Structural Basis for Substrate Specificity in the Escherichia Coli

Structural basis for substrate speciﬁcity in the Escherichia coli maltose transport system

Michael L. Oldhama, Shanshuang Chenb, and Jue Chena,b,1

aHoward Hughes Medical Institute and bDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907

Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved September 27, 2013 (received for review June 14, 2013) ATP-binding cassette (ABC) transporters are molecular pumps that maltose analogs with a modified reducing end are not trans- harness the chemical energy of ATP hydrolysis to translocate sol- ported despite their high-affinity binding to MBP (5, 6). Further utes across the membrane. The substrates transported by different evidence for selectivity through the ABC transporter MalFGK2 ABC transporters are diverse, ranging from small ions to large itself comes from mutant transporters that function independently proteins. Although crystal structures of several ABC transporters of MBP. In the absence of MBP, these mutants constitutively are available, a structural basis for substrate recognition is still hydrolyze ATP and specifically transport maltodextrins (7, 8). lacking. For the Escherichia coli maltose transport system, the se- In this study, we determined the crystal structures of the lectivity of sugar binding to maltose-binding protein (MBP), the maltose transport complex MBP-MalFGK2 bound with large periplasmic binding protein, does not fully account for the selec- maltodextrin in two conformational states. The determination tivity of sugar transport. To obtain a molecular understanding of of these structures, along with previous studies of maltoporin this observation, we determined the crystal structures of the trans- and MBP, allow us to define how overall substrate specificity is porter complex MBP-MalFGK2 bound with large malto-oligosaccha- achieved for the maltose transport system. ride in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms Results two hydrogen bonds with malto-oligosaccharide at the reducing Maltose Transport System and Its Substrates. The maltose transport end. In the outward-facing conformation, the transmembrane sub- system consists of three components: an outer membrane channel, unit MalF binds three glucosyl units from the nonreducing end of maltoporin (also called LamB); a periplasmic substrate-binding the sugar. These structural features explain why modified malto- protein, MBP; and an inner membrane ABC transporter, MalFGK2 oligosaccharides are not transported by MalFGK2 despite their high β binding affinity to MBP. They also show that in the transport cycle, (Fig. 1). Maltoporin is a homotrimer of -barrels, each barrel substrate is channeled from MBP into the transmembrane pathway forming an independent pore to allow diffusion of substrates across the outer membrane (9). In the periplasm, MBP binds the with a polarity such that both MBP and MalFGK2 contribute to the overall substrate selectivity of the system. substrate and undergoes a conformational change from an open form (without substrate) to a closed form (with substrate bound) membrane protein | maltodextrin (10, 11). In the absence of MBP, MalFGK2 rests in an inward- facing conformation, in which the two NBDs are well separated and the transport pathway is open to the intracellular side (12). he ATP-binding cassette (ABC) transporter family contains fi Tmore than 2,000 members sharing a common architecture of This open con guration of the NBDs prevents uncoupled ATP two transmembrane domains (TMDs) that form the translocation hydrolysis despite the high intracellular concentration of ATP. pathway and two cytoplasmic nucleotide-binding domains (NBDs) fi that hydrolyze ATP (1). Importers found in prokaryotes require Signi cance additional soluble proteins that bind substrates with high affinity and deliver them to the TMDs. Some ABC transporters recognize The Escherichia coli maltose transport system selectively im- only a single substrate, whereas others are more promiscuous. For ports malto-oligosaccharides into the cell as nutrients. Here we example, ABC transporters that secrete toxins, hydrolytic enzymes, show that the substrate specificity is conveyed by both the and antibiotic peptides are dedicated to one specific substrate (2), periplasmic binding protein MBP and the ATP-binding cassette but in contrast, the multidrug transporter P-glycoprotein interacts transporter MalFGK2, through crystal structures of MBP-MalFGK2 with more than 200 chemically diverse compounds (3). MRP1, captured in two different conformational states. These structures ABCG2, and TAP also have broad substrate spectra (2). show that the periplasmic binding site (formed by MBP and Regardless of substrate specificity, the ATPase activity of ABC MalG) interacts with only four glucosyl units from the reducing transporters is regulated by the presence of substrates. Thus, end of the polymer, and that the transmembrane-binding site (in substrate binding must generate a signal that enables ATP hy- MalFGK2) binds only three glucosyl units from the opposite, drolysis. Understanding how ABC transporters interact with nonreducing end. The structures essentially lead us to a single their substrates has been a major challenge in the field. concept: that transport selectivity can be explained through + A controversial issue in the ABC transporter field is whether the polarity of substrate binding to the two-component (MBP the transmembrane components contain a well-defined sub- transporter) system. strate-binding site. It has been suggested that for binding pro- fi fi Author contributions: M.L.O., S.C., and J.C. designed research; M.L.O. and S.C. performed tein-dependent ABC transporters, substrate speci city is de ned research; M.L.O., S.C., and J.C. analyzed data; and M.L.O. and J.C. wrote the paper. exclusively by the binding protein, which interacts with the sub- The authors declare no conflict of interest. strate with high affinity. The transmembrane components act as This article is a PNAS Direct Submission. a nonspecific pore for substrate to diffuse through the membrane Escherichia coli Data deposition: The atomic coordinates and structure factors have been deposited in the (4). However, for the maltose transporter, it has Protein Data Bank, www.pdb.org [PDB ID codes 4KHZ (pretranslocation state with mal- been well established that the selectivity of sugar binding to the toheptaose) and 4KI0 (outward-facing state with maltohexaose)]. maltose-binding protein (MBP) does not fully account for the 1To whom correspondence should be addressed. E-mail: [email protected]. selectivity of sugar transport. For example, cyclic maltodextrins, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. maltodextrins containing more than seven glucosyl units, and 1073/pnas.1311407110/-/DCSupplemental.

