Crystal Structures of the ATPase Subunit of the Glucose ... - CiteSeerX

doi:10.1016/S0022-2836(03)00575-8

J. Mol. Biol. (2003) 330, 343–358

Crystal Structures of the ATPase Subunit of the Glucose ABC Transporter from Sulfolobus solfataricus: Nucleotide-free and Nucleotidebound Conformations Gre´gory Verdon1, Sonja V. Albers2, Bauke W. Dijkstra1 Arnold J. M. Driessen2 and Andy-Mark W. H. Thunnissen1* 1

Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Nijenborgh 4, 9747 AG Groningen, The Netherlands 2

Department of Microbiology Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

The ABC-ATPase GlcV energizes a binding protein-dependent ABC transporter that mediates glucose uptake in Sulfolobus solfataricus. Here, we report high-resolution crystal structures of GlcV in different states along its catalytic cycle: distinct monomeric nucleotide-free states and monomeric complexes with ADP-Mg2þ as a product-bound state, and with AMPPNP-Mg2þ as an ATP-like bound state. The structure of GlcV consists of a typical ABC-ATPase domain, comprising two subdomains, connected by a linker region to a C-terminal domain of unknown function. Comparisons of the nucleotide-free and nucleotide-bound structures of GlcV reveal re-orientations of the ABCa subdomain and the C-terminal domain relative to the ABCa/b subdomain, and switch-like rearrangements in the P-loop and Q-loop regions. Additionally, large conformational differences are observed between the GlcV structures and those of other ABCATPases, further emphasizing the inherent flexibility of these proteins. Notably, a comparison of the monomeric AMPPNP-Mg2þ-bound GlcV structure with that of the dimeric ATP-Naþ-bound LolD-E171Q mutant reveals a ^ 208 rigid body re-orientation of the ABCa subdomain relative to the ABCa/b subdomain, accompanied by a local conformational difference in the Q-loop. We propose that these differences represent conformational changes that may have a role in the mechanism of energy-transduction and/or allosteric control of the ABC-ATPase activity in bacterial importers. q 2003 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: ABC-ATPase; ATP-binding cassette; conformational changes; Q-loop; X-ray crystallography

Introduction ATP-binding cassette (ABC) transport systems form a large family of proteins performing the specific and active translocation of molecules across cellular membranes.1,2 These proteins are widespread in all kingdoms of life and their activity is required for various processes essential to cells, such as the uptake of nutrients (e.g. sugars, ions, amino acids), osmosensing and osmoregulation,3 multidrug resistance,4,5 and antigen processing.6 Because of their participation in Abbreviations used: ABC, ATP-binding cassette. E-mail address of the corresponding author: [email protected]

the multidrug resistance of tumour cells,7 a number of these transporters are of high medical interest. In addition, the dysfunction of an ABC transporter forms the molecular basis of several inherited human diseases,8 including cystic fibrosis.9 A feature that unifies the ABC transporters is their molecular organization, consisting minimally of two membrane-spanning domains that form a translocation channel, and two cytosolic ABC-ATPase domains (or ATP-binding cassettes) energizing transport through their ATPase activity. In eukaryotic systems, these domains are fused into one polypeptide chain, whereas their prokaryotic counterparts are usually composed of distinct subunits. Prokaryotic uptake systems use an additional subunit as a scavenger that delivers

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

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the substrate molecules at the entrance of the translocation pore. The ABC-ATPases are the most conserved domains of ABC transport systems. Their primary structures feature a characteristic set of motifs, including most notably the Walker A, Walker B and ABC signature motifs. The Walker A (or P-loop) and B motifs are common to a large group of nucleotide triphosphate-binding proteins and contain residues that participate in binding of ATP and the catalytic cofactor Mg2þ.10 The ABC signature motif is strictly specific to ABC-ATPases, and mutants in this motif have been shown to be defective in ATP-hydrolysis, while retaining their ability to bind ATP.11,12 The crystal structures of the ABCATPases HisP from Salmonella typhimurium, MalK from Thermococcus litoralis, LivG and LolD from Methanococcus jannaschii, and the nucleotide-binding domain of human TAP1 showed that ABCATPases share a common three-dimensional fold and nucleotide-binding site architecture.13 – 17 Moreover, the crystal structures of the Rad50 ATPase domain from Pyrococcus furiosus, MutS from Escherichia coli and the SMC protein head from Thermotoga maritima, all involved in DNA-maintenance, revealed that ABC-ATPase domains are present in other mechano-chemical systems, thus emphasizing their universal function as molecular engines.18 – 20 ATP-hydrolysis by ABC transporters is highly cooperative, suggesting that the two ABC-ATPases in a transporter function as a dimer in the translocation process.21,22 An association/dissociation cycle of this dimer, coupled to binding and hydrolysis of ATP, has been proposed as a key feature of the transport mechanism, allowing chemical energy to be converted into a mechanical stroke. The crystal structure of the ATPase domain of Rad50 in complex with AMPPNP-Mg2þ (or ATP) revealed for the first time the structural organization of such a dimer as a head-to-tail arrangement of the two ABC-ATPase domains, in which two functional active sites form at the dimer interface by juxtaposition of the ABC signature motif of one monomer with the Walker A and B motifs of the other monomer.18 Recently, the structures of the transporter complex BtuCD23 and the ATP-bound LolD-E171Q dimer24 established that a Rad50-like dimer indeed forms the catalytically competent state of transport ABC-ATPases. Although these structures have increased our understanding on the functioning of ABC transport systems, a number of aspects remain unclear. In particular, the mechanism of allosteric control of the ABC-ATPase activity in the bacterial importers has not been elucidated. Whereas isolated transport ABC-ATPases, in the absence of the membrane channel, show a constitutive ATPase activity,25 – 27 in the full bacterial importer ABCATPases are active only upon the binding of a liganded binding-protein to the pore.28,29 This activation most likely happens through conformational changes in the membrane subunits of the

