Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosa

Abstract

The membrane topology of a resistance-nodulationdivision (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (L L-lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N-and C-termini are located in the cytoplasm and the predicted orientation is consistent with the`positiveinside rule'. This topological model can be applied to other RND proteins.z 1999 Federation of European Biochemical Societies.

Transmembrane Molecular Pump Activity of Niemann-Pick C1 Protein

“…The recent topologicaldefinitionof both the MexD and NPCl proteins (11,12) enabled us to compare their topology and secondary structures. NPCl consists of 13 TM domains, three large hydrophilicloops, and a short cytoplasmic tail, whereas MexD is arranged as 12 TM domains with large hydrophilic loops between TM domains 1 and 2 and between TM domains 7 and 8 (12).…”

“…NPCl consists of 13 TM domains, three large hydrophilicloops, and a short cytoplasmic tail, whereas MexD is arranged as 12 TM domains with large hydrophilic loops between TM domains 1 and 2 and between TM domains 7 and 8 (12). The first six TM domains and the first hydrophilic loop are repeated in the carboxyl half of MexD with relatively conserved secondary structure,suggestingan evolutionary duplication (Fig.…”

Alterations of susceptibility of Pseudomonas aeruginosa by overproduction of multidrug efflux systems, MexAB-OprM, MexCD-OprJ, and MexXY/OprM to carbapenems: Substrate specificities of the efflux systems

“…More recently, we have identified amino acid residues involving the recognition of antimicrobial agents, including -lactams, at periplasmic loops in the inner membrane component MexD molecule 14 of MexCD-OprJ. Investigation of the molecular mechanism of substrate recognition by the multidrug efflux system is in progress in our laboratory.…”

An Elegant Means of Self-protection in Gram-negative Bacteria by Recognizing and Extruding Xenobiotics from the Periplasmic Space

“…These transporters consist of the inner membrane spanning the proton-antibiotic antiporter (MexB or MexY), the membrane fusion proteins that are assumed to connect the inner and outer membranes (MexA or MexX) and the outer membrane-associated lipoprotein OprM common to both transporters (3). The inner membrane spanning subunit (RND transporter) has been found to traverse the cytoplasmic membrane 12 times leaving amino and carboxyl termini in the cytoplasm and the TMSs connected by extra membrane loops (5)(6)(7). Although most loops are short, the loops connecting TMS-1 and TMS-2 (loop-1/2) and TMS-7 and TMS-8 (loop-7/8) were found to consist of over 300 amino acid residues largely protruding toward the periplasmic space.…”