18132–18137 | PNAS | November 5, 2013 | vol. 110 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1311407110 Downloaded by guest on October 1, 2021 dimer (Fig. 3A). Maltoheptaose is bound in a groove between the N and C lobes of MBP (Fig. 3B). Clear electron densities are observed only for the first four glucosyl units from the reducing end (g1, g2, g3, and g4) (Fig. 3C). The other three glucosyl units (g5, g6, and g7) are likely disordered. The structures of MBP and the resolved four glucosyl units are essentially identical to those of the isolated MBP bound with maltotetraose [Protein Data Bank (PDB) ID code 4MBP] (10). The overall rmsd for 370 Cα positions is 0.5 Å; the largest dis- placement (1.0 Å) occurs at the interface with MalG. Conse- quently, the contacts between the sugar and MBP are identical to those observed in isolated MBP. The sugar molecule forms 14 direct hydrogen bonds with polar residues and 118 van der Waals interactions with MBP. A prominent feature of the MBP-binding site is the four stacking aromatic residues that form a continuous surface matching the curvature of the left-handed helix of the malto-oligosaccharide (Fig. 3D). In this pretranslocation state, the transmembrane (TM) subunit MalG also interacts directly with the substrate. The third periplasmic loop of MalG (scoop loop) reaches to the reducing end of the sugar by way of a conserved glutamine residue (Q256), hydrogen bonded to the OH6 and ring oxygen of the g1 unit (Fig. 3 B–D). This interaction provides an explanation for a previous finding that maltose analogs with a modified reducing end are not recognized by the transport system despite their high-affinity binding to MBP (6). In isolated MBP, the g1 unit is partially exposed to solvent, and thus some modifications at the reducing Fig. 1. The three components of the maltose transport system. Malto- end are tolerated. On docking to the membrane transporter, g1 oligosaccharides diffuse across the outer membrane through malto- becomes buried at the interface with MalG. Analogs with a porin, bind with MBP in the periplasm, and are transported across the modified g1 structure may interfere with the productive inter- inner membrane by the ABC transporter MalFGK . 2 actions between MBP and MalFGK2 and thus are unable to initiate the transport cycle. Because of the high maltoheptaose concentration used in the Binding of substrate-loaded MBP triggers a conformational crystallization (0.2 mM), the transmembrane-binding site in MalF change of the transporter that brings the NBDs in closer proximity A — also contains a bound substrate (Fig. 3 ). Details of the interactions to one another close enough to permit ATP to make additional between the sugar and the TM subunits will be discussed below in interactions with the protein, which in turn brings about a concerted the context of the higher resolution outward-facing structure. motion that opens the transport pathway to the periplasmic side (outward-facing) (13). Concurrently, the substrate is released from The Transmembrane Binding Site Interacts with Three Glucosyl Units MBP into the transport pathway, while ATP is poised for hydrolysis. From the Nonreducing End. Binding of ATP to the pretranslocation Upon hydrolysis, ADP and inorganic phosphate are released, state promotes formation of the outward-facing