Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate*

The ATPase activity of the maltose transporter (MalFGK2) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK2 are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose- and sucrose-bound sMBP stimulate equal levels of MalFGK2 ATPase activity. Therefore, the ATPase activity of MalFGK2 is coupled to translocation of maltose solely by interactions between MalFGK2 and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the MalFGK2 ATPase was greatly reduced compared with wild-type MBP, indicating that the mutations in sMBP have interfered with important interactions between MBP and MalFGK2. High resolution crystal structure analysis of sMBP shows that in the closed conformation with bound sucrose, three of four mutations are buried, and the fourth causes only a minor change in the accessible surface. In contrast, in the open form of sMBP, all of the mutations are accessible, and the main chain of Tyr62–Gly69 is destabilized and occupies an alternative conformation due to the W62Y mutation. On this basis, the compromised ability of sMBP to stimulate ATP hydrolysis by MalFGK2 is most likely due to a disruption of interactions between MalFGK2 and the open, rather than the closed, conformation of sMBP. Modeling the open sMBP structure bound to MalFGK2 in the transition state for ATP hydrolysis points to an important site of interaction and suggests a mechanism for coupling ATP hydrolysis to substrate translocation that is independent of the exact structure of the substrate.

ATP-binding cassette (ABC) 2 transporters move various substrates across membranes, with substrate movement coupled to the hydrolysis of ATP. Although the ATPase activity of ABC exporters like P-glycoprotein is generally stimulated by substrate binding, the ATPase activity of ABC importers is acti-vated by a peripheral substrate-binding protein and not the free substrate (for recent reviews see Refs. [1][2][3]. However, the mechanism of ATPase regulation is still not fully understood. Here, we use one of the most well studied ABC importers, the Escherichia coli maltose transporter (MalFGK 2 ), to investigate the roles of maltose-binding protein (MBP) and maltose itself in regulation of ATPase activity.
In its resting state MalFGK 2 contains a substrate-binding site that is exposed to the cytoplasm (4). In the periplasm, MBP binds maltose, which stabilizes a change from an "open" to a "closed" conformation, enabling it to stimulate the MalFGK 2 ATPase (5,6). Interactions with closed, maltose-bound MBP lead to exposure of the MalFGK 2 maltose-binding site to the periplasmic side where maltose can move from MBP into an occluded translocation pathway (7,8). After ATP hydrolysis, the transporter returns its binding site to the cytoplasmic face to allow the substrate to enter the cytoplasm. This is known as the alternating access model of maltose transport (4) and may be a common mechanism among ABC transporters (2,9,10).
The structure of a transition state complex between MBP and MalFGK 2 , as well as biochemical data (7,8), indicates that maltose enters the substrate-binding site of MalFGK 2 prior to ATP hydrolysis, but it is unclear how maltose-bound MBP activates the MalFGK 2 ATPase (11) and how ATP hydrolysis is coupled to the movement of maltose across the membrane. Of particular interest are the roles that maltose itself might play in regulating the ATPase activity of MalFGK 2 .
There are two ways maltose could regulate ATP hydrolysis. The first is by stabilizing the closed conformation of MBP, and the second is through direct interactions with MalFGK 2 . Although it is clear from previous studies that substrate-induced domain closure in MBP is critical for robust stimulation of the MalFGK 2 ATPase and substrate transport (6), it is not known whether direct interaction between maltose and MalFGK 2 is also required for ATPase activity.
To address this question, we have used an MBP mutant that is able to bind an alternative substrate, sucrose, with high affinity (12). The sucrose-binding MBP (sMBP) enables us to present the maltose transporter with either maltose or sucrose in equivalent contexts and distinguish whether the substitution influences the ATPase activity of MalFGK 2 . Sucrose is a good alternative substrate for this purpose because experiments by Shuman and co-workers (13,14) have shown that it has a very poor ability to compete for the maltose-binding site in MalFGK 2 , indicating that the change in sugar structure is sufficient to disrupt specific binding interactions with MalFGK 2 .