Crystal Structures of an ABC-ATPase

transporter,30 transduced via intersubunit interactions to the ABC-ATPase subunits such that formation of a productive dimer is stimulated.31 – 34 The exact nature of these conformational changes, however, remains to be determined. In the hyperthermoacidophillic archaeon Sulfolobus solfataricus, a typical substrate binding protein-dependent ABC transporter dedicated to glucose uptake has been identified.35 This transporter consists of five subunits: a membraneanchored glucose-binding protein, GlcS, two membrane-spanning subunits, GlcT and GlcU, and two copies of the ABC-ATPase GlcV. A sequence analysis revealed that GlcV (40 kDa; 353 residues) consists of an ABC-ATPase domain of about 225 residues and, similar to several other ABC-ATPase subunits of prokaryotic uptake systems, a C-terminal extension (125 residues) of unknown function. In vitro, as a free subunit, GlcV exhibits a constitutive ATPase activity, with a rather low affinity for ATP ðKm < 290 mMÞ:27 To further identify and analyze conformational changes participating in the allosteric control of transport ABC-ATPases and the mechanism of energy transduction, we carried out an X-ray crystallographic study aimed at the structural characterization of different states along the catalytic cycle of GlcV. Here, we report the high-resolution crystal structures of two distinct monomeric nucleotide-free forms, one monomeric complex with ADP-Mg2þ that mimics the product-bound state and one with AMPPNP-Mg2þ as a mimic of an ATP-bound form. Analysis of these structures shows several conformational changes that have not been observed previously. Moreover, a comparison with the structures of other transport ABC-ATPases, but most notably that of the ATP-Naþ-bound LolD-E171Q dimer, shows conformational differences that may be of relevance to understand the allosteric properties of transport ABC-ATPases.

Results Structure determinations Wild-type GlcV was purified in a soluble form and crystallized initially in the absence of nucleotide and Mg2þ, as described.27 Preliminary diffraction experiments showed that two crystal forms appear in identical crystallization conditions. These nucleotide-free crystal forms (A and B) both belong to space group P212121 and have related unit cells (Table 1). The structure of GlcV in crystal form A was determined using experimental phases derived from single-wavelength anomalous dif˚ resolution fraction data and refined at 1.65 A (Rfactor ¼ 16:5%; Rfree ¼ 20:9%). Starting from the form A coordinates, the structure of GlcV in crystal form B was solved by molecular replacement, and ˚ resolution (Rfactor ¼ 21:5%; refined at 2.1 A Rfree ¼ 27:5%). These crystal forms A and B feature one and three molecules in the asymmetric unit,

345

Crystal Structures of an ABC-ATPase

Table 1. Data collection and refinement statistics

A. Data Beamline Detector Temperature (K) ˚) Wavelength (A B. Data processing and reduction Space group Unit-cell dimensions ˚) a (A ˚) b (A ˚) c (A No. molecules in the a.u. No. unique reflections Redundancy ˚ )a Resolution range (A Completeness (%)a kI/s(I)la Rsym (%)a,b C. Refinement ˚) Resolution range (A Rfactor/Rfree (%)c No. atoms No. non-protein atoms ˚) rmsd bond lengths (A rmsd bond angles (deg.) ˚ 2) kBl all atoms (A ˚ 2) kBl water molecules (A Ramachandran plot (% residues in) Most favored regions Additionally allowed regions Generously allowed regions Disallowed regions a b c

Nucleotide-free form A

Nucleotide-free form B

GlcV–ADP-Mg2þ

GlcV–AMPPNP-Mg2þ

ID14-EH2, ESRF ADSC Q4 CCD 100 0.933

ID14-EH4, ESRF ADSC Q4 CCD 100 1.00

BM30A, ESRF Mar 345 mm IP 100 0.97

ID14-EH2, ESRF ADSC Q4 CCD 100 0.933

P212121

P212121

P212121

P212121

46.0 48.27 183.04 1 49,451 6 45.0–1.65 (1.69–1.65) 98.1 (90.7) 22.4 (5.9) 6.8 (37.3)

47.03 146.60 178.49 3 69,924 4–5 45.0–2.10 (2.2– 2.10) 95.4 (71.0) 20.5 (3.0) 5.7 (33.6)

47.40 149.04 176.42 3 73,299 4–5 35.0–2.10 (2.15–2.10) 98.9 (98.3) 16.2 (3.9) 7.9 (34.2)

47.09 148.77 176.71 3 94,192 3 45.0–1.95 (2.0–1.95) 95.3 (95.5) 12.2 (2.7) 7.8 (35)

45.0–1.65 16.5/20.9 3248 490 0.016 1.62 17.06 36.45

45.0–2.10 21.5/27.5 9719 675 0.010 1.12 25.11 45.37

35.0–2.10 19.8/25.6 9071 986 0.010 1.10 20.43 35.59

20.0–1.95 18.8/24.0 9078 1045 0.010 1.25 24.86 45.15

92.5 7.2 0.3 0.0

90.6/89.7/91.1 7.0/9.3/8.2 1.0/1.0/0.7 1.3/0.0/0.0

90.7/90.5/89.0 8.3/9.2/10.3 1.0/0.3/0.7 0.0/0.0/0.0

90.2/91.5/90.2 8.8/7.5/9.1 1.0/1.0/0.7 0.0/0.0/0.0

Highest-resolution shell. P P P P RsymP ¼ h i lIðh;iÞ P2 kIlh l= h i kIlh : R ¼ h lFo 2 Fc l= h Fo :

respectively, with similar overall packing geometries. Several experiments were done aimed at obtaining crystals of GlcV in different nucleotidebound states. One such experiment was intended to obtain crystals of GlcV in complex with ADP, Mg2þ and vanadate, to mimic the ATPase transition state, as performed successfully for other ATPases.36 To this end, GlcV was first incubated with ATP and MgCl2 to generate the catalytic product ADP and vanadate was added just prior to the crystallization experiments. Although vanadate is an inhibitor of the ATPase activity of GlcV (data not shown), difference Fourier maps revealed that only ADP and the magnesium ion were bound in the nucleotide-binding site, showing that the crystals contained GlcV only in a product-bound state (Figure 3). GlcV crystals were obtained also in the presence of MgCl2 and ATPgS or AMPPNP. A GlcV complex with nucleotide triphosphate and Mg2þ was formed only with the latter ATP analogue, as evident from the analysis of difference Fourier maps. The structures of the GlcV – ADPMg2þ and GlcV – AMPPNP-Mg2þcomplexes were ˚ (Rfactor ¼ 19:8%; Rfree ¼ 25:6%) and refined at 2.1 A ˚ resolution (Rfactor ¼ 18:8%; Rfree ¼ 24:0%), 1.95 A respectively, and their crystals belong to form