conformation, a key returning the transporter to its resting, inward-facing conformation, intermediate that allows substrate to enter the transmembrane from which substrate is released to the intracellular side. cavity (13). Crystals of the outward-facing transporter in complex The maltose system is highly selective. Linear malto-oligo- with maltohexaose and adenosine 5′-(β,γ-imido)triphosphate (AMP- BIOCHEMISTRY saccharides (or maltodextrins) linked through α-1, 4 glycosidic PNP) diffracted to a resolution of 2.4 Å (Tables S1 and S2). In the bonds are imported by the maltose transport system (Fig. 2A). The outward-facing conformation, MBP is in an open conformation in smallest substrate, maltose, is composed of two glucosyl units, which the two lobes are separated, the substrate-binding site of MalF whereas the largest substrate, maltoheptaose, contains seven glu- is connected to MBP through a large intersubunit cavity, and the cosyl units (5, 14). Closely related carbohydrates, such as glucose, MalK subunits form a closed dimer with AMP-PNP bound at the lactose, sucrose, isomaltose, and cellobiose, are not recognized by interface (Fig. 4A). the maltose transport system (Fig. 2B) (15). In addition, when the The substrate-binding site in MBP does not contain a sugar glucosyl unit at the reducing end is modified, the resulting malto- molecule; instead, it is partially occupied by the MalG scoop loop, dextrin analogs are no longer transported into the cell (6). which makes van der Waals interactions with sugar-stacking residues Y155 and W230 of MBP (Fig. 4A). Removing four residues The Periplasmic Binding Site Recognizes Four Glucosyl Units from the in the scoop loop results in an uncoupled transporter that Reducing End. In the initial step of the transport cycle, the sub- hydrolyzes ATP but does not transport maltose, indicating that the strate is brought to the inner membrane surface by MBP (Fig. 1). scoop loop is essential to the release of substrate from MBP (17). The structure of the complex formed by substrate-bound MBP An electron density map in the TM-binding site shows clear and MalFGK2, the pretranslocation state, was previously de- densities for three sugar rings (Fig. 4B). The curvature of the termined with maltose in the absence of nucleotides (16). To sugar, shown in a green van der Waals surface representation, understand how larger substrates are recognized, we obtained nicely complements the shape of the binding site in MalF (Fig. crystals of the pretranslocation state bound with maltoheptaose 4C). In contrast to the MBP-binding site, where the reducing end and determined the structure to a resolution of 2.9 Å by mo- interacts with the protein, the ordered glucosyl units observed lecular replacement (Fig. 3A and Tables S1 and S2). The struc- inside the transporter are from the nonreducing end. The non- ture of the protein components is identical to that of the reducing glucose (g6) tucks into a crevice on MalF, making three maltose-bound form; MBP is in a closed conformation, MalF direct hydrogen bonds and 60 van der Waals interactions with and MalG pack against each other to encapsulate an occluded the protein (Fig. 4D). The g5 unit is sandwiched between the ar- binding pocket, and the two MalK subunits form a semiopen omatic side chains of Y383 and F436. Single mutations of Y383S,