Using sMBP, we have determined that ATP hydrolysis by MalFGK 2 is not dependent on the exact nature of the substrate, and therefore the coupling of ATPase activity to substrate translocation is due solely to interactions between MBP and MalFGK 2 . Based on these findings and detailed structural analysis of sMBP, we propose that a productive interaction between MalG and the vacated maltose-binding site in MBP is required for ATP hydrolysis. In this manner, substrate translocation from MBP to MalFGK 2 is coupled to ATP hydrolysis without requiring a direct interaction between maltose and MalFGK 2 .

MATERIALS AND METHODS
Cloning of MBP Mutants-Plasmid pDIM-C8MalE, containing sMBP, was kindly provided by Ostermeier and coworkers (12). A 923-bp Kpn21/BclI fragment of this vector, containing the W62Y and E111Y substitutions, was ligated into pLH1, which contains the MBP signal sequence for export to the periplasm. The D14L and K15F mutations were substituted by mutagenic PCR using the following primers: 5Ј-CTGGATTAACGGCCTTTTCGGCTATAACGGTCT-CGC-3Ј and 5Ј-GCGAGACCGTTATAGCCAAAAAG-GCCGTTAATCCAG-3Ј.
To produce intracellularly expressed sMBP and wtMBP with a hexahistidine affinity tag, restriction cut sites for EheI and HindIII were added to excise the two genes (without localization tag) using the following primers: 5Ј-CGCCTCGGCTGG-CGCCAAAATCGAAG-3Ј and 5Ј-CGCCGCATCCGGCAT-TTAAGCTTATTACTTGGTGATACGAG-3Ј. Digested PCR products were then ligated into the multicloning site of pPROEX-HTa (Invitrogen) to introduce an N-terminal hexahistidine tag attached by a tobacco etch virus protease cleavable linker. Cleavage of this linker left an N-terminal glycine-alanine insertion that was common to both the sMBP and wtMBP used in this study.
Expression and Purification-Hexahistidine-tagged sMBP and wtMBP were expressed and purified from HS3309 (MalE Ϫ/Ϫ ) E. coli by Ni 2ϩ -affinity chromatography, removal of the affinity tag by tobacco etch virus protease cleavage, and ion exchange chromatography, as reported previously (15). Both proteins were denatured in 6 M guanidine and dialyzed exhaustively to remove trace sugars before being refolded by dropwise dilution and stored at Ϫ80°C in 50 mM Tris-HCl, pH 8 (15).
Preparation of wt-MalFGK 2 -containing Proteoliposomes-MalFGK 2 was overexpressed from plasmids pNT1SK ϩ and pMR111 in E. coli HS3399 cells, which contain deletions for all transporter components. Membrane fractions were prepared and solubilized as reported previously (15).
Liposomes were prepared from Avanti TM crude E. coli phospholipids, and after homogenization by sonication, the liposomes were combined with MalFGK 2 -containing membranes by detergent dilution (15). The proteoliposomes were frozen at Ϫ80°C under N 2 until used.
ATPase Assays-ATPase measurements were made in a solution of 50 mM Tris-HCl, pH 8.0, 100 mM KCl, and 10 mM MgCl 2 , with proteoliposomes added to a final concentration of 0.1 mg/ml protein. Purified sMBP or wtMBP was added at various concentrations and in the presence or absence of 5 mM maltose or sucrose. ATP hydrolysis at 37°C was measured in vitro by assaying the appearance of inorganic phosphate, using ammonium molybdate, as described previously (15).