B. All these nucleotide-free and nucleotide-bound crystals contain GlcV in the monomeric state, in agreement with gel-filtration experiments performed in the absence or presence of ADP-Mg2þ or AMPPNP-Mg2þ (not shown). Crystallographic data and statistics related to the processing and the quality of the refined models are summarized in Table 1. Overall structure The structure of GlcV consists of two domains connected by a short linker region (Figure 1). The larger N-terminal domain (residues 1 – 223) presents a fold typical of the ABC-ATPases and comprises two subdomains (ABCa/b, residues 1 –88 and 159– 223; ABCa, residues 89 –158). The ABCa/b subdomain contains a solvent-exposed anti-parallel b-sheet (b1, b2, b4, b5) and a larger, mostly buried b-sheet of mixed polarity (b6, b7, b8, b3, b9, parallel and b10, anti-parallel). These b-sheets form a “shell” surrounded by three a-helices. Helix a1 passes through the shell, while helices a5 and a6 flank the larger b-sheet on the other side. This subdomain, which is related structurally to nucleotide-binding proteins like RecA37

346

Crystal Structures of an ABC-ATPase

Figure 1. Three-dimensional structure of the ABC-ATPase GlcV from S. solfataricus in ribbon representation. The locations of the conserved motifs are indicated by different colours: Walker A motif (or P-loop), purple; Walker B motif, turquoise; Q-loop, red; D-loop, blue; H-motif, orange; ABC signature motif, yellow.

and the F1-ATPase,38 constitutes the conserved core of the ABC-ATPase architecture.13,18 Three strandloop-helix regions contain the residues of the Walker A, Walker B and H motifs, which compose most of the nucleotide-binding site. The ABCa subdomain forms an insertion of 70 residues between strands b6 and b7 and contains three a-helices (a2, a3, a4). This a-helical subdomain contains the ABC signature motif, encompassing the N-terminal turn of helix a4 and part of the preceding loop, and the Q-loop18 (residues 89– 95) that forms part of the loop region connecting helix a2 to strand b6 in the ABCa/b-subdomain. The C-terminal domain (residues 243– 353) contains only b-strands, which form an elongated barrel-shaped structure composed of two parts. The lower part (in the orientation of Figure 1) resembles an OB-fold39 and consists of two pairs of anti-parallel b strands (b17, b18 and b15, b19) swapped by another b-strand (b16). The upper part includes a three-stranded anti-parallel b-sheet (b11, b14, b20) and two small anti-parallel b-strands (b12, b13). The overall structure of this domain is very similar to that of the C-terminal ˚ for domain of MalK from T. litoralis (rmsd of 1.6 A a 100 C atoms) in agreement with a sequence identity of , 23% (Figure 2)40 The only structural difference corresponds to the loop region located between strands b13 and b14 of GlcV, where an a-helix is inserted in MalK. This structural similarity with MalK includes the linker region (helices a7 and a8) that connects the N and C-terminal domains, and gives further credence to the notion that the group of transport ABCATPases with a C-terminal extension have a common fold for their C-terminal domains.41 The function of the C-terminal domain in GlcV is not clear. For the E. coli MalK, which shares 27% of identical residues in this region with MalK from T. litoralis, the C-terminal domain was shown to bind the protein MalT, the activator of the mal regulon.42,43 However, such a regulatory system has not been identified for the glucose ABC transporter from S. solfataricus.

The nucleotide-Mg21-binding site The high-resolution structures of the GlcV – ADP-Mg2þ and GlcV –AMPPNP-Mg2þ complexes provide a detailed view of the residues involved in the binding of the nucleotide and the magnesium ion (Figures 3 and 4). Residues of the P-loop (Ser40 to Thr46) provide the majority of the interactions that stabilize the nucleotide in the nucleotide-binding site. As observed in the structures of various nucleotide-bound ATPases and GTPases, these P-loop residues are wrapped around the phosphate groups of the nucleotide and form several hydrogen bonds with the oxygen atoms using their side-chain and main-chain atoms. Additional stabilization of the nucleotide is provided by a stacking interaction between the side-chain of Phe13 and the adenine moiety, a type of interaction observed in the structures of other nucleotide-bound ABC-ATPases. The ribose hydroxyl and adenine amino groups are exposed to the solvent and do not participate in direct contacts with protein residues. In the GlcV –ADPMg2þ complex the Mg2þ is bound through an octahedral coordination geometry involving one b-phosphate oxygen atom, the Thr45 Og atom and four water molecules. The coordinating water molecules make additional contacts with oxygen atoms of the a and b-phosphate groups, and the sidechains of residues Gln89 (Q-loop), Asp165 (Walker B) and Glu166 (the putative catalytic base). The configuration of the nucleotide-binding site is very similar in all three molecules of GlcV present in the asymmetric unit and comparable to those observed in the structures of the ADP-Mg2þbound LivG, LolD and cTAP1. However, a major difference concerns the strictly conserved Q-loop glutamine residue. In the GlcV – ADP-Mg2þ structure, this glutamine residue makes a water-bridged contact with the Mg2þ, while in the structures of LolD and LivG with bound ADP-Mg2þ, it is found outside the nucleotide-binding site due to a different position of the ABCa subdomain relative to the ABCa/b subdomain.15,16 In the structure of

Figure 2. Structure-based sequence alignment of GlcV from S. solfataricus (GlcVS.s) with the ABC-ATPases MalK from T. litoralis (MalKT.l), LolD from M. janasschii (LolDM.j), BtuD from E. coli (BtuDE.c) and HisP from S. typhimurium (HisPS.t). Residue numbers and secondary structure elements refer to GlcV. The colour scheme used to highlight residues of the conserved ABC-ATPase motifs is similar to that in Figure 1.

