«INAUGURALDISSERTATION zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophischen-Naturwissenschaftlichen Fakultät der ...»
On the Interaction of Cationic Compounds with ABCTransporters and Lipid Membranes
Erlangung der Würde eines Doktors der Philosophie
der Universität Basel
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Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultaet auf Antrag von Prof. Dr. Anna Seelig Prof. Dr. Joachim Seelig Prof. Dr. Dagmar Klostermeier Basel, den 22. Juni. 2010 Prof. Dr. Eberrhard Parlow Table of Contents 1 Introduction
4 The Activity of Sav1866 in Lipid Vesicles and Detergent Micelles
1.1 Integral Membrane Proteins Integral Membrane proteins (IMPs) are permanently attached to biological membranes and are simultaneously in contact with two distinct aqueous compartments (Gohon and Popot 2003). IMPs are amphipatic and requiere therefore the lipid bilayer and the water phase for stabilization (von Heijne 2006). IMPs can be divided into many different classes such as transporters, channels, receptors, enzymes, structural membrane-anchoring domains, proteins involved in accumulation and transduction of energy, and proteins responsible for cell adhesion. The main function of all IMPs is the translocation of various substances or signals through the membrane. This can be achieved either by active (transporters) or passive (channels/pores) transport or translocation, respectively.
In order to study IMPs in terms of structure and activity they have to be purified. For purification purposes detergents are used to disintegrate the lipid membrane to get access to the IMP (Rigaud and others 1997). However the stabilization of IMPs by detergents or detergents mixtures is still a semi-empirical task. Compared to soluble proteins the amphipaticity of IMPs makes them difficult to study. The database shows that in March 2010 only 234 unique structures of membrane proteins have been solved. However the structures of more than 30’000 soluble proteins have already been solved.
Since most of the commercially available drugs target IMPs the interest in this research area has increased in the past years (Overington and others 2006). In the next chapter one of the largest superfamiliy of IMPs, the so called ATP binding cassette-transporters are discussed. Further, the focus will concentrate on the two structurally related, specific ABC-transporters of which one is of eukaryotic (P-glycoprotein) and the other of prokaryotic (Sav1866) origin, respectively. The two transporters will be compared in terms of structure and function.
1.2 ABC-Transporters ATP-binding cassette transporters (ABC-transporters) are integral membrane proteins and are found in every organism from archae to man (Holland 2003). A common feature of all ABC-transporters is the energy dependent transport of substances across cell membranes that is driven by ATP hydrolysis (Holland and Blight 1999; Schneider and Hunke 1998). ABC-transporters are structurally related but can transport structurally unrelated substances. The maltose transporter from E.coli transports maltose (Davidson and others 1996) from one aqueos compartement to the other. On the other hand Pglycoprotein (P-gp) transports various amphipathic substances from one membrane leaflet to the other (Seelig 1998). Transported substances are referred to as allocrites or substrates. However the word substrate has to be taken with care as in a strict nomenclature ATP is the proper substrate.
ABC-transporters consist of two transmembrane domains (TMDs) to which the substrate binds and two nucleotide binding domains (NBDs) where ATP is hydrolyzed. The energy from the NBDs is transferred over the coupling helices to the TMDs (Dawson and Locher 2006). The NBDs are highly conserved among different ABC-transporters as the energy source is ATP. On the other hand the TMDs are less conseved as they are responsible for the transport of the various structurally unrelated substrates.
In both eukaryotes and prokaryotes the ABC-transporters are responsible for import as well as export of substances. In bacteria ABC-transporters export drugs and antibiotics, whereas ABC-importers mediate the uptake of nutrients. The third class of ABCtransporters are involved in mRNA translation and DNA repair (Davidson and others 2008). Bacterial ABC-transporters usually are organized as separate subunits or half transporters and assemble at/in the membrane to become fully active. Eukaryotic transporters in contrast are generally expressed as fully functional transporters (Lage 2003).
In man ABC-transporters belong to a class of IMPs which are often involved in clinical severe problems. Mutations in almost half of the 48 human ABC-transporters cause a severe disease in humans (Dean and Annilo 2005). Two of the best studied ABCtransporters in human are P-glycoprotein (P-gp, MDR1, ABCB1) and the cystic fibrosis transduction regulator (CFTR) as both contribute to significant clinical problems.
