Portal de Periódicos da CAPES

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

2000; Springer Nature; Volume: 19; Issue: 22 Linguagem: Inglês

10.1093/emboj/19.22.5951

1460-2075

Kay Diederichs,

Bacterial Genetics and Biotechnology

Article15 November 2000free access Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis Kay Diederichs Kay Diederichs Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Joachim Diez Joachim Diez Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Gerhard Greller Gerhard Greller Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Christian Müller Christian Müller School of Bioscience, Cardiff University, 10 Museums Avenue, PO Box 911, Cardiff, CF10 3 US Search for more papers by this author Jason Breed Jason Breed Astra Zeneca, Mereside, Macclesfield SK10 4TG, Cambridge, CB3 0AX UK Search for more papers by this author Christoph Schnell Christoph Schnell Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Clemens Vonrhein Clemens Vonrhein Global Phasing Ltd, Sheraton House, Castle Park, Cambridge, CB3 0AX UK Search for more papers by this author Winfried Boos Winfried Boos Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Wolfram Welte Corresponding Author Wolfram Welte Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Kay Diederichs Kay Diederichs Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Joachim Diez Joachim Diez Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Gerhard Greller Gerhard Greller Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Christian Müller Christian Müller School of Bioscience, Cardiff University, 10 Museums Avenue, PO Box 911, Cardiff, CF10 3 US Search for more papers by this author Jason Breed Jason Breed Astra Zeneca, Mereside, Macclesfield SK10 4TG, Cambridge, CB3 0AX UK Search for more papers by this author Christoph Schnell Christoph Schnell Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Clemens Vonrhein Clemens Vonrhein Global Phasing Ltd, Sheraton House, Castle Park, Cambridge, CB3 0AX UK Search for more papers by this author Winfried Boos Winfried Boos Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Wolfram Welte Corresponding Author Wolfram Welte Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany Search for more papers by this author Author Information Kay Diederichs1, Joachim Diez1, Gerhard Greller1, Christian Müller2, Jason Breed3, Christoph Schnell1, Clemens Vonrhein4, Winfried Boos1 and Wolfram Welte 1 1Fachbereich Biologie, Universität Konstanz, M656, D-78457 Konstanz, Germany 2School of Bioscience, Cardiff University, 10 Museums Avenue, PO Box 911, Cardiff, CF10 3 US 3Astra Zeneca, Mereside, Macclesfield SK10 4TG, Cambridge, CB3 0AX UK 4Global Phasing Ltd, Sheraton House, Castle Park, Cambridge, CB3 0AX UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5951-5961https://doi.org/10.1093/emboj/19.22.5951 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 Å 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. Introduction ABC transporters are found in all eubacterial, archaeal and eukaryotic species studied to date and most probably represent the largest family of homologous proteins. In Escherichia coli, an estimated 5% of the whole genome encodes them (Linton and Higgins, 1998). ABC transporters are modular mechanical machines that couple ATP hydrolysis to the physical movement of molecules through membranes. Several subclasses can be defined according to the direction of substrate translocation, substrate specificity and subunit organization. Prominent family members are P-glycoprotein involved in multiple drug resistance, the gated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) involved in the inherited disease cystic fibrosis, sterol transporters, eye pigment precursor importers and protein exporters. Even though the array of substrates seems endless and the molecular architecture can be rather diverse, the ATPase module is an essential and conserved subunit of all transporters. Its sequence features are the main basis for the identification of new family members (Holland and Blight, 1999). A subfamily of ABC transporters are binding protein-dependent systems that are ubiquitous in eubacteria and archaea, where they catalyse the high-affinity uptake of small polar substrates into the cell. One of the best studied examples is the E.coli maltose–maltodextrin system (Boos and Lucht, 1996; Boos and Shuman, 1998). It consists of a binding protein (MalE) as its major substrate recognition site, located in the periplasm. Two homologous hydrophobic membrane proteins (MalF and MalG) form a heterodimeric translocation pore with a dimer of the ATP-hydrolysing subunit (MalK) associated from the cytoplasmic side. Formation of the MalEFGK2 transport complex therefore couples ATP hydrolysis with active transport of substrate. The E.coli MalK (E.c.MalK) and its Salmonella typhimurium