348

Crystal Structures of an ABC-ATPase

Figure 3. Stereo views of the nucleotide-binding site of GlcV in (a) nucleotide-free form A, (b) nucleotide-free form B, (c) the GlcV – ADP-Mg2þ complex and (d) the GlcV – AMPPNP-Mg2þ complex. For the complexes, a portion of a 2Fo 2 Fc simulated annealing electron density omit map63 is shown, contoured at 1s and covering the nucleotide, the magnesium ion and its coordinating water molecules. Residues and ligands in the nucleotide-binding site are shown in ball-and-stick representation. The P-loop is coloured in purple.

ADP-Mg2þ-bound cTAP1, this residue is positioned closer to the Mg2þ, like in GlcV, but its side-chain is disordered.17 In the GlcV – AMPPNP-Mg2þ structure the ATP analogue is bound in a mode similar to that with ADP but with additional interactions made by the

three oxygen atoms of the g-phosphate group. Two of the oxygen atoms form a hydrogen bond with the side-chains of the P-loop residues Ser40 and Lys44. The third oxygen atom coordinates the Mg2þ, thus substituting a water molecule as equatorial ligand. In two of the three GlcV molecules

349

Crystal Structures of an ABC-ATPase

Conformational flexibility of GlcV in its nucleotide-free state

Figure 4. A representation of the interactions stabilizing the nucleotide, the magnesium ion and its coordinating water molecules (red dots) in (a) the GlcV– ADP-Mg2þ complex and (b) the GlcV – AMPPNP˚ and residue colouring Mg2þ complex. Distances are in A is identical with that used in Figures 1 and 2.

present in the asymmetric unit, the Q-loop Gln89 makes a water-bridged contact with the Mg2þ, as observed in the GlcV – ADP-Mg2þ structure. In the third molecule, however, the side-chain of this glutamine has a different conformation and its O1 atom is in direct contact with the Mg2þ, thus substituting a second equatorial water ligand. Such a direct interaction of the Q-loop glutamine with the Mg2þ is also observed in the dimeric structures of the Rad50 –AMPPNP-Mg2þ and LolD-E171Q –ATP-Naþ complexes.18,24 In fact, in the third GlcV molecule the overall binding mode of AMPPNP-Mg2þ is very similar to that of AMPPNP-Mg2þ in Rad50 and ATP-Naþ in LolD, if one excludes the contacts formed across the dimeric interface in these latter complexes (Figure 6(c)). An important difference, though, is noted for a highly conserved histidine residue in GlcV and LolD, His199 and His204, respectively, which forms part of the so-called H-motif.1 This histidine residue has a different position and side-chain conformation in the GlcV monomer, not allowing the formation of a hydrogen bond with the g-phosphate group, similar to that occurring in the LolD dimer. In addition, no putative nucleophilic water molecule could be observed ˚ in front of the g-phosphate group in the 1.95 A resolution structure of the GlcV – AMPPNP-Mg2þ complex.

Pairwise structural alignments of all the molecules in the two nucleotide-free crystal forms (A and B) highlight a significant conformational flexibility in the GlcV protein. Two of the three form B molecules show very similar conformations (rmsd ˚ for 223 ˚ for all Ca atoms; rmsd of 0.17 A of 0.41 A a C atoms of the ABC-ATPase domain), which differ appreciably from that of the form A molecule ˚ and 1.0 A ˚ for all Ca atoms; rmsd (rmsd of 1.22 A ˚ and 0.73 A ˚ for 223 Ca atoms in the ABCof 0.75 A ATPase domain). Differences with the form A molecule are even more pronounced in the third ˚ for all Ca atoms; form B molecule (rmsd of 1.35 A ˚ for 223 Ca atoms in the ABCrmsd of 0.98 A ATPase domain). Further analysis revealed that these structural differences consist of rigid-body rotations of the ABCa subdomain and C-terminal domain with respect to the ABCa/b subdomain, as well as of structural rearrangements in the Q-loop and P-loop regions. Compared to the form A molecule, the ABCa subdomains in the form B molecules have rotated by 4– 88 towards the nucleotide-binding site in the ABCa/b subdomain (similar to what is shown in Figure 5). This rotation, which is correlated to the slight differences in crystal packing geometry between the form A and form B crystals, occurs about a hinge located at the C-terminal end of the ABCa subdomain, involving residues in the loop between helix a4 and strand b7. At the N-terminal end of the ABCa subdomain, a significant change in flexibility of the Q-loop is observed. In the form A molecule, the Q-loop is ordered and completely resolved in density. Its conformation is stabilised by van der Waals contacts between the side-chain of the Q-loop Trp91 and the side-chains of Met86 and Arg153, as well as by two hydrogen bonds from the guanidinium group of Arg153 to the backbone carbonyl groups of Trp91 and Ala92 (Figure 7). The side-chain of the Q-loop Gln89 is not involved in any hydrogen-bonding contact and occupies a position distant from the nucleotide-binding site (Figure 5(b)). In contrast, in the three form B molecules, the Q-loop shows considerable disorder, as is evident from weak and discontinuous electron density for this region. Since the Q-loop is not involved in any crystal packing contact, its disorder must be a direct consequence of the re-orientation of the ABCa subdomain, probably by affecting the contacts with neighbouring residues and/or by introducing strain in its backbone. In addition to the structural change in the Q-loop, a peculiar switch-like rearrangement occurs in the P-loop. Whereas in all the form B molecules the P-loop has a conformation identical with that observed in the nucleotide-bound molecules, in the form A molecule the P-loop assumes a “closed” conformation deviating from the typical “open” geometry associated with a