Oldham et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18133 Downloaded by guest on October 1, 2021 Fig. 2. Substrate specificity of the maltose transport system. (A) The maltose transport system takes up linear malto-oligosaccharides containing two to seven glucosyl units linked through the α-1, 4 bonds. The glucosyl unit with a free anomeric carbon (C1) is able to form an open chain with an aldehyde group through isomerism, and thus is defined as the reducing end (shown in red). The opposite end, the nonreducing end, is shown in green. To be consistent with the nomenclature used in the MBP studies, the glucosyl units are identified from the reducing end as g1, g2, g3, and so on. The structure of two large substrates, maltohexaose and maltoheptaose, are shown. (B) Examples of closely related carbohydrates that are not recognized by the maltose transport system.

F436L, or F436S reduces the transport activity to approximately residues (26). In addition, their equatorial hydroxyl groups 1% of that of the WT protein (18), underscoring the importance form hydrogen bonds with charged residues inside the channel. of these aromatic stacking interactions in the TM-binding site. The remaining glucosyl units protrude into the vestibule and The g5 unit also forms hydrogen bonds with S433 and Y325. The g4 the channel outlet without contacting the protein. The speci- unit is loosely attached through van der Waals interactions and ficity of the channel is conveyed by the overall hourglass shape a water-mediated hydrogen bond with the protein. No clear elec- and the internal binding site, which allows malto-oligosachrides tron density was observed for glucosyl units g1–g3, suggesting that to slide through while maintaining their left-handed helical these units do not form any specific interactions with the protein. conformation (26). The periplasmic protein MBP functions as the primary receptor Discussion of the inner membrane transport system. It binds linear malto-oli- With the addition of the crystal structures presented in this work, gosaccharides from maltose to amylose, cyclic maltodextrins, and we now have atomic pictures of how malto-oligosaccharides in- various maltodextrin derivatives (6). As described beautifully by teract with maltoporin, MBP, and MalFGK2. By synthesizing the Quiocho et al. (10), the excellent shape-complementarity, extensive structural information with decades of functional data, we are hydrogen-bonding, and aromatic stacking interactions account for beginning to understand how each component contributes to the the high-affinity binding of MBP to sugar substrates. overall substrate specificity of the transport system. The substrate specificity of the transport system is further re- fi The outer membrane channel maltoporin allows nonspeci c stricted by the inner membrane transporter, MalFGK2. In the permeation of ions and monosaccharides (19–24). For larger pretranslocation state, the scoop loop of MalG restricts mod- molecules, maltoporin preferentially conducts maltodextrin over ifications at the reducing end. In the outward-facing state, the other oligosaccharides (20). Consistent with the millimolar af- binding site in MalF recognizes the sugar from the nonreducing finity for malto-oligosachrides (25), maltoporin contains defined end. The shape-complimentarity and five direct protein–sugar binding sites for only three consecutive glucosyl units. In the hydrogen bonds confer specificity for the α-1, 4 linkage of the crystal structure of maltoporin bound with maltohexaose, the malto-oligosaccharide. Compared with MBP, in which the sub- pyranose rings of the g2, g3, and g4 units pack against a con- strates of various sizes are involved in 12–14 direct hydrogen tinuous apolar surface, the “greasy slide,” formed by aromatic bonds with the protein (10), the TM-binding site appears to be

