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Microbiology 150 (2004), 2493-2495; DOI  10.1099/mic.0.27312-0
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Microbiology 150 (2004), 2493-2495; DOI  10.1099/mic.0.27312-0
© 2004 Society for General Microbiology

MICROBIOLOGY COMMENT

Sequence similarity between multidrug resistance efflux pumps of the ABC and RND superfamilies

Se H. Kim, Abraham B. Chang and Milton H. Saier, Jr

Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA

Correspondence
Milton H. Saier Jr
(msaier{at}ucsd.edu)

Supplementary material related to this article is available from Microbiology Online.

The LmrA multidrug resistance (MDR) efflux pump of Lactococcus lactis is a member of the ATP-binding cassette (ABC) superfamily (TC 3.A.1). LmrA catalyses drug export in a process driven by ATP hydrolysis. Since a primary form of energy (ATP) drives transport, such a system is called a primary active transporter.

Recently, Venter et al. (2003)Down used molecular genetic techniques to cleave the ATP-hydrolysing domain from the integral membrane transporter domain of LmrA and demonstrated that the latter could catalyse drug transport driven by the transmembrane proton electrochemical gradient. Substrate : proton symport or antiport is a general characteristic of simple carriers also called secondary active transporters. Their observations suggested that the primary active transporter, LmrA, was derived from a secondary carrier by superimposition of the ATP-hydrolysing subunit onto the carrier during evolution as suggested previously (Saier, 2000aDown).

There are four superfamilies of secondary active transporters that include members that catalyse drug export using a drug : cation antiport mechanism. These are the MF (TC 2.A.1), RND (TC 2.A.6), DMT (TC 2.A.7) and MOP (TC 2.A.66) superfamilies (Saier, 2000bDown; Saier & Paulsen, 2001Down). We were curious to know if LmrA could be shown to exhibit sufficient sequence similarity to the members of any one of these superfamilies to suggest a functional or an evolutionary connection.

Fig. 1Down shows a binary alignment of an extended region of LmrA with a portion of the MexB MDR efflux pump of the RND superfamily (Tseng et al., 1999Down). These two sequences exhibit 28 % identity and 42 % similarity for this 98 residue position alignment. A shorter segment including the first 63 residue positions gave 32 % identity and 47 % similarity. Using the GAP program with 500 random shuffles (Devereux et al., 1984Down), comparison scores of 9·5 standard deviations (SD) and 11·6 SD were obtained for the longer and shorter binary comparisons, respectively. The probability of obtaining such a score by chance is less than 10–20. These comparison scores are sufficient to establish that the sequence similarity observed for these regions within the two proteins did not arise by chance.



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  		Fig. 1. Binary alignment of internal regions of LmrA of Lactococcus lactis (ABC superfamily) and MexB of Pseudomonas aeruginosa (RND superfamily). The GAP program was used to derive the alignment (Devereux et al., 1984Down). Symbols below the alignment have significance as follows: asterisk, identity; colon, close similarity; period, more distant similarity; a large dot ({bullet}) above the alignment signifies the exclusive occurrence of conservative substitutions in all of the 22 proteins, arbitrarily selected for comparison, 11 from the ABC superfamily, 11 from the RND superfamily (see our website, http://www-biology.ucsd.edu/~msaier/supmat).

 
As shown by the two shaded transmembrane segments (TMSs) depicted in Fig. 2Down(a), the region in LmrA which shows similarity to MexB includes the hairpin structure corresponding to TMSs 3 and 4. This region is homologous to the hairpin structure in MexB corresponding to TMSs 2 and 3 (Fig. 2bDown). Since both proteins have their N and C termini in the cell cytoplasm, this means that the regions of homology in the two proteins have opposite orientations in the bacterial cytoplasmic membrane.



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  		Fig. 2. Schematic diagrams showing the topologies of LmrA (a) and the first half of the internally duplicated MexB (b). The hatched transmembrane segments are included within the regions showing the greatest degree of sequence similarity between the two proteins.