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bound nucleotide (Figure 3). The switch from an open to a closed conformation involves mainly residues Gly41, Ala42 and Gly43, which move ˚ for the Ca atom of over a large distance (6.5 A Ala42) to form an additional a-helical turn at the N terminus of helix a1, thus filling the cavity normally occupied by the a and b-phosphate groups of the nucleotide. Structural rearrangements underlying this switch include the flipping of peptide bonds Pro39-Ser40 and Ala42-Gly43. The position and orientation of the conserved P-loop lysine are not affected. Finally, the C-terminal domain and the linker region of GlcV have rotated as one rigid body by 6 –128 relative to the ABCa/b-subdomain, if one compares the nucleotide-free form B molecules with the form A molecule (Figure 5(a)). This movement occurs about a hinge that includes residues of the loop region between strand b10 and helix a7. Like the re-orientation of the ABCa

Crystal Structures of an ABC-ATPase

subdomain, the rotation of the C-terminal domain results from small differences in crystal packing, indicative of a strong inherent flexibility of the GlcV protein. Conformational changes in GlcV associated with nucleotide-Mg21-binding While the GlcV molecules show significant conformational differences in the nucleotide-free form B crystal, structural alignments of the molecules in the nucleotide-bound form B crystals reveal that in the presence of ADP-Mg2þ or AMPPNP-Mg2þ bound in the nucleotide-binding site, the ABCATPase domains assume a single conformation. The backbone conformations are very similar ˚ to 0.54 A ˚ for 223 Ca (rmsd varying from 0.21 A atoms of the ABC-ATPase domain), as are the conformations of the side-chains in the nucleotidebinding sites, except for the Q-loop glutamine

Figure 5. Stereo views of a superposition of the nucleotide-free crystal form A and AMPPNP-Mg2þ-bound structures of GlcV. (a) Superposition of the Ca backbones of the nucleotide-free (blue line) and nucleotide-bound (orange line) structures. (b) View of the nucleotide-binding site and the Q-loop region (the nucleotide-free structure is shown with semi-transparent colours and the phosphate groups of AMPPNP are drawn with sticks).

Crystal Structures of an ABC-ATPase

residue, as described above. Overall, this nucleotide-bound conformation is comparable to that of the nucleotide-free form B molecule deviating most from the form A molecule (rmsd varying ˚ to 0.45 A ˚ for 223 Ca atoms of the from 0.29 A ABC-ATPase domain). However, a significant difference is observed for the Q-loop. In the nucleotide-bound crystals of GlcV, the Q-loop is not disordered, but adopts a new conformation, which is different from that observed in the nucleotide-free crystal form A molecule (Figures 5 and 7). Compared to the form A conformation, the Trp91 side-chain has swung to the other side of the ˚ , to be Q-loop, moving over a distance of about 8 A lodged in a hydrophobic pocket lined by the Val87, Leu93, Gln146, Gln147, and Ala150 sidechains. Concomitantly, the Gln89 side-chain has flipped from an outward location into a position where it binds the Mg2þ in the nucleotide-binding site. The conformational change of the Q-loop results in the loss of the hydrogen bond between the carbonyl group of Trp91 and the guanidinium group of Arg153. At the nucleotide-binding site, slight side-chain displacements and reorientations are observed that lead to the binding of nucleotide and the Mg2þ. In addition, small displacements are observed for residues that surround the binding pocket of the Trp91 side-chain in the nucleotide-bound conformation, as well as of residues in the D-loop, which forms a non-covalent link between the Trp91-binding pocket and the nucleotide-binding site. Hence, the interactions of GlcV with the Mg2þ and nucleotide stabilize both directly and indirectly the new conformation of the Q-loop, reducing its flexibility and thus tightening the overall conformation of the ABCATPase domain. Comparisons with other transport ABCATPase structures The GlcV – ADP-Mg2þ and GlcV –AMPPNPMg2 þ structures represent conformations of a nucleotide-bound ABC-ATPase in a monomeric, non-productive state. Other crystal structures of isolated ABC-ATPases in various non-productive states are those of HisP (ADP-bound, no Mg2þ), LolD (ADP-Mg2þ-bound), LivG (nucleotide-free and ADP-Mg2þ-bound), cTAP1 (ADP-Mg2þbound) and MalK (diphosphate-bound).13 – 17 LolD is so far the only transport ABC-ATPase for which a structure mimicking the productive ABC-ATPase dimeric state is available (ATP-Naþ-bound LolDE171Q mutant).24 The overall fold of the ABCATPase domain of GlcV is very similar to these other transport ABC-ATPases, in agreement with the high degrees of sequence identity (49% with MalK, 40% with LolD, 32% with HisP, 28% with LivG; 26% with cTAP1). However, substantial differences are observed for the relative orientations of the ABC subdomains, as well as for the conformation of the Q-loop. To further define these differences and evaluate their functional sig-

