Periplasmic binding protein structure and function. Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine

The three-dimensional structure of the native unliganded form of the Leu/Ile/Val-binding protein (Mr = 36,700), an essential component of the high-affinity active transport system for the branched aliphatic amino acids in Escherichia coli, has been determined and further refined to a crystallographic R-factor of 0.17 at 2.4 A resolution. The entire structure consists of 2710 non-hydrogen atoms from the complete sequence of 344 residues and 121 ordered water molecules. Bond lengths and angle distances in the refined model have root-mean-square deviations from ideal values of 0.05 A and 0.10 A, respectively. The overall shape of the protein is a prolate ellipsoid with dimensions of 35 A x 40 A x 70 A. The protein consists of two distinct globular domains linked by three short peptide segments which, though widely separated in the sequence, are proximal in the tertiary structure and form the base of the deep cleft between the two domains. Although each domain is built from polypeptide segments located in both the amino (N) and the carboxy (C) terminal halves, both domains exhibit very similar supersecondary structures, consisting of a central beta-sheet of seven strands flanked on either side by two or three helices. The two domains are far apart from each other, leaving the cleft wide open by about 18 A. The cleft has a depth of about 15 A and a base of about 14 A x 16 A. Refining independently the structure of native Leu/Ile/Val-binding protein crystals soaked in a solution containing L-leucine at 2.8 A resolution (R-factor = 0.15), we have been able to locate and characterize an initial, major portion of the substrate-binding site of the Leu/Ile/Val-binding protein. The binding of the L-leucine substrate does not alter the native crystal structure, and the L-leucine is lodged in a crevice on the wall of the N-domain, which is in the inter-domain cleft. The L-leucine is held in place primarily by hydrogen-bonding of its alpha-ammonium and alpha-carboxylate groups with main-chain peptide units and hydroxyl side-chain groups; there are no salt-linkages. The charges on the leucine zwitterion are stabilized by hydrogen-bond dipoles. The side-chain of the L-leucine substrate lies in a depression lined with non-polar residues, including Leu77, which confers specificity to the site by stacking with the side-chain of the leucine substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of the RNA 2'-phosphotransferase from Aeropyrum pernix K1

In the final step of tRNA splicing, the 2'-phosphotransferase catalyzes the transfer of the extra 2'-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2'-phosphotransferase protein from Aeropyrum pernix K1 at 2.8 Angstroms resolution. The structure of the 2'-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2'-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2'-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2'-phosphotransferase.     

The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis

The three-dimensional structure of the maltose- or maltodextrin-binding protein (Mr = 40,622) with bound maltose has been obtained by crystallographic analysis at 2.8-A resolution. The structure, which has been partially refined at 2.3 A, is ellipsoidal with overall dimensions of 30 x 40 x 65 A and divided into two distinct globular domains by a deep groove. Although each domain is built from two peptide segments from the amino- and carboxyl-terminal halves, both domains exhibit similar supersecondary structure, consisting of a central beta-pleated sheet flanked on both sides with two or three parallel alpha-helices. The groove, which has a depth of 18 A and a base of about 9 x 18 A, contains the maltodextrin-binding site. We have previously observed the same general features in the well-refined structures of six other periplasmic receptors with specificities for L-arabinose, D-galactose/D-glucose, sulfate, phosphate, leucine/isoleucine/valine, and leucine. The bound maltose is buried in the groove and almost completely inaccessible to the bulk solvent. The groove is heavily populated by polar and aromatic groups many of which are involved in extensive hydrogen-bonding and van der Waals interactions with the maltose. All the disaccharide hydroxyl groups, which form a peripheral polar surface approximately in the plane of the sugar rings, are tied in a total of 11 direct hydrogen bonds with six charged side chains, one Trp side chain, and one peptide backbone NH, and five indirect hydrogen bonds via water molecules. The maltose is wedged between four aromatic side chains. The resulting stacking of these aromatic residues on the faces of the glucosyl units provides a majority of the van der Waals contacts in the complex. The nonreducing glucosyl unit of the maltose is involved in approximately twice as many hydrogen bonds and van der Waals contacts as the glucosyl unit at the reducing end. The binding protein-maltose complex shows the best example of the extensive use of polar and aromatic residues in binding oligosaccharides. The tertiary structure of the maltodextrin-binding protein, along with the results of genetic studies by a number of investigators, has also enabled us for the first time to map the different regions on the surface of the protein involved in the interactions with the membrane-bound protein components necessary for transport of and chemotaxis toward maltodextrins. These sites permit distinction of the "open cleft" (without bound sugar) and closed (with bound sugar) conformations of the binding protein by the chemotactic signal transducer with which the maltodextrin-binding protein interacts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1, complexed with an alginate tetrasaccharide at 1.6-A resolution