18134 | www.pnas.org/cgi/doi/10.1073/pnas.1311407110 Oldham et al. Downloaded by guest on October 1, 2021 be transported by these mutants (28, 29), in agreement with the observation that the TM-binding site interacts with the substrate at the nonreducing end. Because MBP and MalG interact with up to four glucosyl units from the reducing end and MalF recognizes the glucosyl units from the opposite end, the substrate of the transport system is restricted to linear malto-oliogosaccharides with the α-1, 4 linkage (Fig. 5A). A remaining question concerns the size limitation for the substrates. Although MBP binds maltodextrins as large as amylose, only dextrins up to the size of maltoheptaose can be transported (5). We speculate that the limiting factor for the size of the substrate may lie in the pretranslocation state, in which the substrate is occluded in a small cavity of ∼2,400 Å3. By superpositioning the four glucosyl units observed in the MBP-binding site with those in the structure of glycogen synthase (PDB ID code 3RT1), we modeled a maltoheptaose molecule into the binding cavity of the pretranslocation state (Fig. 5B). The cavity appears to be large enough to accommodate only seven glucosyl units. Larger malto- oligosaccharides, although able to bind to MBP, may hinder the interactions between MBP and MalFGK2, and thus do not stimulate ATPase activity (Fig. 5C) and are not transported across the inner membrane. In summary, the substrate speciﬁcity of the maltose transport system is conveyed by both the periplasmic binding protein MBP and the membrane transporter MalFGK2. The structural information described herein correlates beautifully with decades of functional data. It would be premature to propose the generality of this study, given that crystal structures of other ABC transporters with substrates bound to the TMDs are not yet available. Nonetheless, the maltose system serves as an elegant example of how evolution has ﬁne-tuned each component of the transport system to select the very nutrients needed by bacteria for survival. Methods

Expression and Puriﬁcation of MalFGK2 and MBP. MalFGK2 and MBP were expressed and puriﬁed as described previously (13, 30).

Crystallization of the Pretranslocation State with Maltoheptaose. MalFGK2 [10 mg/mL in 0.06% UDM) and MBP were mixed at a 1:1.25 molar ratio in the presence of 0.2 mM maltoheptaose. Crystals were grown by mixing the protein sample with the reservoir solution containing 30% PEG 400, 100 mM

NaCl, 10 mM MgCl2, and 100 mM sodium Hepes (pH 7.5), at a 1:1 ratio in

sitting drops at 20 °C. BIOCHEMISTRY

Crystallization of the Outward-Facing State with Maltohexaose. MalFGK2 [10 mg/mL in 0.06% n-undecyl-β-D-maltopyranoside (UDM)] and MBP were mixed at a 1:1.25 molar ratio in the presence of 0.2 mM maltohexaose, 2 mM AMP-

PNP, and 2 mM MgCl2. Crystals were grown by mixing the protein sample with the reservoir solution containing 27% PEG 400, 200 mM NaCl, 50 mM MgCl2, and 100 mM sodium Hepes (pH 7.5), at a 1:1 ratio in sitting drops at 20 °C.

Fig. 3. Structure of the pretranslocation state in complex with malto- Nanodisc Reconstitution and ATPase Activity Assays. Puriﬁed WT maltose heptaose. (A) Ribbon representation and a surface slab view. The four transporter was reconstituted into lipidic nanodiscs for use in ATPase glucosyl units of maltoheptaose are shown in stick models (red and gray). activity assays as described previously (31). ATPase activities in the pres- Color code: MBP, purple; MalG, yellow; MalF, blue; MalK, red and green. ence of different malto-oligosaccharides (CarboSynth and Elicityl Oligo- (B) Stereoview of the binding site. MBP is shown in a surface representa- Tech) were determined using an ATP/NADH coupled assay (32, 33). tion, the scoop loop of MalG is shown in a ribbon representation, and the Reaction mixtures (100 μL) contained 50 mM Hepes (pH 8.0), 94 nM μ μ μ glucosyl units are shown in stick models (red and gray) and as van der MalFGK2,15 M MBP (when applicable), 60 g/mL pyruvate kinase, 32 g/mL

Waals dot surfaces (green). (C) Stereoview of the Fo-Fc map contoured at lactate dehydrogenase, 4 mM phosphoenolpyruvate, 0.3 mM NADH, 10 mM μ 2σ with the sugar molecule omitted in the phase calculation. (D) Stereo- MgCl2, 1 mM ATP, and 400 M malto-oligosaccharide (when applicable). The view of the detailed interactions between the sugar and residues in MBP rate of decrease in absorbance at 340 nm from NADH consumption was and MalG. Hydrogen bonds are indicated by dashed lines. recorded with the 8453 UV-Vis Diode Array System (Agilent Technologies) at 25 °C. All measurements were performed in triplicate from the same protein preparations.