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nificance, we superimposed the various structures for comparative analysis. The nucleotide-bound conformation of the ABC-ATPase domain of GlcV shows the greatest similarity to that of monomeric ADP-Mg2þ-bound LolD (Figure 6(a)). Alignment ˚ for 206 of their backbones yields an rmsd of 1.3 A a equivalent C atoms in the ABC-ATPase domain (calculated with the GlcV molecule in which the Q-loop glutamine has a direct interaction with the Mg2þ). A small difference in orientation of the ABCa subdomains relative to the nucleotidebinding core is observed, which is, however, accompanied by a substantial difference in backbone conformation of the Q-loop (Figures 6(a) and 7). This difference involves changes in backbone dihedral angles, and a peptide bond flip of the residues in the þ 2 and þ 3 positions after the conserved glutamine residue (Trp91-Ala92 for GlcV and Phe92-Asn93 for LolD). In fact, the Q-loop in ADP-Mg2þ-bound LolD has a conformation with features of both the nucleotide-free form A and nucleotide-bound form B structures of GlcV, with the main-chain carboxyl groups of the þ 2 and þ 3 residues both interacting with a conserved arginine residue (Arg158, the equivalent of Arg153 in GlcV), as occurring in the form A structure of GlcV, while the side-chain of the þ 2 residue is in a position similar to that in the nucleotide-bound structures of GlcV. Another difference occurs at the C terminus of the b-strand that precedes the Q-loop. In LolD, this region is displaced relative to its position in the structures of GlcV, causing a breakage of inter-strand hydrogen bonds and a shift of the Q-loop glutamine residue away from the Mg2þ. Structural comparison of GlcV with MalK, HisP, LivG and cTAP1 reveals significant differences in orientation of the ABCa subdomains, especially with ATP-bound HisP, ADP-Mg2þ-bound cTAP1 and di-phosphate-bound MalK. The largest conformational differences are observed if GlcV is compared to the LolD-E17lQ mutant in the ATPNaþ-bound dimeric state (Figure 6(b) and (c)). Their ABCa subdomains show a difference in orientation of , 208, displacing the signature motif ˚ , while in the nucleotide-binding site the by , 10 A side-chains of the H-motif histidine residues show significant differences in position and rotamer conformation. These differences are similar to those revealed previously in the LolD structures of the ADP-Mg2þ-bound monomer and the ATP-Naþbound dimer,24 and may be associated with the ATP-dependent dimerization and activation of isolated ABC-ATPases. However, the structural changes predicted to occur upon dimerization of the nucleotide-bound structures of GlcV, as based on the comparison with the ATP-Naþ-bound structure of LolD-E17l, point to a different involvement of the Q-loop region (Figures 6 and 7). In the monomer to dimer transition of LolD, the rotation of the a-helical subdomain is mediated mainly by a relocation of the conserved glutamine residue and the C terminus of the b-strand that precedes the Q-loop. In the nucleotide-bound structures of

352

Crystal Structures of an ABC-ATPase

Figure 6. Stereo superposition of the Ca backbone of the GlcV – AMPPNP-Mg2þ complex (green lines) versus that of (a) monomeric ADP-Mg2þ-bound LolD (purple line) and (b) dimeric ATP-Naþ-bound LolD-E171Q (purple line). Only the ABC-ATPase domains are shown. (c) Stereo view of the aligned nucleotide-binding sites of GlcV –AMPPNP-Mg2þ (green) and ATP-Naþ-bound LolD-E171Q (purple).

GlcV, however, the Q-loop glutamine residue is already in contact with the metal ion, and the predicted change in Q-loop conformation associated with dimerization would involve localized dihedral angle changes in residues that follow Gln89 (i.e. Trp91 and Ala92).

Discussion Our work has led to the determination of several high-resolution crystal structures of an ABCATPase, GlcV, in different states along its catalytic cycle: two distinct nucleotide-free states, a

Crystal Structures of an ABC-ATPase

353

Figure 7. Stereo views of the different Q-loop conformations observed in the GlcV and LolD structures: (a) GlcV in nucleotide-free form A; (b) GlcV– AMPPNP-Mg2þ complex; (c) LolD– ADP-Mg2þ complex; (d) LolD-E171Q – ATP-Naþ dimer. The orientation of the different Q-loops is based on a structure alignment using the Ca backbones of the ABCa/b-subdomains (see also Figure 6).

product-bound state (ADP-Mg2þ) and ATP-like bound state (AMPPNP-Mg2þ). In all these structures, GlcV is present in a monomeric state, in agreement with the results of gel-filtration experiments. Biochemical analysis of GlcV mutants, however, has shown that GlcV, like other transport ABC-ATPases, forms dimers in solution in an ATP-Mg2þ-dependent manner (our unpublished results). The current GlcV structures, including the AMPPNP-Mg2þ-bound form, thus represent nonproductive conformations of an ABC-ATPase. The conformation of the ABC-ATPase domains in the catalytically competent, ATP-Mg2þ-bound GlcV dimer is expected to resemble closely that of LolD in the ATP-Naþ-bound LolD-E171Q dimer. This is evident from the high level of overall sequence identity and the considerable structural conservation we observe if GlcV is compared with monomeric LolD. Moreover, the residues that form the dimeric interface in the LolD dimer are strictly conserved in GlcV. On the other hand, GlcV has an extra C-terminal domain that is not present in

LolD, which could have an effect on the organization of the GlcV dimer. To analyse this, a dimeric model of GlcV was built by using the LolD-E171Q dimer as a template (Figure 8). The result shows that the C-terminal domains can be accommodated easily in the dimeric model, and do not obstruct association of the ABC-ATPase domains. Furthermore, if this dimer is superimposed on the two ABC-ATPase subunits of the BtuCD transport complex,23 it is clear that both C-terminal domains would be fully exposed in the cytoplasm and would not interfere with the binding of the ABCATPase domains to the membrane-spanning subunits, thus further augmenting the reliability of the dimeric model. Comparison of the monomeric GlcV structures with this model suggests the conformational rearrangements that are necessary to change GlcV from a non-productive state into its productive, ATP-bound state. As shown previously for LolD, these rearrangements include a large reorientation of the ABCa subdomain and the repositioning of side-chains in the

354

Crystal Structures of an ABC-ATPase

Figure 8. Stereo views of a model of an ATP-Mg2þ-bound GlcV dimer. The structure was built by homology modeling using the SWISS-MODEL server (http://swissmodel.expasy.org/SWISS-MODEL.html). The dimeric structure of ATP-Naþ-bound LolD-E171Q was used as a template. The relative orientations of the C-terminal domains are based on the structure of the GlcV– AMPPNP-Mg2þ complex. (a) View toward the ABC-ATPase domains, the C-terminal domains are at the back; (b) A 1808 degrees rotated view toward the C-terminal domains.