Sphingomonas sp. A1 possesses a high molecular weight (HMW) alginate uptake system composed of a novel pit formed on the cell surface and a pit-dependent ATP-binding cassette (ABC) transporter in the inner membrane. The transportation of HMW alginate from the pit to the ABC transporter is mediated by the periplasmic HMW alginate-binding proteins AlgQ1 and AlgQ2. We determined the crystal structure of AlgQ2 complexed with an alginate tetrasaccharide using an alginate-free (apo) form as a search model and refined it at 1.6-A resolution. One tetrasaccharide was found between the N and C-terminal domains, which are connected by three extended hinge loops. The tetrasaccharide complex took on a closed domain form, in contrast to the open domain form of the apo form. The tetrasaccharide was bound in the cleft between the domains through van der Waals interactions and the formation of hydrogen bonds. Among the four sugar residues, the nonreducing end residue was located at the bottom of the cleft and exhibited the largest number of interactions with the surrounding amino acid residues, suggesting that AlgQ2 mainly recognizes and binds to the nonreducing part of a HMW alginate and delivers the polymer to the ABC transporter through conformational changes (open and closed forms) of the two domains.     

Open conformation of a substrate-binding cleft: 19F NMR studies of cleft angle in the D-galactose chemosensory receptor.

The Escherichia coli D-galactose and D-glucose receptor is a two-domain structure with a sugar-binding site at the interface between domains. The structure of the closed cleft containing bound D-glucose has been determined crystallographically, but the open cleft remains to be characterized. The present study illustrates a generalizable approach that is used to detect and analyze both the open- and closed-cleft conformations in solution. A 19F nucleus located inside the cleft is monitored by 19F NMR. When the cleft is occupied by D-glucose, the 19F nucleus is found to be inaccessible to the aqueous paramagnetic probe Gd-EDTA, verifying that the occupied cleft is closed in solution and inaccessible to bulk solvent. When the cleft is empty, the 19F nucleus becomes accessible to the paramagnet such that the distance of closest approach is r less than or equal to 10 A, indicating that the empty cleft opens at least transiently by an angle theta greater than or equal to 18 +/- 3 degrees.     

The N-terminal substrate-binding domain of ClpA unfoldase is highly mobile and extends axially from the distal surface of ClpAP protease

ClpAP is a barrel-like complex consisting of hexameric rings of the ClpA ATPase stacked on the double heptameric ring of ClpP peptidase. ClpA has two AAA+ domains (Dl and D2) and a 153-residue N-domain. Substrate proteins bind to the distal surface of ClpA and are unfolded and translocated axially into ClpP. To gain insight into the functional architecture of ClpA in the ATPgammaS state, we have determined its structure at 12A resolution by cryo-electron microscopy. The resulting model has two tiers, corresponding to rings of Dl and D2 domains: oddly, there is no sign of the N-domains in the density map. However, they were detected as faint diffuse density distal to the Dl tier in a difference image between wild-type ClpAP and a mutant lacking the N-domain. This region is also accentuated in a variance map of ClpAP and in a difference imaging experiment with ClpAP complexed with ClpS, a 12kDa protein that binds to the N-domain. These observations demonstrate that the N-domains are highly mobile. From molecular modeling, we identify their median position and estimate that they undergo fluctuations of at least 30A. We discuss the implications of these observations for the role of N-domains in substrate binding: either they effect an initial transient binding, relaying substrate to a second site on the Dl tier where unfolding ensues, or they may serve as an entropic brush to clear the latter site of non-specifically bound ligands or substrates bound in non-productive complexes.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

