Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis

Abstract

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

Fig. 1. Ribbon representation of the T.l.MalK dimer. The A- and B-molecules are coloured yellow and blue, respectively, except for both regulatory domains which are coloured grey. Labels indicate the numbers of strands and helices according to the secondary structure assignment given in Figure 3. This figure and the following structural diagrams were made with MOLSCRIPT (Kraulis, 1991) if not indicated otherwise. (A) The side view shows the extended dumb-bell shape resulting from the two regulatory domains on either end and the central ATPase domain dimer. The pseudo 2-fold symmetry axis is oriented vertically and runs through the centre of the dimer. The strong involvement of helices 2 and 4 in dimerization is seen. The bottom part of the dimer is supposed to interact with the membrane translocation pore MalFG. (B) The bottom view along the pseudo 2-fold axis shows the deviation from 2-fold symmetry. The helical layer of one monomer is seen in contact with the two upper layers containing the nucleotide-binding site of the other monomer. Gln88 residues from both monomers are shown to demonstrate their close apposition. The A- and B-viewing directions are indicated.

Fig. 1. Ribbon representation of the T.l.MalK dimer. The A- and B-molecules are coloured yellow and blue, respectively, except for both regulatory domains which are coloured grey. Labels indicate the numbers of strands and helices according to the secondary structure assignment given in Figure 3. This figure and the following structural diagrams were made with MOLSCRIPT (Kraulis, 1991) if not indicated otherwise. (A) The side view shows the extended dumb-bell shape resulting from the two regulatory domains on either end and the central ATPase domain dimer. The pseudo 2-fold symmetry axis is oriented vertically and runs through the centre of the dimer. The strong involvement of helices 2 and 4 in dimerization is seen. The bottom part of the dimer is supposed to interact with the membrane translocation pore MalFG. (B) The bottom view along the pseudo 2-fold axis shows the deviation from 2-fold symmetry. The helical layer of one monomer is seen in contact with the two upper layers containing the nucleotide-binding site of the other monomer. Gln88 residues from both monomers are shown to demonstrate their close apposition. The A- and B-viewing directions are indicated.

Fig. 2. A-view along the interface perpendicular to the pseudo symmetry axis. From top to bottom the three layers are seen: antiparallel sheet; mixed sheet with a P-loop; and helix 1 and helical layer. Colouring is as in Figure 1 except that the conserved regions Walker-A, Walker-B, signature motif, D-loop, switch from monomer A and the ‘lid’ region from monomer B are marked by red colouring with yellow outlines. Labels indicate the numbers of strands and helices according to the numbering given in Figure 3.

Fig. 3. Structural alignment of HisP from S.typhimurium (Hung et al., 1998) and of T.l.MalK, as well as sequence alignment with E.c.MalK. Secondary structure elements of T.l.MalK are indicated and numbered. Regions that are conserved in all three sequences are shaded yellow, those that are conserved only between the two MalKs are shaded blue.

Fig. 4. Stereo bottom view (C_α chain) of the ATPase domain dimer. The A- and B-chains are coloured green and blue, respectively. The B-chain has been superimposed with the A-chain and is shown in red. The good matching of the sheets in the upper two layers is seen, while helices 2–4 are shifted against each other.

Fig. 5. Comparison of the A- and B-view. In the A-view (A), the interface appears to be more narrow than in the corresponding B-view (B) due to the upward shift of the loop containing the ‘lid’ region, which has been clarified by including side chains of residues 90, 93, 96 and 104. Notably, B-His95 approaches the pyrophosphate to within hydrogen-bonding distance in the A-view, while A-Tyr93 plays this role in the B-view. Arg47 shows a difference in side chain conformation in the two views. Helices 2 and 3 are shifted outwards in the B-monomer by ∼3 Å as compared with the A-monomer.

Fig. 5. Comparison of the A- and B-view. In the A-view (A), the interface appears to be more narrow than in the corresponding B-view (B) due to the upward shift of the loop containing the ‘lid’ region, which has been clarified by including side chains of residues 90, 93, 96 and 104. Notably, B-His95 approaches the pyrophosphate to within hydrogen-bonding distance in the A-view, while A-Tyr93 plays this role in the B-view. Arg47 shows a difference in side chain conformation in the two views. Helices 2 and 3 are shifted outwards in the B-monomer by ∼3 Å as compared with the A-monomer.

Fig. 6. The two pyrophosphate-binding sites. Hydrogen-bonding partners of the pyrophosphates as seen in the A- and B-view are shown. Labels A- and B- in front of the residues refer to the two monomers. The bond length in angstroms is given as a number. This figure was made with ISIS-DRAW.

Fig. 7. B-factors of the C_α atoms of the A- (yellow) and B- (blue) monomer of T.l.MalK. Regions of high asymmetry are marked by bars and labelled with numbers. The A-chain is significantly more disordered in the ‘lid ’region (1), the loop between helices 2 and 3 (2) and the signature motif region (3). The regulatory domains have fairly similar B-factors.