nucleotide-binding site. The ABCa subdomain reorientation requires a substantial flexibility of the GlcV protein, but our analysis of the GlcV structures and their comparison with those of other, related transport ABC-ATPases shows that such flexibility exists. The subdomain reorientation is necessary to reshape the protein surface involved in dimerization, while bringing the signature motif into a position that enables it to bind the g-phosphate group of ATP across the dimeric interface. Concurrent conformational changes at the nucleotide-binding site, including most notably that of the H-motif histidine side-chain, ensure proper stabilization of the g-phosphate group and binding of the nucleophilic water molecule, as required for catalysis. The AMPPNP-Mg2þ-bound GlcV structure, where these extra stabilizing interactions with the g-phosphate group are absent and the potential-binding site for the nucleophilic water is empty, supports the notion that ABCATPases are not active in their monomeric form. The reason for the inability of AMPPNP to induce dimer formation of GlcV is not clear. Similar observations have been reported for LivG and LolD,24,44 showing that AMPPNP is not a true substrate analogue for transport ABC-ATPases. However, in

the AMPPNP-Mg2þ –GlcV complex, the binding mode and interactions of AMPPNP with most amino acid residues in the nucleotide-binding site are very similar to that of ATP in the ATP-Naþbound LolD dimer, not considering the contacts formed across the dimer interface. The similarities include the direct contact of the conserved Q-loop glutamine with the Mg2þ, which is stabilized by the presence of the g-phosphate group. Nevertheless, binding of AMPPNP-Mg2þ to GlcV apparently generates insufficient energy to induce the conformational changes that lead to dimer formation, which must be related to its single chemical difference with ATP, the replacement of the P –O – P bonds by P – NH – P bonds in the b – g phosphate linkage. This replacement has two main consequences for the properties of AMPPNP versus ATP. In the AMPPNP-Mg2þ – GlcV the imino group in the P – NH –P bond makes a close contact with the backbone amide group of Gly41 (Figure 4(b)) but, since it can function only as hydrogen bond donor, the formation of a hydrogen bond is precluded. In the ATP-Mg2þ-bound state of GlcV, however, it is expected that a hydrogen bond is formed from the backbone amide group of Gly41 to the oxygen atom in the P –O –P linkage. In

355

Crystal Structures of an ABC-ATPase

agreement with this view, a small difference is observed in the geometries of the P – NH – P and P –O – P bonds, relative to the Gly41 amide group, if one compares the GlcV and LolD structures (Figure 6(c)). Secondly, in ATP the oxygen atoms of the g-phosphate group will carry a slightly weaker negative charge than in AMPPNP, due to the stronger electro-negativity of the oxygen atom in the b – g-phosphate linkage as compared to nitrogen. This last difference could be an important factor that determines the different effect on GlcV dimerization of ATP and AMPPNP, as studies with LolD and LivG show that dimer formation of transport ABC-ATPases depends critically on the electrostatic properties of the nucleotide-binding site.24,44 An important difference between the GlcV and LolD structures is observed for the Q-loop region. In both GlcV and LolD, this region undergoes conformational rearrangements coupled to reorientations of the ABCa-subdomain. However, the precise nature of these rearrangements is different for each protein. In the monomer to dimer transition of LolD, the structural rearrangements consist essentially of a shift of the conserved glutamine residue toward the Mg2þ, in conjunction with a repositioning of the C terminus of the b-strand that precedes the Q-loop. On the basis of these observations, the Q-loop glutamine residue in LolD has been proposed to function as a g-phosphate sensor,15,16 changing position to coordinate the Mg2þ as soon as the g-phosphate group of ATP is bound in the nucleotide-binding site, which, in turn, triggers the reorientation of the ABCa subdomain as required for dimer formation. In GlcV, we observe a shift of the Q-loop glutamine residue toward the Mg2þ, but it occurs between nonproductive nucleotide-free and nucleotide-bound structures. The direct interaction of Gln89 with the Mg2þ occurs only in the GlcV – AMPPNP-Mg2þ complex, showing that this direct interaction is both Mg2þ and g-phosphate-dependent. However, the structure shows that the formation of this interaction is not coupled to a large reorientation of the ABCa subdomain. We propose that, in solution, the interactions of the ABC signature motif with the g-phosphate groups in the dimer are probably the most important source of energy that drives the reorientation of the ABCa subdomains. In the transporter complex, on the other hand, the subdomain reorientation will be influenced by the contacts of the ABC-ATPases with the membranespanning domains. As shown in the structure of BtuCD,23 the interface with the transmembrane subunits involves residues of both ABC subdomains that are located around the Q-loop. It is conceivable that this interface serves to relay the conformational changes from one subunit to the other, as required in the energy transduction mechanism of the transporter.23,24 In GlcV, the predicted change in Q-loop conformation upon the transition from a non-productive to a productive ATP-bound state consists of localized dihedral angle changes

in residues downstream from Gln89; the conserved glutamine residue itself remains bound to the Mg2þ. Considering this, we propose that within the intact transporter the role of the conserved Q-loop glutamine residue, and its interaction with Mg2þ, is to keep the two ABC subdomains in close proximity to each other, restricting their motional freedom. While in solution isolated ABC-ATPases can approach each other freely, thus increasing the chance of dimer formation, it is conceivable that, when embedded in the full transporter, the reorientation of the ABCa subdomains must follow a precisely defined path, to prevent a misalignment of the two ABC-ATPases. This hypothesis is supported by the results of several mutational studies focussed on the role of the Q-loop and Q-loop glutamine residue in bacterial importers. For instance, mutations of Q-loop residues in HisP, including the conserved glutamine residue, led to defective transporters in vivo, whereas the isolated mutant proteins in solution could still bind ATP.11 Similarly, in MalK, mutations of the conserved Q-loop glutamine residue into a glutamate and lysine residue reduced the ATPase activity of the isolated proteins only weakly, whereas they produced defective transporters when integrated in the membrane protein complex.45 Caution is required when assessing the significance of findings on isolated ABC-ATPases for the functioning of these protein modules in full ABC transporter complexes. Nevertheless, we wish to propose that the AMPPNP-Mg2þ-bound structure of GlcV represents a possible conformation of the ABC-ATPases during the resting state of the transporter, unsuitable for the formation of the productive dimer, but still able to bind ATP and Mg2þ. In the transporter the transmembrane subunits could be responsible for “freezing” the ABCATPases in such a non-productive conformation. Binding of the liganded substrate-binding protein may then trigger certain conformational changes in the transmembrane subunits, that lead to the stabilization of a dimerization-competent conformation of ABC-ATPases, thus explaining the allosteric control of the ATPase activity in bacterial importers.