Uptake of iron from hemoglobin and the haptoglobin-hemoglobin complex by hemolytic bacteria

The abilities of Staphylococcus aureus and Streptococcus pyogenes to remove iron from mouse 59Fe hemoglobin that was either in free form or complexed with human haptoglobin, were evaluated. 59Fe hemoglobin from the amphibian Taricha granulosa was also used in free form or complexed with the amphibian's hemoglobin-binding proteins. Contrary to what was reported from a study using pathogenic Escherichia coli, haptoglobin failed to exhibit a bacteriostatic influence when complexed with hemoglobin. In our study, more 59Fe was removed by the bacteria from the haptoglobin-hemoglobin complex than from free mouse hemoglobin. The hemoglobin and hemoglobin-plasma protein complexes of Taricha were stripped of 59Fe at similar rates and extents by both bacterial species.     

Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein

A maltodextrin-binding protein from Pyrococcus furiosus (PfuMBP) has been overproduced in Escherichia coli, purified, and crystallized. The crystal structure of the protein bound to an oligosaccharide ligand was determined to 1.85 A resolution. The fold of PfuMBP is very similar to that of the orthologous MBP from E. coli (EcoMBP), despite the moderate level of sequence identity between the two proteins (27 % identity, 46 % similarity). PfuMBP is extremely resistant to heat and chemical denaturation, which may be attributed to a number of factors, such as a tightly packed hydrophobic core, clusters of isoleucine residues, salt-bridges, and the presence of proline residues in key positions. Surprisingly, an attempt to crystallize the complex of PfuMBP with maltose resulted in a structure that contained maltotriose in the ligand-binding site. The structure of the complex suggests that there is a considerable energy gain upon binding of maltotriose in comparison to maltose. Moreover, isothermal titration calorimetry experiments demonstrated that the binding of maltotriose to the protein is exothermic and tight, whereas no thermal effect was observed upon addition of maltose at three temperatures. Therefore, PfuMBP evidently is designed to bind oligosaccharides composed of three or more glucopyranose units.     

Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1 at 2.0A resolution

Sphingomonas sp. A1 possesses a high molecular mass (average 25,700 Da) alginate uptake system mediated by a novel pit-dependent ABC transporter. The X-ray crystallographic structure of AlgQ2 (57,200 Da), an alginate-binding protein in the system, was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 18.3% for 15 to 2.0 A resolution data. The refined structure of AlgQ2 was comprised of 492 amino acid residues, 172 water molecules, and one calcium ion. AlgQ2 was composed of two globular domains with a deep cleft between them, which is expected to be the alginate-binding site. The overall structure is basically similar to that of maltose/maltodextrin-binding protein, except for the presence of an N2-subdomain. The entire calcium ion-binding site is similar to the site in the EF-hand motif, but comprises a ten residue loop. This calcium ion-binding site is about 40 A away from the alginate-binding site.     

Location of the sugar-binding site of L-arabinose-binding protein. Sugar derivative syntheses, sugar binding specificity, and difference Fourier analyses.