 
In order to determine if the residues conserved between LmrA and MexB were also conserved among other members of each of these two families, 10 homologues of each of these two proteins were selected for further analysis (see Table S1, available from Microbiology Online). The proteins listed include the well-characterized Escherichia coli AcrB protein of the RND superfamily and the E. coli MsbA protein of the ABC superfamily. The high-resolution structures of both of these proteins have been solved by X-ray crystallography (Chang, 2003Down; Chang & Roth, 2001Down; Murakami et al., 2002Down). A multiple alignment was generated as shown in Fig. S1 (available from Microbiology Online) and a phylogenetic tree based on this alignment revealed the relationships of these sequences to each other (Fig. S2, available from Microbiology Online). Only one residue, a proline, was fully conserved in all 22 proteins, but a glutamate was fully conserved in 21 of the 22 proteins, and many positions exhibited only conservative substitutions. In fact, most of the conserved positions between LmrA and MexB shown in Fig. 1Up proved to be well-conserved between the 22 members of these two families arbitrarily selected for analysis. All such residue positions are indicated by a large dot above the binary alignment in Fig. 1Up. We conclude that the many hydrophobic, hydrophilic and semi-polar residues that are conserved between LmrA and MexB are characteristic of both families of drug efflux pumps. It is probable that these conserved residues are important for structure and/or function in both protein families.

The fact that the two homologous hairpin structures are of opposite orientation in the membrane is worthy of comment. Many secondary carriers are built of duplicated units including uneven numbers of TMSs (3 or 5 TMSs duplicated to give 6 or 10 TMSs). In several such cases, opposite orientation of the two halves has been unequivocally demonstrated (for a review, see Saier, 2003Down). In addition, altering the phospholipid composition of the membrane can cause reversible inversion either of an entire transmembrane protein domain (Bogdanov et al., 2002Down; Wang et al., 2002Down) or of an integral membrane N-terminal {alpha}-helical hairpin structure within a larger protein domain (Zhang et al., 2003Down). It is therefore clear that the position of a transmembrane protein hairpin is determined not only by its amino acid sequence, but also by its position in the protein and its interaction with other membrane macromolecules.

REFERENCES

Bogdanov, M., Heacock, P. N. & Dowhan, W. (2002). A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition. EMBO J 21, 2107–2116.[CrossRef][Medline]

Chang, G. (2003). Structure of MsbA from Vibrio cholera: a multidrug resistance ABC transporter homolog in a closed conformation. J Mol Biol 330, 419–430.[CrossRef][Medline]

Chang, G. & Roth, C. B. (2001). Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 293, 1793–1800.[Abstract/Free Full Text]

Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387–395.

Murakami, S., Nakashima, R., Yamashita, E. & Yamaguchi, A. (2002). Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587–593.[CrossRef][Medline]

Saier, M. H., Jr (2000a). Vectorial metabolism and the evolution of transport systems. J Bacteriol 182, 5029–5035.[Free Full Text]

Saier, M. H., Jr (2000b). A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64, 354–411.[Abstract/Free Full Text]

Saier, M. H., Jr (2003). Tracing pathways of transport protein evolution. Mol Microbiol 48, 1145–1156.[CrossRef][Medline]

Saier, M. H., Jr & Paulsen, I. T. (2001). Phylogeny of multidrug transporters. Semin Cell Dev Biol 12, 205–213.[CrossRef][Medline]

Tseng, T.-T., Gratwick, K. S., Kollman, J., Park, D., Nies, D. H., Goffeau, A. & Saier, M. H., Jr (1999). The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J Mol Microbiol Biotechnol 1, 107–125.[Medline]

Venter, H., Shilling, R. A., Velamakanni, S., Balakrishnan, L. & van Veen, H. W. (2003). An ABC transporter with a secondary-active multidrug translocator domain. Nature 426, 866–870.[CrossRef][Medline]

Wang, X., Bogdanov, M. & Dowhan, W. (2002). Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition. EMBO J 21, 5673–5681.[CrossRef][Medline]

Zhang, W., Bogdanov, M., Pi, J., Pittard, A. J. & Dowhan, W. (2003). Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition. J Biol Chem 278, 50128–50135.[Abstract/Free Full Text]




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