Transmembrane Domains (TMDs) of ABC transporters

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1 Transmembrane Domains (TMDs) of ABC transporters Most ABC transporters contain heterodimeric TMDs (e.g. HisMQ, MalFG) TMDs show only limited sequence homology (high diversity) High degree of conservation of secondary structural elements Structural Elements in TMDs: Large hydrophobic stretches (α-helical), separated by more hydrophilic regions (loops) at the aqueous phase (i.e., at the periplasmic and cytoplasmic faces of the membrane) coupling helices interact the nucleotide-binding domains (NBDs) N- and C-termini are located at the cytoplasmic face of the membrane most TMDs have (per subunit) 6 TM helices, 3 periplasmic and 2 cytoplasmic loops (there are however a number of exceptions, e.g. type II importers) 1

2 TMD architectures Exporter Importer (type I) Importer (type II) Hollenstein, K. et al. (2007) Curr. Opin. Struct. Biol. 17, Type I importer: smaller substrates (ions, sugars, amino acids, ) which are delivered to the TMD by specific BPs Type II importer: larger metal chelates (e.g., Vit B12) Type III importer: energy coupling factor (ECF) transporters see assignment 2 2

3 The coupling helices are part of the TMDs. They extend into the cytoplasm to establish critical contacts with the ABC subunits (NBDs). These contacts are important as they are essential for facilitating the ATPinduced switch from inward-to-outward or outward-to-inward facing TMDs. Locher, K. (2009) Phil. Trans. R. Soc. B 364,

4 Crystal structures of full ABC transporters Type I Type II Type II Hollenstein, K. et al. (2007) Curr. Opin. Struct. Biol. 17,

5 The EAA loop Although TMDs display much lower sequence similarities than do the ABC subunits, there is a highly conserved stretch of amino acids (20 residues) in all TMDs near their C termini (the so-called: EAA loop - because the sequence starts with glutamate-alanine-alanine) The EAA loop contains mostly hydrophilic residues (consistent with its exposure to the cytosol) Mutations in the loop can lead to complete loss of substrate uptake in MalFG: mutations in EAA loop of either MalF or MalG do not overly affect transport, whereas mutations in both subunits result in a completely defective transport system EAA loops are absent in ABC-type exporters (this suggests that the loops must be important for a functional connection between TMDs and the ABC subunits) 5

6 Interaction of TMDs with binding proteins Binding stoichiometry: Usually assumed to be 1 binding protein per TMD dimer Some TMDs are fused to BPs (i.e. they have two binding proteins - one per TMD subunit) 6

7 Noteworthy/peculiar observations on the maltose transport system: Maltose-binding protein (MBP) independent mutants of MalFG retain specificity for maltose (certain mutations in MalFG lead to transport which does not involve the binding protein. Maltose is presumably recognized by MalFG, suggesting a binding site within the TMDs. Also note that transport with these mutants still requires MalK and ATP hydrolysis!) Lactose is transported in addition to maltose when MalF bears a L334W mutation (at periplasmic side): transport requires MBP, although MBP does not bind lactose transport of maltose and lactose is not mutually exclusive (suggests different binding sites for both substrates) 7

8 The ATP hydrolyzing subunit(s) The NBDs (ABC subunits) are the engines for substrate translocation through the membrane Highly conserved (amino acid sequence; motifs), in contrast to the TMDs For maltose transport (MalK) Diederichs et al. (2000) EMBO J. 19,

9 Ribbon representation of the MalK from Thermococcus litoralis MalFG-binding region The side view shows the extended dumb-bell shape resulting from the two (grey) regulatory domains (β-barrel) on either end and the central (N-terminal) ATPase domain dimer. The bottom part of the dimer is thought to interact with the membrane translocation pore MalFG. 9

10 The bottom view along the pseudo 2-fold axis. Gln88 residues from both monomers are shown in red to demonstrate their close apposition. 10

11 A-view showing three layers of secondary structural motifs 11

12 Structure and sequence alignment 12

13 Detailed A-view 13

14 Detailed B-view 14

15 Detailed view of the pyrophosphate binding sites 15

16 Based on crystal structures of a various NDB domains, the assembly of NDB subunits can be generalized as shown below P-loop = Walker A motif LSGGQ = signature motif 16

17 Structure of cyanocobalamine Vitamin B12 transport Corrin ring (aromaticity highlighted in yellow) Involved, for instance, in the methylation of homocysteine to methionine 17

18 Vitamin B12 transport in E. coli Taken from: Locher (2004) Curr. Opin. Struct. Biol. 14,

19 Translocation pathway: Large cavity that opens to periplasmic space (big enough to permit corrin ring to bind) Cavity has no structural resemblance to binding sites of B12-dependent enzymes It has been argued that such type II importers implement an inert translocation pathway (sometimes called Teflon pathway) because the TMDs have essentially no affinity for the cognate substrate) Cavity is closed at the cytoplasmic end of the membrane (two loops function as gates) 19

20 Possible mechanism of vitamin B12 transport Taken from: Locher (2004) Curr. Opin. Struct. Biol. 14, The tilting mechanism for these TMDs parallels those of lactose permease (LacY) and the G-3-P transporter (GlpT) 20

21 Proposed mechanism: 5 years later Locher, K. (2009) Phil. Trans. R. Soc. B 364, What are the differences? What questions remain? 21

22 Newer insights into the structure and mechanism of ABC-type transporters are going to be discussed in the context of assignment 2! 22

23 Regulation of transport in ABC-type systems Feedback Inhibition: Desirable at high internal concentrations of substrate accumulation of substrate at high and even toxic concentrations could be the result of an impairment of metabolism of the substrate (e.g., through mutations in genes that code for enzymes that metabolize the substrate) feedback mechanism is observed with His transport (transport and ATPase activity is inhibited by histidine) G-3-P transport (transport inhibited by phosphate, which is a product of G-3-P metabolism) 23

24 Proposed model for maltose transport and repression Here: ABC subunits serve as regulators tetramer inducer monomer MalT: transcriptional activator of all mal genes 24

25 Important features of maltose transport and repression All mal genes are constitutively expressed when MalK is absent Overproduction of MalK leads to repression Repression is a consequence of the formation of a MalT(monomer):MalK:ATP complex (note that ATP is not hydrolyzed in this case!) Repression occurs when maltose levels outside the cell are low 25