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Introduction

The problem of drug resistance is a main obstacle in the struggle between human being and infectious diseases. One of the most important bacterial resistant mechanisms is the multidrug efflux pumps situated on the membranes, which can be classified into two major types based on the energy source: primary transport system, such as ATPª²binding cassette (ABC) transporter superfamily utilizes the energy produced by ATP hydrolysis to efflux diverse compounds; while secondary transport system pumps different substrates via the proton motive force (PMF) providing by transmembrane electrochemical proton gradient (¦¤¦ÌH ) or the sodium motive force supplying by ion gradients (¦¤¦ÌNa ). Despite the totally different evolutionary trait and energy sources, both can transport a broad spectrum of structurally unrelated substrates. To date, due to the highly hydrophobic nature, the crystallization of the structure on most of the membrane transporters is yet to be cracked down, albeit recently, a few have been partially crystallized. Therefore, the most important analysis approach is still protein engineering methods involving siteª²directed mutagenesis and chemical modification. And currently there have developed some improvements on these methods.

Bacterial multidrug efflux pump systems

ATPª²binding cassette (ABC) transporter

To date, LmrA is the only wellª²characterised bacterialª²origin ABC transporter, which is encoded by chromosomally located lmrA gene in Lactococcus lactis. In general, active ABC proteins demand minimal two transmembrane domains (TMDs) and two ABC units (nucleotideª²binding domain, NBD). The former usually consists of six transmembrane segments (¦Áª²helices); the latter, a 200¡«250 amino acids, contains three conserved motifs Walker A, ABC signature and Walker B. Therefore, functional structural module is (TMDª²NBD)2. Comparing with functional ABC transporter such as human multidrug resistance Pª²glycoprotein (Pª²gp), LmrA has only two domains, but homologous to each of the two halves of the Pª²gp, suggesting that it belongs to "half ABC transporter" and functions as a homodimer, which has been identified by various studies£Û1¡«3£Ý.

Although the wideª²substrate transport mechanism of LmrA still remains unclear, two hypotheses have been proposed: hydrophobic vacuum cleaner model shows that toxic hydrophobic compounds are directly extruded from the inner leaflet of the membrane into the external water phase£Û4£Ý; and the flippase model suggests that LmrA first recognizes substrates in the inner leaflet of the membrane then flip them to the outer leaflet from where they diffuse into the exterior. Possibly, substrateª²binding resulting in conformational changes, which modulate the interaction between NBDs and ATP, triggers the transport. The binding and hydrolysis of ATP by one site lead to the movement of drugª²binding site from inner membrane to the outside due to the prevention of ATP hydrolysis at the other site, so the drugs can be translocated between the highª² and lowª²affinity binding sites, which forms a transport cycle£Û5£Ý. So far, the confirmed substrates of LmrA include anticancer drugs (vinca alkaloids and anthracyclines), DNA intercalators, toxic peptides, fluorescent membrane probes and dyes as well as clinically important antibiotics such as aminoglycosides, lincosamides, macrolides, quinolones, streptogramins and tetracyclines£Û6£Ý. Hence, the unusually broad antibiotic profile of LmrA and possible gene transfer of lmrA to other bacteria enable the nonª²pathogenic L.lactis potentially threatened£Û7£Ý.

Some other bacterial ABC transporters are also identified, such as the DrrAB doxorubicin/daunorubicin transporter of Streptomyces prucetius; the MsbA and BtuCD transporters of Escherichia coli, which efflux lipid A and vitamin B12, respectively. Albeit it seems that these pumps have little connection with bacterial resistance, the determination of the structure of MsbA to 4.5¬þ£Û8£Ý and BtuCD to 3.2¬þ deepens our understanding on multidrug transport mechanism.

Secondary transport system

On the basis of size and primary energy as well as secondary structure, the system is divided into four families: the major facilitator superfamily (MFS), the small multidrug (SMR) family, the resistanceª²nodulationª²cell division (RND) family, and the multidrug and toxic compound extrusion (MATE) family.

(1) Major facilitator superfamily

So far, more than twenty MFS families have been recognised according to the degrees of sequence simiª²larity. The clinically important multidrug transporters are in the family 2 and 3, which possess 14 and 12 TMSs (transmembrane segments), respectively. They are also referred to as the DHA14 and DHA12 families due to the ability of catalysing drug: H antiport. Multiple sequence alignment suggested that Nª²terminal region is related to proton translocation producing energy for transport since the high similarity among them of the proteins; while Cª²terminal halves may contribute to substrate recognition and binding because of the less similarity among different proteins. Additionally, it is noticeable that the majority of the bacterial drug exporters belong to MFS.