The sugar-binding site of the L-arabinose-binding protein, an essential component of the high affinity L-arabinose uptake system in Escherchia coli, is located deep in a cleft formed by the asymmetric contributions from both of the two similar domains. The site was unambiguously identified with the electron-rich substrate analog 6-bromo-6-deoxy-D-galactose in a difference Fourier analysis. The observation that the original native structure might have been solved with bound L-arabinose necessitated the synthesis of a heavy atom analog, its structure consistent with the known sugar-binding specificity of the protein. Difference Fourier maps (3.5 A) of crystals soaked in 46 mM analog showed a peak 3.5 times background, which is attributed to the -CH2Br moiety of the analog. Superposition of a difference map onto a 2.8-A native electron density map indicated that the difference peak is 6 to 7 A from the reactive single cysteine (Cys-64) and partially coincident with an "extraneous" density found in the native map. This "extraneous" peak was previously attributed to a bound L-arabinose molecule, and its presence accounts for the early failures of difference Fourier analyses of crystals soaked in or co-crystallized with L-arabinose to locate the sugar-binding site.     

Crystal structure of the cell division protein FtsA from Thermotoga maritima

Bacterial cell division requires formation of a septal ring. A key step in septum formation is polymerization of FtsZ. FtsA directly interacts with FtsZ and probably targets other proteins to the septum. We have solved the crystal structure of FtsA from Thermotoga maritima in the apo and ATP-bound form. FtsA consists of two domains with the nucleotide-binding site in the interdomain cleft. Both domains have a common core that is also found in the actin family of proteins. Structurally, FtsA is most homologous to actin and heat-shock cognate protein (Hsc70). An important difference between FtsA and the actin family of proteins is the insertion of a subdomain in FtsA. Movement of this subdomain partially encloses a groove, which could bind the C-terminus of FtsZ. FtsZ is the bacterial homologue of tubulin, and the FtsZ ring is functionally similar to the contractile ring in dividing eukaryotic cells. Elucidation of the crystal structure of FtsA shows that another bacterial protein involved in cytokinesis is structurally related to a eukaryotic cytoskeletal protein involved in cytokinesis.     

A salt-bridge motif involved in ligand binding and large-scale domain motions of the maltose-binding protein

The uptake of nutrients is essential for the survival of bacterial cells. Many specialized systems have evolved, such as the maltose-dependent ABC transport system that transfers oligosaccharides through the cytoplasmic membrane. The maltose/maltodextrin-binding protein (MBP) serves as an initial high-affinity binding component in the periplasm that delivers the bound sugar into the cognate ABC transporter MalFGK(2). We have investigated the domain motions induced by the binding of the ligand maltotriose into the binding cleft using molecular dynamics simulations. We find that MBP is predominantly in the open state without ligand and in the closed state with ligand bound. Oligosaccharide binding induces a closure motion (30.0 degrees rotation), whereas ligand removal leads to domain opening (32.6 degrees rotation) around a well-defined hinge affecting key areas relevant for chemotaxis and transport. Our simulations suggest that a "hook-and-eye" motif is involved in the binding. A salt bridge between Glu-111 and Lys-15 forms that effectively locks the protein-ligand complex in a semiclosed conformation inhibiting any further opening and promoting complete closure. This previously unrecognized feature seems to secure the ligand in the binding site and keeps MBP in the closed conformation and suggests a role in the initial steps of substrate transport.     

Crystal structures of the maltodextrin/maltose-binding protein complexed with reduced oligosaccharides: flexibility of tertiary structure and ligand binding

The structure of the maltodextrin or maltose-binding protein, an initial receptor for bacterial ABC-type active transport and chemotaxis, consists of two globular domains that are separated by a groove wherein the ligand is bound and enclosed by an inter-domain rotation. Here, we report the determination of the crystal structures of the protein complexed with reduced maltooligosaccharides (maltotriitol and maltotetraitol) in both the "closed" and "open" forms. Although these modified sugars bind to the receptor, they are not transported by the wild-type transporter. In the closed structures, the reduced sugars are buried in the groove and bound by both domains, one domain mainly by hydrogen-bonding interactions and the other domain primarily by non-polar interactions with aromatic side-chains. In the open structures, which abrogate both cellular activities of active transport and chemotaxis because of the large separation between the two domains, the sugars are bound almost exclusively to the domain rich in aromatic residues. The binding site for the open chain glucitol residue extends to a subsite that is distinct from those for the glucose residues that were uncovered in prior structural studies of the binding of active linear maltooligosaccharides. Occupation of this subsite may also account for the inability of the reduced oligosaccharides to be transported. The structures reported here, combined with those previously determined for several other complexes with active oligosaccharides in the closed form and with cyclodextrin in the open form, revealed at least four distinct modes of ligand binding but with only one being functionally active. This versatility reflects the flexibility of the protein, from very large motions of interdomain rotation to more localized side-chain conformational changes, and adaptation by the oligosaccharides as well.     

