus; Haemophilus) actinomycetemcomitans

 
8.A.1.3.5

Uncharacterized MFP of 346 aas and 1 N-terminal TMS. Possibly functions with an RND pump.

UP of Parvularcula oceani

 
8.A.1.3.6

Uncharacterized protein of 572 aas with two TMSs, one at residue 170 (the beginning of the MFP domain) and the last one at the C-terminus of the protein.  This first 160 aas are hydrophilic and are not homologous to most other MFPs. The gene encoding this protein (OUT43425) is adjacent to an ABC protein (OUT43424 of 719 aas)  that is homoloous to the entirety of the leukotoxin export protein, LtxB, of 707 aas with an M-C domain order (TC# 3.A.1.109.8), that functions with an MFP, LpxD (TC# 8.A.1.3.4), and a TolC-like protein for the export of the toxin from the cytoplasm to the external medium in one energy-coupled step.

UP of Pelagibacteraceae bacterium TMED13 (marine metagenome)

 
Examples:

TC#NameOrganismal TypeExample
8.A.1.4.1

Mesenterecin Y105 (bacteriocin) secresion accessory protein, MesE  of 457 aas (cluster 4). Functions with MesD; TC#3.A.1.112.8.

Firmicutes

MesE of Leuconostoc mesenteroides

 
8.A.1.4.2Competence factor transport accessory protein, ComB Competence peptide ComB of Streptococcus pneumoniae
 
Examples:

TC#NameOrganismal TypeExample
8.A.1.5.1Open reading frame, Orf2 Bacteriocins Orf2 of Lactobacillus gasseri
 
Examples:

TC#NameOrganismal TypeExample
8.A.1.6.1Acridine efflux pump constituent, AcrAMultiple drugsAcrA of E. coli (P0AE06)
 
8.A.1.6.2

The multidrug efflux pump constituent, MdtA [may form a complex with MdtB and MdtC (2.A.6.2.12), both RND proteins. All three proteins appear to be required for resistance (Baranova and Nikaido, 2002).

Bacteria

MdtA of E. coli (P76397)

 
8.A.1.6.3

Multidrug resistance protein MdtE (YhiU).  Functions with MdtF (TC# 8.A.1.6.3).

Bacteria

MdtE of E. coli

 
8.A.1.6.4

Cation translocating membrane fusion protein of 295 aas

Planctomycetes

MFP of Rhodopirellula baltica

 
Examples:

TC#NameOrganismal TypeExample
8.A.1.7.1

MFP, AaeA, that functions with AaeB, a γ-hydroxybenzoate efflux pump, a member of the Aromatic Acid Exporter Family (TC# 2.A.85) (Van Dyk et al., 2004). Several aromatic carboxylic acids serve as inducers of yhcRQP operon expression.

Bacteria

AaeA of Escherichia coli (P46482)