homologue (S.t.MalK), which share 95% identical residues, have been subject to intense analysis ever since their discovery (Bavoil et al., 1980; Shuman and Silhavy, 1981). Studies of enzymatic activity as an ATPase (Morbach et al., 1993; Davidson et al., 1996) have been performed. The homodimeric subunit interaction has been analysed (Davidson and Sharma, 1997; Kennedy and Traxler, 1999), and the requirements for its assembly in the transport complex (Davidson and Nikaido, 1991; Panagiotidis et al., 1993; Lippincott and Traxler, 1997) have been recognized. Mutational analysis for domain interactions with its cognate membrane components (Mourez et al., 1997) as well as cross-linking studies (Hunke et al., 2000a) have been reported. Mutational analysis has also defined the functional importance of conserved regions such as Walker-A, Walker-B and the switch region for ATP binding, as well as the signature motif region and the helical domain for coupling of ATP hydrolysis to transport (for a review see Schneider and Hunke, 1998), and has revealed a remarkable versatility of MalK to interact with different regulatory proteins. According to other studies, MalK is able to interact with unphosphorylated EIIAGlc, a subunit of the phosphotransferase (PTS)-type glucose transporter, leading to the inhibition of maltose transport (Dean et al., 1990; Vandervlag and Postma, 1995), a phenomenon called inducer exclusion. In addition, the C-terminus of MalK is able to affect mal gene regulation (Kühnau et al., 1991) by interacting with and inactivating MalT, the specific gene activator of mal gene expression (Panagiotidis et al., 1998), demonstrating a link between transport of substrate and gene regulation. Binding protein-dependent ABC transporters have also been found in thermophilic bacteria (Herrmann et al., 1996; Sahm et al., 1996). Recently, we described an ABC transporter for maltose/trehalose in the hyperthermophilic archaeon Thermococcus litoralis (Xavier et al., 1996). This transport system has several unusual properties: it shows a high affinity (Km of ∼20 nM) at 85°C, the optimum growth temperature of this organism, and it recognizes its very different substrates, maltose and trehalose, with equal affinity but does not bind larger maltodextrins. Its cognate binding protein has been purified (Horlacher et al., 1998) and its crystal structure has been solved (J.Diez, K.Diederichs, G.Greller, R.Horlacher, W.Boos and W.Welte, manuscript submitted). The T.litoralis MalK (T.l.MalK) has been heterologously expressed in E.coli and its biochemical properties have been studied. Its sequence, size (372 residues) and biochemical properties reveal its close relationship to the E.c.MalK protein. It optimally hydrolyses ATP at 85°C and exhibits a Km of 150 μM for ATP at this temperature (Greller et al., 1999). Little is known about how ATP hydrolysis, presumably via a series of protein conformational changes (Ehrmann et al., 1998), is coupled to the mechanism of transport. Thus, structural information about the translocating complex as well as the ATP-coupling structures is needed. As to the subclass of importers, the only known atomic structure is that of HisP, the ATP-hydrolysing subunit of the histidine transporter of S.typhimurium (Hung et al., 1998). Here we present the crystal structure of T.l.MalK, the energy-coupling subunit of the trehalose/maltose transporter of T.litoralis, at 1.9 Å resolution. Results Purification and crystal structure analysis of T.l.MalK Instead of a C-terminally His-tagged T.l.MalK as published previously (Greller et al., 1999), we constructed an N-terminally His-tagged MalK that appeared to crystallize more easily. The recombinant protein was purified from the soluble cellular extract of strain BL21, which lacks several proteases (Studier and Moffatt, 1986), as described previously. The ATPase activity corresponded to that of the previously published construct containing a C-terminal His tag. The crystals comprise two molecules (termed A and B) per asymmetric unit, and the structure was solved by multiwavelength anomalous diffraction (MAD) analysis of an HgCl2 derivative. As only one atom of Hg per molecule of MalK was bound, the phasing power was low. Furthermore, despite cryogenic conditions, the isomorphous and anomalous signal strongly decreased with exposure time, presumably due to radiation damage. The crystallographic analysis therefore first employed single anomalous diffraction (SAD) phasing at the peak wavelength, and was then extended to include all four wavelengths. To stabilize the heavy atom refinement process in the four-wavelength case, the Hendrickson– Lattman coefficients of the SAD phases were introduced as external restraints. The phasing power values given by SHARP (de la Fortelle and Bricogne, 1997; Table I) are much higher for the isomorphous than for the