Phases were determined by molecular replacement with the wild-type proteins (PDB codes 1ANF and 1OMP). Rigid body refinement of the two isolated domains was carried out first to capture any domain movements relative to the wild-type structures. Structures were refined using CNS (  (12). The sMBP molecule has four point mutations, D14L, K15F, W62Y, and E111Y, all within the substrate binding cleft. Although wild-type MBP (wtMBP) has a dissociation constant (K D ) of 1 M for maltose and no ability to bind sucrose (6), sMBP has a K D for maltose of 24 M and for sucrose of 6.6 M (12). These values were confirmed for our sMBP constructs using fluorescence titrations (data not shown). Furthermore, we measured substrate-induced conformational changes in sMBP, in solution, by small angle x-ray scattering. Sucrose-induced changes in the conformation of sMBP were identical to changes seen in wtMBP (supplemental Fig. S1), with the ligand-bound and unliganded conformations of sMBP clearly matching the ligand-bound and unliganded conformations of wtMBP, respectively (data not shown). We also observed that sMBP could complement the growth of wtMBP-deficient E. coli on M9 maltose minimal media (data not shown). Although growth with sMBP was 3-4 times slower compared with that observed with wtMBP, in control experiments with no binding protein, there was no growth. Therefore, sMBP interacts productively with MalFGK 2 to promote maltose transport.

Sucrose-binding MBP-sMBP is a mutant form of MBP developed by Ostermeier and co-workers
sMBP stimulates MalFGK 2 with Bound Sucrose or Maltose-MalFGK 2 has a binding site that is relatively specific for maltodextrins (13), and binding of the substrate to this site may be important for stimulation of the MalFGK 2 ATPase. To determine the importance of specific interactions between maltose and MalFGK 2 , we used sMBP to present MalFGK 2 with either maltose or sucrose as a transport substrate and measured the resulting stimulation of ATPase activity in vitro. Consistent with literature findings, MalFGK 2 -containing proteoliposomes showed a low level of basal activity (Fig. 1). This activity was unaltered by the addition of 5 mM maltose or sucrose, confirming that free sugar cannot stimulate the transporter in the absence of MBP (data not shown; see Ref. 11). When 20 M sMBP was added to wild-type MalFGK 2 , no statistically signif-icant increase in activity was observed. However, in the presence of either 5 mM maltose or sucrose, sMBP stimulated a 3-fold increase in ATP hydrolysis over background (Fig. 1A, bars with diagonal lines). We have therefore observed that, with respect to ATPase activation, MalFGK 2 cannot distinguish sucrose from maltose. To demonstrate that the equivalence of maltose-and sucrose-bound sMBP was not limited to 20 M sMBP concentration, a range of concentrations, from 1 to 100 M, was tested (Fig. 1B). Across this concentration range, maltose-and sucrose-bound sMBP stimulate the MalFGK 2 ATPase to similar levels; the overall trend (in both cases a proportional increase in MalFGK 2 ATPase activity as sMBP concentration is raised) suggests that the mechanism for stimulation is the same, irrespective of the sugar with which MalFGK 2 comes into contact.
Although maltose-and sucrose-bound sMBP both stimulate the MalFGK 2 ATPase to the same extent, the absolute levels of ATPase activity produced by sMBP were much lower than those produced by wtMBP. For example, in the presence of maltose, the level of ATPase stimulation by 20 M wtMBP was 40-fold higher than the stimulation produced by the same concentration of either maltose-or sucrose-bound sMBP. In addition, unliganded sMBP did not produce a significant increase in MalFGK 2 ATPase, in contrast to unliganded wtMBP that consistently produces a 2-fold stimulation ( Fig.  1A) (11,15). Therefore, although the substitution of sucrose for maltose did not influence stimulation of the MalFGK 2 ATPase, when compared with wtMBP the mutations in sMBP have drastically compromised its overall ability to stimulate the MalFGK 2 ATPase.
Structural Analysis of Open and Closed sMBP-To determine how the mutations in sMBP disrupt its ability to stimulate MalFGK 2 , we solved the crystal structures of sMBP in both the sucrose-bound and substrate-free forms to resolutions of 2.0 and 1.5 Å, respectively (Table 1). In both forms sMBP adopts a wild-type fold, with main chain atoms differing from wtMBP by a root mean square deviation for C␣ positions of 0.48 Å in the closed form and 0.55 Å in the open form.