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.     

Structure of the L-leucine-binding protein refined at 2.4 A resolution and comparison with the Leu/Ile/Val-binding protein structure

The three-dimensional X-ray structure of the leucine-binding protein (36,900 Mr and 346 residues), an active transport component of Escherichia coli, has been determined by the method of molecular replacement, using the refined structure of the Leu/Ile/Val-binding protein (344 residues) as the model structure. The two amino acid-binding proteins have 80% sequence identity and, although both crystallize in the same space group, they have very different unit cell dimensions. The rotation function yielded one significant peak, which subsequently led to a single self-consistent translation function solution. The model was first refined by the constrained least-squares method, with each of the two domains of the molecule treated separately to allow for any small change in the relative orientation of the two domains. The model was then modified in order to reflect the 72 changes in amino acid side-chains and two insertions in going from the Leu/Ile/Val-binding protein sequence to that of the L-leucine-binding protein. Final structure refinement, using the restrained least-squares technique, resulted in an R-factor of 0.20 for 13,797 reflections to a resolution of 2.4 A. The model is comprised of 2600 protein atoms and 91 solvent molecules. The L-leucine-binding protein structure is, as expected, very similar to the Leu/Ile/Val-binding protein structure; both are in the unliganded conformation with the cleft between the two domains wide open and easily accessible. The superimposing of the structures yields a root-mean-square difference of 0.68 A in the alpha-carbon atoms of the 317 equivalent residues. The five regions of the leucine-binding protein structure that differ by more than 1.6 A from the Leu/Ile/Val-binding protein structure are far from the major portion of the ligand-binding site, which is located in one domain of the bilobate protein. Between the structures, there are three differences in the amino acid side-chains that form the major portion of the substrate-binding sites. These substitutions, by themselves, fail to clearly explain the differences in the specificities for branched aliphatic amino acids.     

A mutant lactose repressor with altered inducer and operator binding parameters

The lactose repressor protein from the mutant Escherichia coli BG185 contains valine at position 81 instead of alanine. Spectroscopic, chemical and direct binding measurements demonstrate that the BG185 protein exhibits properties similar to the wild-type repressor-inducer complex. Kinetic measurements of inducer binding to BG185 repressor yielded rate constants that were more than two orders of magnitude smaller than those observed for wild-type repressor; these results suggest that the structural transitions required for inducer binding are markedly impaired by the mutation. The fluorescence spectral shift in response to inducer binding was identical for mutant and wild-type proteins. This identity indicates direct effects of inducer binding on the tryptophan(s) near the sugar binding site rather than environmental changes consequent to conformational shifts. Analogy to the bacterial sugar binding proteins suggest that the Ala to Val change at position 81 in BG185 repressor yields a molecule that is fixed in a closed, sugar-binding conformation.     

The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.     

Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution

ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole. We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method. As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region. The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function. In the closed ligand-bound form observed here, D-allose is buried at the domain interface. Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity. Details of the ligand-binding cleft reveal the features that determine substrate specificity. Extensive hydrogen bonding as well as hydrophobic interactions are found to be important. Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar. In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms. Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds. The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity. Thus, ALBP can function as a low affinity transporter for D-ribose. The significance of these results is discussed in the context of the function of allose and ribose transport systems.