less selective and of lower afﬁnity. Consistently, the Km values for MBP-independent mutants (1 mM) that recognize the substrate Data Collection. Crystals were looped out of the drop and directly frozen in liquid nitrogen. X-ray diffraction data were collected at the Advanced only through the TM-binding site are 1,000-fold higher than that Photon Source (beamline 23-ID-D) at 100 K. Diffraction images were of the WT transporter (1 μM) (27). In addition, analogs with processed and scaled with the HKL-2000 program package (HKL Re- a modiﬁed reducing end that are not recognized by WT MBP can search) (34).

Oldham et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18135 Downloaded by guest on October 1, 2021 with translation/libration/screw (TLS) parameters generated by the TLSMD server (39). TLS tensors were analyzed and anisotropic B factors were derived with TLSANL (40).

Figure Preparation. All ﬁgures were prepared with PyMOL software (www.pymol. org). The dimensions of the cavity shown in Fig. 5B were calculated using VOIDOO (41). The volume of the cavity was determined by CASTp (42) with a 2.5-Å probe.

Structure Deposition. The atomic coordinates and structure factors have been deposited in the PDB under ID codes 4KHZ (pretranslocation state with maltoheptaose) and 4KI0 (outward-facing state with maltohexaose).

Fig. 4. Structure of the outward-facing conformation in complex with maltohexaose. (A) Ribbon representation and a surface slab view. Color code is as in

Fig. 3. (B)StereoviewoftheFo-Fc map contoured at 3σ with the sugar molecule omitted in the phase calculation. (C) Stereoview of the binding site. MalF and fi MalG are shown in surface representations, and the glucosyl units are Fig. 5. Substrate speci city is conveyed by both MBP and MalFGK2.(A) shown in stick models (red and gray) and as van der Waals dot surfaces Schematic model of substrate recognition in the pretranslocation state and (green). (D) Stereoview of the detailed interactions between the sugar and the outward-facing state. The reducing end of the malto-oligosaccharide is in residues in MalF and MalG. Hydrogen bonds are indicated by dashed lines. red, and the nonreducing end is in green. The glucosyl units bound by protein are shown as solid circles, and dashed circles represent the glucosyl units not observed in the crystal structures. (B) Stereoview of a modeled maltoheptaose bound to the pretranslocation state. The substrate-binding cavity at the in- Structure Determination. The structures were determined by molecular terface of MBP and MalFGK2 is shown as a mesh (cyan). (C) The ATPase of replacement in PHASER (CCP4) (35) using the pretranslocation and out- MalFGK2 reconstituted into lipidic nanodiscs. M3, maltotriose; M6, malto- ward-facing states as search models (PDB ID codes 3PV0 and 3RLF). The hexaose, M7, maltoheptaose; M10, a mixture containing 65% maltodecaose, models were improved by manual building in Coot (36) and initially re- 24% maltononaose, 7% maltooctaose, 2% maltoheptaose, and 2% impuri- fined with CNS (37). Further refinement was performed in REFMAC5 (38) ties. Data columns represent mean ± SD values from triplicate measurements.

18136 | www.pnas.org/cgi/doi/10.1073/pnas.1311407110 Oldham et al. Downloaded by guest on October 1, 2021 ACKNOWLEDGMENTS. We thank the staff at Advanced Photon Source sample by mass spectrometry. This work was supported by National beamline 23-ID-D for assistance with data collection. We also thank Jia Institutes of Health Grant GM070515 (to J.C.). J.C. is an investigator of the Ren and Yu Xia (Purdue University) for analyzing the maltodecaose Howard Hughes Medical Institute.

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