-2Introduction Because P-gp (MDR1, ABCB1) contributes to multi-drug resistance (MDR) of human cancer cells they are important in clinics and thus probably the best studied ABCtransporters in eukaryotes (Gottesman and Ambudkar 2001). The term MDR is used for a cross-resistance phenotype against unrelated drugs and has to be destinguished from single-drug resistance (SDR). P-gp is over-expressed in cancer cells which make drug treatment very difficult. The applied drugs remain ineffective since they are exported out of the membrane therefore not reaching the cytosol (Ling 1997; Nervi and others). A close description of the substrate pathway can be found in chapter 1.4.
The other example of a clinically relevant ABC-transporter is the human cystic fibrosis transduction regulator (CFTR) (Sheppard and Welsh 1999). Only one point mutation in the CFTR protein causes a miss-folding and destroys the protein function which causes the disease called cystic fibrosis. As CFTR is involved in chloride transport, which is coupled with H20 transport, the CFTR patients suffer under thick mucus especially in the lung.
Clinically relevant ABC-transporters in prokaryotes are involved in MDR of bacteria (Van Bambeke and others 2000). The first evidence that antibiotic resistances is caused by active efflux of drugs was found in 1980 (McMurry and others 1980). Most prokaryotic drug transporter belong to the class of secondary active drug transporters.
However several ABC-transporters are involved in multiple drug export. Such efflux systems are thought to have an important role in bacterial MDR (Li and others 2004;
Lomovskaya and Totrov 2005; Poole 2005).
1.3 P-glycoprotein, a Human ABC-Transporter P-gp is the best studied ABC-transporter. P-gp is able to transport a variety of different substances across the lipid bilayer and is therefore involved in many different tasks.
Moreover human P-glycoprotein (P-gp, ABCB1, MDR1) accounts for significant clinical problems (for review (Ambudkar and others 1999; Litman and others 2001)).
Since P-gp is also involed in multi drug resistance (MDR) of cancer cells and cancer is the second most common death cause in first world, there is great interest in this protein (Bronchud MH 2000). The phenomenon of MDR was first described in the scientific
-3Introduction literature in 1970 (Biedler and Riehm 1970). The classical MDR phenotype is characterized by cross resistance patterns against a variety of drugs. P-gp, however, was originally isolated from Chinese hamster ovary (CHO) cells in 1976 (Juliano and Ling 1976). In 1979 P-gp was purified (Riordan and Ling 1979) and its encoding gene was identified as MDR1 or ABCB1 (Shen and others 1986), and also found to be overexpressed in multidrug-resistant human and mammalian cell lines (Kartner and others 1983); (Kartner and others 1985).
The main functions of P-gp are (i) the protection of various tissues against environmental toxins (Schinkel 1997) or (ii) the absorption of molecules in the intestinal barrier. For toxin protection, P-gp is expressed at the apical surface of endothelial cells in tissues like the blood-brain barrier, blood testis barrier, blood nerve or fetal maternal barrier (Ceckova-Novotna and others 2006; Cordon-Cardo and others 1990; Holash and others 1993; Schinkel and others 1996; Tanaka and others 1994; Tatsuta and others 1992;
Thiebaut and others 1987; Thiebaut and others 1989). For substrate uptake it is expressed at the intestinal barrier.
P-gp has as most of the ABC-transporters two times six transmembrane helices in the TMDs and the two NBDs where ATP binds to. Like other ABC-transporters P-gp has typical conseved sequence motifes in the NBDs, which are listed: The Walker A (GXSGCGKST) and Walker B (ILLLDEA) motifs, the signature region (LSGGQ), and the A, D, H and Q structural loops (Gottesman and others 1996). These conserved residues play important role in positioning the MgATP, in the activation of the catalytic water molecule and in the signal transduction between the NBDs and the TMDs (Carrier and others 2007).