Materials and Methods Determination of GlcV nucleotide-free structures Procedures for the purification of GlcV, its crystallization and the determination of experimental phases have been reported.27 Briefly, experimental phases were obtained from single-wavelength anomalous diffraction (SAD) data collected on an iodide derivative of nucleotide-free crystal form A and improved by density modifi˚ resolution. Subsequently, these phases cation to 2.1 A ˚ resolution were combined with data collected to 1.65 A on a similar iodide derivative at beamline ID14-EH2 (ESRF, Grenoble). After phase extension, improved phases were obtained from an average of six refined dummy atom models, as produced by the program

356 ARP/wARP.46 The protein main chain could be traced automatically (333 residues in four chains) using the ARP/wARP alternated building and refinement routines.47 After addition of the side-chains to the model, the refinement was carried out using the program CNS48 and monitored with the conventional Rfactor and an Rfree set that included 8% of the data.49 The refinement was alternated with manual model rebuilding, performed with the programs O50 and QUANTA (Accelrys, San Diego). Water molecules were placed automatically using the solvent building protocol implemented in ARP/wARP, and further checked manually. The refinement was completed using the program REFMAC5,51 including TLS refinement.52 The quality of the structure was assessed using the program PROCHECK53 and the ADIT validation server of the Protein Data Bank.54 Diffraction data for the nucleotide-free crystal form B were collected at beamline ID14-EH4 (ESRF, Grenoble) ˚ resolution and reduced using the programs to 2.1 A DENZO and SCALEPACK.55 Structure factors were calculated using programs from the CCP4 software suite.56 The starting model was obtained by molecular replacement performed with the program AmoRe,57 using the ˚ ). crystal form A structure as a search model (15 – 3.5 A The refinement was performed as described for the structure of GlcV in nucleotide-free crystal form A, without any use of non-crystallographic symmetry restraints. Crystallization and structure determination of the nucleotides complexes GlcV (4.25 mg/ml in 20 mM Mes buffer (pH 6.5), 150 mM NaCl, 5% (v/v) glycerol, 5 mM MgCl2) was incubated overnight at room temperature with 5 mM ATP. Vanadate was added to 5 mM just prior to the crystallization experiment. Crystals suitable for data collection were grown by microseeding in hangingdrop, vapour-diffusion set-ups. Drops were made of equal volumes of protein solution and reservoir solution containing 15% (w/v) PEG 3350, 15% (w/v) PEG 400, 0.1 M Tris buffer (pH 8.3), 0.5 M NaI, 15% glycerol. Diffraction data were collected at beamline BM30A ˚ resolution. Difference Fourier (ESRF, Grenoble) to 2.1 A maps revealed that only ADP and a magnesium ion were bound in the nucleotide-binding site of the three molecules present in the asymmetric unit; no vanadate ion could be identified in the electron density maps during the whole refinement procedure. Crystals of the GlcV– AMPPNP-Mg2þ complex were obtained using a similar procedure. GlcV was incubated overnight at room temperature in the presence of 5 mM AMPPNP. Diffraction data were collected at beamline ˚ resolution. Initial phases ID14-EH2 (ESRF) to 1.95 A were calculated from the GlcV – ADP-Mg2þ structure, including only the protein atoms, and the refinement was conducted as described above. Size-exclusion chromatography experiments Samples for size-exclusion chromatography analysis (20 ml, 0.5 mg/ml of GlcV in 25 mM Mes (pH 6.5), 200 mM NaCl, 5% glycerol, 1 mM MgCl2) were incubated overnight and at room temperature in the absence and presence of ADP, AMPPNP or ATP (0.1 mM). The experiments were performed using a PC 3.2/30 Sephadex 75 column mounted on a SMART system (Pharmacia). The column was equilibrated and eluted with the solution used to prepare the samples, including

Crystal Structures of an ABC-ATPase

the corresponding nucleotide. the elution of the protein was monitored with the absorption profiles recorded at 254 nm and 280 nm. Miscellaneous Structure superpositions were performed with the program LSQKAB from the CCP4 suite.56 Structure Figures were prepared using MOLSCRIPT, Bobscript and Raster3D.58 – 60 The sequence alignment was generated using ClustalW61 at the EBI server† and edited manually. The program Alscript62 was used for presentation of the sequence alignment. Protein data bank accession numbers Atomic coordinates of the GlcV structures (nucleotidefree crystal forms A and B, and the complexes GlcV – ADP-Mg2þ and GlcV – AMPPNP-Mg2þ) have been deposited with the RCSB Protein Data Bank with the entry codes 1OXS, 1OXT, 1OXU and 1OXV.

Acknowledgements We are grateful to Edward Mitchell, Michel Roth, and other staff scientists for assistance during data collections at beamlines ID14-EH2, ID14-EH4 and BM30A (ESRF, Grenoble). G.V. is a recipient of an Ubbo Emmius fellowship (University of Groningen). This research was supported by the EC with a TMR grant (ERBFMBIC971980) for S.V.A.

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Edited by D. Rees (Received 12 February 2003; received in revised form 9 April 2003; accepted 10 April 2003)