Architecture and mechanism of ABC transporters

Despite their large number and enormous substrate diversity, ABC transporters share a common modular architecture. Members of this family invariably consist of two membrane spanning domains (TMD) that form the translocation pathway through the membrane, and two cytoplasmic nucleotide binding (ABC) domains that energize the transport reaction through binding and hydrolysis of ATP. These four domains can be arranged in many different ways. In bacteria and archaea the four domains are usu-ally separate polypeptides, whereas in higher organisms these polypeptides are fused in different ways. The membrane spanning domains are poorly conserved and vary con-siderably in the number of predicted α-helices This is likely to reflect the diversity of

1.3 ATP binding cassette transporters

Figure 1.3: Structures of ABC transporters Transmembrane spanning domains (TMD) are shown in red, intracelluar domains (transmission interfaces) in yellow and ABC cassettes in blue. A) MsbA from Escherichia coli, B) MsbA from Vibrio cholera, C) Btu(CD)2 from Escherichia coli, with binding protein BtuF in green.

transported substrates. What brings the different transporters together to a large family is the conserved ATP-binding cassette (ABC) engine that is common to all transporters of this class and shows highly conserved features. Among those are the Walker A and B motifs (Gx₄GKT and Rx_4-12h₄D, with h being any hydrophobic residue), signature motif or C-loop (LSGGQ/E), Q-loop, D-loop, and switch II. Because of this conservation, it is assumed that all ABC cassettes drive the transport through the membrane spanning domains by a common mechanism.

A first step in the determination of high resolution structures of ABC transport com-plexes was MsbA from E. coli (Chang and Roth, 2001) (Figure 1.3A). MsbA is closely related to the N- and C- terminal part of MDR1 and forms a homodimeric complex within the membrane that builds the functional unit of the transporter. In Gram-negative bac-teria, where MsbA transports lipid A, a major component of the outer membrane, the loss of MsbA activity leads to a lethal accumulation of lipid A in the inner leaflet of the cytoplasmic membrane (Zhou et al., 1998). However, in the MsbA structure important parts in the ATPase domains are missing due to disorder of the crystals, and the large opening angle between the two monomers is likely to be artificially caused by the crystal lattice. A more suitable model of MsbA presents the structure of MsbA from Vibrio cholera. The model is much more complete and the ABC cassettes are close enough to interact with each other (Chang, 2003) (Figure 1.3B). The first structure of a binding protein dependent ABC transporter was that of the vitamin B₁₂ transporter Btu(CD)₂ (Locher et al., 2002) (Figure 1.3C). This structure has been determined to a resolution of 3.2 ˚A and presents the most detailed structure of an ABC transporter available so

From biochemical and structural studies it is known that ABC cassettes work in pairs of either homo- or heterodimers. Two ATPase domains form two composite ATP-binding active sites in their dimer interface. The ATP binding site is formed by the Walker A motif of one monomer and the signature motif of the other and vice versa (Hopfner et al., 2000; Chen et al., 2003). A monomeric ABC ATPase can be subdivided into two lobes:

lobe I containing the P-loop that is the major ATP binding site formed by the Walker A motif and lobe II containing the signature motif. These two lobes are connected by a shared β-sheet that contains the Walker B motif. The signature motif is remote to the Walker A and B motifs of the same monomer. In the functional dimer the signature motif binds to the ATP γ-phosphate which results in the engagement and alignment of Walker A, B and the signature motif (see Figure 1.4) (Hopfner and Tainer, 2003). This may lead to a reorientation of lobe I and II within one monomer, thereby generating force. Furthermore, the D-loop (close to the Walker B motif) presents the attacking wa-ter via a main chain carbonyl to the other monomer, likely linking the ATP hydrolysis in one monomer to the other one. Additionally, the activation of the attacking water is likely to be controlled by the switch II including a conserved His (sometimes replaced by Q or S). Another important substructure of ABC cassettes is the Q-loop a loop with a conserved glutamine which binds the catalytic magnesium ion and the nucleophilic attacking water molecule. The residues adjacent to the glutamine of the Q-loop interact tightly with the L-loop (Locher et al., 2002) of the transmembrane subunits and form the transmission interface. It is supposed to be involved in the allosteric activation of the ABC ATPase by the TMD and the transmission of the ATP-dependent conformational changes back to the TMDs (Hopfner and Tainer, 2003). Mutations or deletions in the transmission interface cause severe transport defects as known from the deletion of F508 in CFTR, being responsible for 70 % of the cystic fibrosis cases.

Roughly, a transport cycle may involve the following course of events: 1) substrate binding or binding of a liganded binding protein induces conformational changes in the TMDs 2) these changes are transmitted via the L-loop to the Q-loop of the ATPase inducing engagement of the ATPase dimer 3) the conformational rearrangement caused by the engagement is now transmitted back to the L-loop of the TMDs via the Q-loop which then opens the channel for substrate translocation 4) ATP hydrolysis brings the system back to the ground state.

1.3 ATP binding cassette transporters

Figure 1.4: ABC ATPase dimer structure Structural model of MalK fromE. coli (Chen et al., 2003). Two composite active sites are formed in the dimer interface where two ATP molecules are bound.