Well defined electron density for sucrose was seen in the binding site of the sMBP sucrose structure; the electron density clearly defines each hydroxyl group of sucrose and does not fit maltose ( Fig. 2A). Like maltose-bound wtMBP, sMBP binds sucrose through hydrogen bonds with each of the two sugar rings. The first nonreducing glucose unit is common to both maltose and sucrose and occupies an identical binding pocket in wtMBP and sMBP (Fig. 2B). The second sugar ring differs between maltose and sucrose, being an ␣-1,4-linked reducing glucose in maltose and an ␣-1,2-linked fructose in sucrose; as a result, sucrose adopts a 90°bend compared with maltose. This bend allows the C3 hydroxyl to hydrogen bond with residue W62Y, which was likely selected for this purpose (Fig. 2B). The

Energetic Coupling in the Maltose Transporter
bend also creates a cavity in the binding site and separates the sugar from residues 14 and 15 (Fig. 2C). The D14L, K15F, and E111Y mutations modify this hydrophobic cavity by removing what would otherwise be unmatched buried charges or polar groups.
We also solved the crystal structure of the open conformation of sMBP to 1.5 Å resolution and were surprised to find a significant change in the structure of the ligand-binding site. The W62Y mutation is able to adopt an alternative conformation that displaces the main chain segment from residues Tyr 62 to Gly 69 (Fig. 3). This difference was evident from clear electron density for the Tyr 62 side chain in two different places, one of which necessarily displaces Phe 67 and is therefore incompatible with the wild-type main chain conformation (Fig. 3C). The only way this change can be accommodated is for residues 62-69 to partially extend into the substrate binding cleft. The occupancies of the two conformations of residues 66 -69 were set such that the temperature factors for the wild-type conformation are similar to the main chain average, as is the case with open, wild-type MBP (5). On this basis, the occupancy of the wildtype conformation is estimated at 0.4 and that of the alternative conformation is 0. 6.
Both the open and closed conformations of MBP are involved in maltose transport (8, 14 -16). To understand why sMBP has such a compromised ability to stimulate the MalFGK 2 ATPase, we compared its surface in both the open and closed conformations to that of wtMBP. The changes in sMBP necessary to support sucrose binding require only side chain substitutions, most of which are buried in the sugar-binding site and are not surface accessible in the closed form of the protein. As a result, the surface morphology of closed sMBP is virtually unaltered from closed wtMBP (Fig. 4A), with only a slight perturbation caused by the exposure of a methyl group on D14L (supplemental Fig. S2).
In contrast to the closed state, the open conformation of sMBP fully exposes all four binding site mutations to the solvent (Fig. 4B) as well as the alternative and partially disordered conformations for residues 62-69, caused by the W62Y mutation (Fig. 3). To summarize, our structural analysis found that in the sucrose-bound closed form, sMBP closely mimics the surface morphology of wtMBP, but open unliganded sMBP displays a drastically altered sugar-binding site.  (Table 1); coordinates for wtMBP and maltose were from PDB code 1ANF (25). A, bound sucrose (dark green) and the mutated residues (pale green) are shown, along with 2F o Ϫ F c electron density for the sucrose (blue mesh) contoured at 2. The electron density map was calculated using phases from the partially refined structure, prior to the addition of sucrose to the binding site. B, hydrogen bonding interactions between sucrose and sMBP (top) are compared with those between maltose and wtMBP (bottom). Hydrogen bonding interactions to the first glucose ring are the same for both proteins. C, comparison of the ligand-binding sites of sMBP and wtMBP. The molecular surface that sMBP and wtMBP have in common is shown in gray; carbon atoms from maltose and wtMBP are shown in orange and yellow, respectively, and those from sucrose and sMBP are shown in dark green and pale green. The conformation of sucrose creates a cavity that would normally be filled with atoms from the second glucose unit of maltose, to which three charged residues (Asp 14 , Lys 15 , and Glu 111 ) would be hydrogenbonded, as illustrated in B. The mutations in sMBP (pale green) convert these three charged residues to neutral residues. APRIL 9, 2010 • VOLUME 285 • NUMBER 15

JOURNAL OF BIOLOGICAL CHEMISTRY 11293
Altered Interactions between Open sMBP and MalFGK 2 -To investigate how the mutations in open sMBP could cause such a drastic defect in its ability to stimulate the MalFGK 2 ATPase, we replaced wtMBP with sMBP in the crystal structure of MBP-MalFGK 2 that corresponds to the transition state for ATP hydrolysis (7,8). The backbone positions of sMBP fit the MBP component of the trapped transition state to a root mean square deviation of 0.84 Å.