P-gp can transport a wide variety of substrates and the question how the individual substrates are recognized is still matter of debate. In general there are three different approaches used to get insight into the substrate recognition. If a crystal structure is available computer models can predict what the possible recognition sites are. Further mutational studies of the binding region can give insight where the specific substrate binds to. Probably the most successful approach is the close investigation of the transported substrates in order to find the possible recognition patterns. The mechanism proposed in 1998 by Seelig based on this approach is well accepted (Seelig 1998). The
-4Introduction investigation of 100 different substrates in terms of structures led to the conclusion that the substrate recognition has to be via hydrogen-bonding. All the substrates investigated had specific H-bond acceptor patterns. This modular recognition theory can explain the wide variety of transported substrates.
Probably one of the most difficult parts in P-gp research field is the fact that the substrates are amphipatic. The substrate first incorporates into the membrane and is then flipped to the other site of the lipid bilayer by the transporter under ATP hydrolisis. The measurement of a drug concentration in the membrane is hardly feasible. Binding constants are measured of a substrate from water to the membrane. The binding constant of a substance to the membrane can be measured by various techniques, probably most sophisticated is the use of isothermal titration calorimetry as this technique does not need labelling of the substance. However it should be mentioned at this point that incorporation of a charged substrate into a charged membrane has to be evaluated carefully as the incorporation behavior is governed by hydrophobic and electrostatic contributions. The topic is explained in more detail in chapter 6 (DTAC).
1.4 Sav1866, a Bacterial ABC-transporter Most prokaryotic drug transporters belong to the class of secondary active transporters particular drug-proton exchange systems (Paulsen and others 1996). However several drug transporting systems utilize the energy of ATP hydrolysis to drive drug efflux (Lage 2003). The high-resolution structure of SAV1866 is the first reported for an ABC exporter (Dawson and Locher 2006; Dawson and Locher 2007). Sav1866 is a close homolog of the multidrug ABC transporter P-glycoprotein (see previous chapter).
Therefore the structure raised great interest since at the time the structure of SAV1866 was solved there was no high resolution structure of P-gp available. Sav1866 provided the basis for structural models for P-glycoprotein (O'Mara and Tieleman 2007). However nowadays the high resolution structure of P-glycoprotein is solved (Aller and others 2009). Figure 1 shows the structure of Sav1866 in a ADP bound outward facing conformation. The approximate location of the lipid bilayer and the substrate pathway are depicted.
-5Introduction Figure 1. High resolution structure of the bacterial ABC-transporter Sav1866. The structure of Sav1866 was solved to a 3 Å resolution in detergents micelles (C12E8) in the year 2006. The two sbdomains are depicted in green and blue. The dotted line depicts the approximate location of the lipid bilayer.
Sav1866 originates from Staphylococcus aureus a gram positive bacterium invloved in multidrug resistance (Huet and others 2008). Sav1866 can transport structurally unrelated substrates similar to P-glycoprotein (Velamakanni and others 2008). Therefore it is thougth to be invloved in multidrug-resistance of Staphylococcus aureus which makes it clinically relevant. Generally, Staphylococcus aureus is involved in severe antibiotic resistance in hospitals. Very well known is for example the MRSA strain, which was called in previous times methicillin-resistant staphylococcus aureus strain, nowaday called multi-resistant staphylococcus aureus strain, as it is resistent against all kind of beta-lactam-antibiotics. However the mechanism is very well known and originates from a modified penicillin binding protein. Sav1866 is not involved.
For crystallisation purposes the ABC transporter was overexpressed in E.coli and solubilized in a detergent mixture (Dawson and Locher 2006). The crystallization step was performed in C12E8. The crystal showed a very good diffraction pattern and the structure was solved to a 3 Å resolution. The structural motifs of Sav1866 are typical for ABC-transporters. The Sav1866 dimer consits of 2 transmembrane domains (TMDs) containing 6 -helices each for drug translocation and 2 nucleotidbinding domains (NBDs) as P-gp. Usually ABC-importers contain a short coupling helix that contacts a
-6Introduction single NBD. Sav1866, as an ABC exporter, has two intracellular coupling helices, one contacting the NBDs of both subunits and the other interacting with the opposite subunit (Rees and others 2009). A major improvement was the mode of action the autors propose.