anomalous signal, which is atypical for a MAD experiment and is more reminiscent of a multiple isomorphous replacement (MIR) analysis. Table 1. Statistics on data reduction and MAD phasing for MalK data sets Native HgCl2 peak inflection remote high remote low Wavelength (Å) 0.8424 1.0080 1.0089 1.0163 1.0000 Resolution a (Å) 1.86 (1.88−1.86) 2.65 (2.68−2.65) 2.65 (2.68−2.65) 2.65 (2.68−2.65) 2.65 (2.68−2.65) Redundancy a 4.6 (3.0) 4.0 (4.0) 4.0 (4.1) 4.0 (3.2) 4.0 (4.1) No. of unique observations a 82 668 (2430) 29 141 (963) 29 172 (965) 29 158 (934) 29 183 (963) Completeness (%) 99.0 (93.8) 99.0 (99.8) 99.1 (100.0) 99.0 (96.8) 99.1 (99.8) R-meas b 3.9 (28.0) 4.2 (13.9) 4.1 (16.3) 4.0 (17.5) 4.0 (15.8) R-mrgd-F b 4.1 (27.2) 3.7 (10.8) 3.8 (12.9) 4.0 (15.8) 3.8 (12.2) Isomorphous phasing – – 5.61 6.45 3.41 power (acentric reflections) Anomalous phasing – 1.88 1.23 1.13 0.25 power (acentric reflections) Figure of merit – 0.69 aThe values for the highest resolution shell are given in parentheses. bR-mrgd-F as defined by Diederichs and Karplus (1997). After initial automatic ARP/wARP (Lamzin and Wilson, 1993) model building, model refinement was continued using standard procedures at 1.9 Å. Although T.l.MalK was crystallized in the presence of ADP, there was only clear density for a pyrophosphate molecule. At the expected position of the adenosine group, the map shows elongated density that could result from superposition of several adenosine groups. The occupancy of the pyrophosphate was determined as 0.7. The missing density for the nucleoside can thus be attributed to disorder and low occupancy. Obviously, the structure deviates from the ATP-binding conformation. Comparison with known structures Comparison of the structures of the ATPase domain of T.l.MalK and HisP reveals an almost identical fold (for the A and the B monomer, r.m.s.d. values of 1.71 Å for 153 Cα-coordinates and 1.95 Å for 206 Cα-coordinates at a cut-off of 3.8 Å were found, respectively). The only significant difference is an additional helix–strand motif after strand 7 in HisP as compared with T.l.MalK. Rad50 is a protein involved in DNA double strand repair and is found in all kingdoms of life. It forms a heterodimer with its N- and C-terminal fragments, termed Rad50cd (Hopfner et al., 2000), which, upon binding of ATP, dimerizes with another Rad50cd heterodimer and becomes active as an ATPase. Rad50cd shares the homology regions with ABC-type ATPases and is structurally similar to the monomers of T.l.MalK and HisP, although the r.m.s.d. values are much higher than between the latter. With respect to the mode of dimerization, all three published structures differ from each other so that in contrast to the view of Hopfner et al. (2000), the Rad50cd dimer cannot be used as a model for the nucleotide-binding domains of ABC transporters. General description of the structure T.l.MalK consists of two domains. The N-terminal 223 residues form an α/β-type ATPase domain as found in the ABC transporter family (Armstrong et al., 1998; Hung et al., 1998; Linton and Higgins, 1998). Its overall shape resembles an ellipsoidal planeconvex lens with a longer (∼55 Å) and a shorter (∼40 Å) axis. Residues 224–372 (regulatory domain) form a barrel with a diameter of ∼20 Å and a height of 45 Å. In the asymmetric unit, the ATPase domains of two adjacent MalK molecules are apposed with part of their flat faces to form a globular dimer with significant deviations from 2-fold symmetry (Figure 1). Attached to opposite poles of the dimer are the regulatory domains, resulting in an extended dumb-bell-shaped molecule with a long axis of ∼120 Å. In the following, the bottom and top of T.l.MalK will be referred to on the basis of the orientation given in Figure 1A. As will be shown later, the bottom part contains the residues involved in interaction with MalFG. The contact interface between the ATPase domains extends along the 2-fold axis and forms an angle of ∼35° with the long axis of the dimer (see Figure 1B). When viewed along the interface perpendicular to the pseudosymmetry axis, the ATPase domains are composed of three layers (Figure 2). An antiparallel β-sheet formed by strands 2, 1, 4, 5 and 6 forms the top layer (see Figure 3 for the numbering of the secondary structure elements). The middle layer contains a mixed β-sheet formed by the parallel strands 10, 3, 9, 8 and 7, and the antiparallel strand 11, which connects to the regulatory domain. A final and essential part of the middle layer is the P-loop and helix 1, which follow strand 3 and contain the Walker-A motif. They are located between the two sheets. The P-loop contains a pyrophosphate molecule bound at the interface. Comparison with the similar ATPase structures of HisP and Rad50cd complexed with ATP (Hung et al., 1998; Hopfner et al., 2000) shows that the two phosphates are positioned similarly to the α and β phosphates of ATP. The two sheets of the upper two layers approach each other at roughly a right angle. The last and bottom layer consists of helices 2–6, of which helices 2 and 4 form most of the interface with the other ATPase domain (Figure 1). Figure 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. Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint We find that the majority of the regions conserved in ABC-type ATPases, i.e. the Walker-A, the Walker-B and the switch region (Linton and Higgins, 1998; Schneider and Hunke, 1998; Hopfner et al., 2000), are placed around the pyrophosphate group in or near the parallel sheet of the middle layer (Figure 2). Two conserved regions that contain residues known to be involved in interaction with the translocation pore MalFG are at the bottom of the helical layer (signature motif and D-loop). Another less well conserved region is around a highly conserved glutamine residue at position 88 in T.l.MalK. Hopfner et al. (2000) have used the term 'Q-loop' for this region, while we prefer the descriptive term 'lid' in order to convey its location near the nucleotide-binding site (Figure 2). The MalK dimer Given the two non-crystallographic MalK molecules in the P21212 crystal lattice, several choices of dimers are possible in principle. One rationale to select the biologically relevant dimer was that it should bury a large surface area from solvent. When arranging the buried surfaces of possible dimers in decreasing order, the four largest values are 2755, 1715, 1075 and 719 Å2. In none of these does the arrangement of the nucleotide-binding domains resemble the proposed dimer of HisP (Hung et al., 1998). Further more, the dimer with the largest buried surface is in accord with other data, as will be discussed below. We therefore strongly favour this dimer, although it is different from that proposed for HisP, which buries 915 Å2, and from that of Rad50cd (Hopfner et al., 2000), which buries 2631 Å2. Although the T.l.MalK dimer interface is formed by many apolar residues (22), mainly with aromatic side chains (10), there are a majority of polar residues (29). Interestingly, there is no direct intermolecular hydrogen bond. The cavities in the interface and the immobilized solvent molecules (25) indicate that the interaction energy is not predominantly hydrophobic. Deviations from 2-fold symmetry Inspection of Figure 1B shows that the A-regulatory domain can be superimposed with its B-domain partner by a clockwise rotation of ∼170° around the pseudo 2-fold axis. A least squares superposition of the ATPase domains with a cut-off of 3.8 Å superimposes 200 Cα atoms with an r.m.s.d. value of 1.7 Å (Figure 4). While the sheets closely match each other, significant displacements are found for the loop between strand 7 and helix 2 (containing the 'lid' region) as well as for helices 2 and 3. The latter are shifted in the B-monomer by up to 3 Å outwards compared with the corresponding helices of the A-monomer (see Figure 4). Thus, the upper and middle layer, upon a rotational displacement from ideal 2-fold symmetry, appear to behave virtually as a rigid structure rotating around the pseudo 2-fold axis, while the helices (notably helices 2 and 3) show conformational plasticity of the interface. Figure 4.Stereo bottom view (Cα 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. Download figure Download PowerPoint The asymmetry is also obvious when the dimer is viewed upright in two opposite directions along an axis oriented perpendicular to the 2-fold axis and along the interface (Figures 1B and 5). We distinguish between the A-view, which has the A-monomer on the right and the B-monomer on the left, and the B-view, with the two monomers arranged oppositely. In the A-view (Figure 5A), the interface appears more narrow as B-His95 Nδ1 of the 'lid' region approaches an oxygen of the β-phosphate of the A-pyrophosphate to within hydrogen-bonding distance (Figure 6). In the B-view (Figure 5B), the 'lid' of the A-molecule is seen to be shifted by ∼6 Å towards the bottom, thus opening the interface and interrupting the interaction of the B-His with the pyrophosphate. This shift brings A-Tyr93 OH of the 'lid' within hydrogen-bonding distance of another β-phosphate-bonded oxygen from B-pyrophosphate (Figure 6); this proximity is absent in the A-view. Due to the high B-factors in this region (Figure 7), a hydrogen-bonding distance is not satisfactory evidence for existence of the bond. Figure 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. Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint Figure 7.B-factors of the Cα 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. Download figure Download PowerPoint Lys42, which is highly conserved among nucleoside triphosphate-binding proteins, also shows slight asymmetry with respect to its Nϵ hydrogen bonds with the phosphate oxygens of the β-phosphate of the pyrophosphate molecule due to an altered side chain conformation. While in the B-molecule one oxygen is within hydrogen-bonding distance, in the A-molecule there are two oxygens (Figure 6). Arg47 shows two different side chain conformations. In the A-molecule, it forms a salt bridge with A-Glu53. In contrast, in the B-molecule, a hydrogen bond between the NH2 group and the Oγ1 of B-Thr44 is formed (Figure 6). All three residues are conserved in HisP, E.c.MalK and T.l.MalK (Figure 3). The distances between the Cα atoms of Glu139 in the conserved signature motif at the bottom end of helix 4 and of Leu170 in the conserved D-loop motif near the bottom (Figures 1B, 2 and 3) provide another example for the deviation from 2-fold symmetry. While A-Glu139Cα and B-Leu170Cα are 13.6 Å apart, the distance between B-Glu139Cα and A-Leu170Cα is only 8.8 Å. Furthermore, the deviations from 2-fold symmetry manifest themselves in the B-factors (Figure 7). The 'lid', the loop between the two asymmetric helices 2 and 3 and the signature motif at the N-terminal end of helix 4 near the bottom have significantly higher values in the A- than in the B-monomer. The regulatory domain The regulatory domain contains the 149 C-terminal residues of T.l.MalK, which form a small connecting domain of 22 residues (helices 7 and 8), an antiparallel sheet of three β-strands and a five-stranded β-barrel with shear number 10 (see Figures 1A and 3). A structure-based search using the DALI-server (Holm and Sander, 1993) indicated a weak similarity to an N-terminal fragment of phenylalanyl-tRNA and aspartyl-tRNA synthetases. Less similarity of the barrel also exists to the C-terminal aminoacyl-tRNA-binding domains of bacterial elongation factors EF-Tu (Kjeldgaard and Nyborg, 1992; Nyborg et al., 1996), the C-terminal barrel domain of bacterial methionyl-tRNA formyltransferase (Schmitt et al., 1996) and other proteins of the so-called OB-fold (Murzin, 1993). These domains function as binding modules for aminoacyl-tRNA, carbohydrates and DNA. In EF-Tu, they are connected to a nucleoside triphosphatase domain, a combination reminiscent of MalK. The sequences of the regulatory domains of T.l.MalK and E.c.MalK are not very similar except for a region in the connecting domain and another between strands 15 and 16 (Figure 3). This is in accordance with the fact that interactions with regulatory proteins such as MalT and EIIAGlc are unknown in T.litoralis. The existence of the C-terminal domain in T.l.MalK indicates, however, that the ATPase is also involved in regulatory circuits. Indirect evidence for a similar fold of both regulatory domains are the few conserved regions that are shared by a larger number of members of the bacterial ABC-type ATPase domains, which also possess an extension of ∼110 residues at the C-terminus of their ATPase domain. In S.t.MalK, the regulatory functions have indeed been mapped roughly to a segment of 106 residues at the C-terminus (Schmees and Schneider, 1998). Discussion The T.l.MalK dimer is in accord with restrictions by other data relevant for the dimer structure The A85C mutant of S.t.MalK has been shown, under in vitroconditions (i.e. reconstituted with MalFG in liposomes), to form a disulfide-linked dimer by forming a bond between the two pseudosymmetry-related cysteines (Hunke et al., 2000a). This means that the backbones of both monomers in the vicinity of this residue must approach each other in the dimer interface to a distance of less than ∼6 Å. In T.l.MalK, the alanine residue is conserved and corresponds to residue Ala91 in the 'lid' region. A-Ala91 and B-Ala91 are placed 17 Å apart. However, the 'lid' region has high and asymmetric B-factors (80 and 50 Å2 for the A- and B-chains, respectively; Figure 7), and the backbones of the two T.l.MalK monomers at another nearby residue, Gln88, are indeed placed only 8.5 Å apart (Figure 1B). This shows that the T.l.MalK dimer can meet the strong distance restraint of the cross-linking result after a subtle structural rearrangement, and thus provides evidence that the crystal structure is close if not identical to one conformation of the MalK dimer in the transport complex. Gln88 is conserved in most of the ABC-type ATPase domains (Schneider and Hunke, 1998). Among these are the two nucleotide-binding domains of the cystic fibrosis-related gated chloride channel CFTR. According to Hopfner et al. (2000) and Hung et al. (1998), it may coordinate Mg2+ and the water molecule that attacks the γ-phosphate bond. In our structure, the glutamine residues are placed too far away from the nucleotides to suggest such a role. In support of this finding, Walter et al. (1992) state that mutants of S.t.MalK in the corresponding Gln82 residue do not have significantly reduced ATPase activity. According