The binding of sMBP to the transporter transmembrane (MalFG) domains was not obviously compromised across the exterior surface of the binding protein, including contacts between sMBP and the MalF P2 arm (17,18). However, mutations D14L, W62Y, and E111Y disrupted interactions of the maltose-binding site with residues 253-258 of MalG, which occupy the maltose-binding site in the transporter transition state (Fig. 5). These residues include an invasive structure known as the MalG P3 "scoop loop," named for its probable role in excluding maltose from the ligand-binding site in MBP (8). A previous study showed that a 31-residue insertion into this loop did not affect assembly of MalFGK 2 but abolished transport by the system by disrupting interactions with MBP (19). In the case of the interaction with sMBP, D14L clashes with Asn 254 of MalG, whereas W62Y and E111Y remove stabilizing van der Waals and hydrogen bond interactions. In addition, the alternative conformation adopted by residues 62-69 will interfere with MalG interactions. Altogether, the mutations in the open conformation of sMBP would be expected to disrupt interactions with the MalG P3 loop in the transition state for ATP hydrolysis.
In summary, the mutations in sMBP have a drastic effect on its ability to stimulate MalFGK 2 ATPase activity. Structural analysis indicates that this effect is due to a disruption of interactions between residues 253 and 258 of MalG and the empty sugar-binding site of MBP as it occurs in the transition state for ATP hydrolysis. The magnitude of this effect shows that these interactions are critical for stimulation of the MalFGK 2 ATPase.

DISCUSSION
We observed that a sucrose-binding mutant of MBP was able to stimulate the ATPase activity of MalFGK 2 with either maltose or sucrose present as substrate. Although the level of stimulation by sMBP was only 2-3% of that produced by wtMBP, we believe the system is operating along the same reaction pathway as the fully wild-type system. The ATPase measurements were carried out in a well characterized proteoliposome system in which the ATPase activity of MalFGK 2 is tightly coupled to interactions with MBP. Because MalFGK 2 is wild type, and sMBP adopts the same open and closed structures as wtMBP, the conformational changes in the system as a whole will be similar for sMBP and wtMBP. In addition, sMBP is able to mediate growth on minimal maltose media, showing that the sMBP-stimulated ATPase activity is associated with maltose transport in vivo.
The activation by sMBP was indistinguishable between maltose and the nonphysiological substrate sucrose. The available evidence suggests that sucrose is unable to interact with the maltose-binding site in MalFGK 2 . For example, in transport assays using MBP-independent MalFGK 2 mutants, sucrose was incapable of competitively inhibiting the transport of maltose (13) indicating that the substrate-binding site of MalFGK 2 has little, if any, affinity for sucrose. This can be explained using the MalFGK 2 structure in complex with maltose (8); modeling sucrose into the same position as maltose results in clashes with MalF residues 383, 433, and 436, including steric clashes with backbone atoms. Because sucrose is unable to occupy the maltose-binding site of MalFGK 2 , the observation that maltoseand sucrose-bound sMBP have equal abilities to stimulate MalFGK 2 demonstrates that specific binding of the carbohydrate by MalFGK 2 is not important for activation of its ATPase. Therefore, it is the substrate-induced conformational change in MBP, but not the identity of the substrate itself, that is critical for stimulation of the MalFGK 2 ATPase.
In principle, ATP-dependent transporters should couple ATP hydrolysis to the actual movement of substrate. Our results with sMBP show that direct interactions with the substrate are not required for stimulation of the MalFGK 2 ATPase, and therefore coupling of ATP hydrolysis to substrate translocation must depend solely on interactions between MBP and MalFGK 2 . In this regard, the very strong defect in the ability of sMBP to stimulate the MalFGK 2 ATPase indicates that a critical interaction between MBP and MalFGK 2 has been disrupted by the mutations.
Both the open and closed conformations of MBP interact with MalFGK 2 during the catalytic cycle (3,7,8,15). The surface of closed sMBP is almost identical to that of wtMBP, suggesting that this conformation is not responsible for the reduced ability of sMBP to stimulate the MalFGK 2 ATPase. In fact, Leu 14 (Asp 14 in wtMBP) is the only mutant residue that causes a change in the exposed surface of closed sMBP and could therefore alter interactions with MalFGK 2 . Although functional genetic screens have demonstrated that the region around residue 14 is important for the interaction of MBP and MalFGK 2 (20,21), the only change found at residue 14 in the genetic screens was a mutation to tyrosine, a much larger residue that cannot be buried in the ligand-bound conformation of MBP and would therefore produce a large change in the surface of the closed conformation. In contrast, the D14L mutation is mostly buried and almost isosteric (supplemental Fig. S2), resulting in only a very small change in the surface, namely a 2-Å extension of an existing hydrophobic patch (supplemental Fig. S2). Therefore, the small effect of the D14L mutation on the surface of closed sMBP does not provide a convincing explanation for the profound effect of the mutations on the ability of sMBP to stimulate MalFGK 2 .
The surface of open sMBP, on the other hand, is drastically altered by the exposure of mutant residues in the sugar-binding site and the creation of an area of conformational instability due to the W62Y mutation. On this basis, the profound defect in sMBP is most likely due to a disruption of interactions between the open, rather than the closed, conformation of MBP. This conclusion is consistent with an important role for open MBP in stabilization of the transition state for ATP hydrolysis (8,15).
In fact, the reduced activation of MalFGK 2 ATPase by sMBP coincides with a disruption of interactions between sMBP ligand-binding site residues and the invasive MalG P3 loop of In addition, the main chain residues 62-69 are partially disordered; the alternative conformation for these residues is shown in magenta. Coordinates for open and closed wtMBP correspond to 1OMP (5) and 1ANF (25), respectively.  (8)). Residues 254 -257 of MalG (cyan) extend into the sugar-binding site, making contacts with wtMBP residues 14, 62, and 111 (yellow). The contacts made between MalG and the ligand-binding site would be affected by the mutations in sMBP. In addition, interaction with the MalG P3 loop would be disrupted due to disorder in sMBP residues 62-69, as outlined in Fig. 3. A role for the MalG P3 loop in energetic coupling is consistent with its position in MalFGK 2 (Fig. 6). The P3 loop is connected to MalG helices 15 and 16, which extend from the scoop loop to the MalG C terminus, located in a hydrogen bond network equidistant between the two ATP-binding sites of MalK 2 .
Our data indicate that in addition to extracting maltose from the MBP sugar binding cleft (8), interactions between the MalG P3 loop and MBP also play a direct role in promoting ATP hydrolysis. These interactions do not depend on the specific chemical identity of the substrate, and therefore a similar mechanism might be operative in multidrug exporters and other ABC transporters that couple ATP hydrolysis to the transport of diverse substrates.