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University of Groningen
Structure and function of substrate-binding proteins of ABC-transportersBerntsson, Ronnie Per-Arne
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Chapter 1
General introduction tosubstrate-binding proteins
Ronnie P-A Berntsson, Sander HJ Smits, Lutz Schmitt, Dirk JanSlotboom and Bert Poolman
1.1 Introduction
Substrate-binding proteins (SBPs) are a class of proteins or domains that are oftenassociated with membrane protein complexes for transport of substrate or signaltransduction. SBPs were originally found to be associated with ATP bindingcassette (ABC)-transporters (Wilkinson, 2002), but have more recently been shownto be part of other membrane protein complexes as well, such as tripartite ATP-independent periplasmic (TRAP)-transporters (Gonin et al., 2007; Mulligan etal., 2009) as well as being domains within two-component regulatory systems(Neiditch et al., 2006), Guanylate cyclase-atrial natriuretic peptide receptors (Felderet al., 1999; Misono, 2002), G-protein coupled receptors and ligand-gated ionchannels (Armstrong and Gouaux, 2000). SBPs vary in size from roughly 25 to 70kDa, and despite little sequence similarity their overall three dimensional structuralfold is highly conserved. All proteins of the SBP family are composed of twodistinct domains (Quiocho and Ledvina, 1996), although some members contain anextra domain as an exception from the common rule (Tame et al., 1994). A schematicoverview of membrane protein systems containing SBPs is shown in Figure 1.1. Thetypical SBP fold as seen in the three-dimensional structures is also found amongtranscriptional regulators, such as the lac-repressor (Felder et al., 1999).
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Figure 1.1. Schematic overview of SBP-dependent membrane proteins. A-B: visualizes ABC-transporters. A: ABC-importer with the SBP in the periplasm (in Gram-negative prokaryotes), or with alipid-anchored SBP (in Gram-positive prokeryotes). The nucleotide-binding domain (NBD) hydrolyzesATP to drive the transport of the substrate over the membrane. B: ABC-importer in a prokaryote withthe SBD fused to the TMD. Some transporters have more than one SBD fused to the transmembranedomain (TMD), illustrated by light gray icons. C: TRAP transporter that can have either lipid-anchoredor periplasmic SBP. D: Schematic of a Guanylate Cyclase-Atrial Natriuretic Peptide Receptor, with aSBD, a single transmembrane helix and a intracellular domain (ICD). E: Ligand-gated ion channel,based on the ionotropic glutamate receptors (tetramer structures), with at the top the ATD domainsinvolved in the oligomerization of the protein, below the ATD the SBDs (in these proteins often termedLBD). F: G-protein coupled receptor with a cytoplasmic domain (CTD). The schematic is based on themetabotropic glutamate receptors which have been hypothesized to be functional dimers (Muto et al.,2007) G: Schematic of a two-component sensor kinase. Schematic based on data available for the quorumsensing complex LuxPQ (Neiditch et al., 2006).
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General introduction to substrate-binding proteins 3
1.1.1 SBP dependent transport proteins
ABC-transporters exist in all three kingdom of life and transport a large variety ofsubstrates across biological membranes. Based on the directionality of transport,ABC transporters can be classified as exporters of importers. Both consist oftwo nucleotide-binding (NBD) and two transmembrane domains (TMD) (Higgins,1992; Biemans-Oldehinkel, Doeven, and Poolman, 2006) (Fig. 1.1A-B). In thecase of ABC importers, a fifth domain is part of the functional unit, the SBP.The NBDs power transport through binding and hydrolysis of ATP, whereas theTMDs form the translocation pathway. But without SBPs ABC importers cannottransport their substrate, because SBPs bind their ligand with high affinity anddeliver it to the translocator, where the substrate is released into the translocationpore upon ATP binding (Khare et al., 2009). Substrate-binding proteins (SBPs) arelocated in the periplasm of Gram-negative bacteria or lipid-anchored or fused tothe TMD in the case of Gram-positive bacteria and Archaea (Heide and Poolman,2002). ABC importers with fused substrate-binding proteins can also be found inGram-negative bacteria however less frequently than in Gram-positives (Heide andPoolman, 2002). In addition to the classic SBP dependent systems, only recently anew family of prokaryotic vitamin-uptake ABC transporters that is not dependingon a SBP (Rodionov et al., 2006, 2009) was identified. Here, an integral membraneprotein binds the substrate with high affinity (pM to nM range) and it will beinteresting to see whether this binding mechanism resembles that of SBPs.
In contrast to ABC exporters, ABC importers have been found only in bacteria andArchaea, and can be identified by the highly conserved EAA motif in the TMDs(Hunke et al., 2000; Mourez et al., 1997). Due to the recently solved crystal struc-tures of various ABC importers (Locher et al., 2002; Hvorup et al., 2007; Kadaba etal., 2008; Oldham et al., 2007; Hollenstein et al., 2007; Khare et al., 2009), we knownow that the EAA motif is located in a cytoplasmic loop, which forms the couplinghelix (Hollenstein et al., 2007) implicated in NBD-TMD communication. In thelast decade new membrane protein complexes were discovered that contained aSBP. This family of transporters was called tripartite ATP-independent periplasmic(TRAP)-transporters (Kelly and Thomas, 2001). They are secondary transportersbut rely on SBPs to import their ligands (Fig. 1.1C). The membrane componentof these proteins usually consists of one larger and one smaller subunit (Kellyand Thomas, 2001). The transport of substrate is driven by an electrochemical iongradient (H+ or Na+ gradient). In striking contrast to ABC-transporters, the same
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4 Chapter 1
TRAP-transporter may function as both importer and exporter. Furthermore, it hasbeen shown that the SBP of the sialic acid transporter, SiaPQM, is also required forthe ligand export (Mulligan et al., 2009).
1.1.2 SBP dependent channel, signal and regulator proteins
Bacterial histidine sensor kinase complexes, part of two-componant sensor kinasesystems, also have substrate binding proteins (Fig. 1.1G). A good example is theautoinducer-2 (AI-2) binding protein, LuxP, which upon binding of AI-2 interactswith the histidine sensor kinase, LuxQ, an integral membrane protein. The sensingof AI-2 plays a large role in the quorum sensing of bacteria (Neiditch et al., 2006).Guanylate cyclase-atrial natriuretic peptide receptors are a class of mammalianreceptors that are responsible for body fluid homeostasis as well as for controland regulation of blood pressure. They consist of an extracellular ligand-bindingdomain (the SBD), homologous to bacterial SBPs (Felder et al., 1999), one singlemembrane spanning helix and an intracellular domain (Fig 1.1D). Functionallythese proteins form homodimers or homotetramers (Potter and Hunter, 2001).
Two other membrane protein complexes that contain SBPs are the mammalianglutamate receptors (Fig. 1.1E-F). Ionotropic glutamate receptors (iGluR) belongto the family of ligand-gated ion channels, with two well-studied examples beingGluA2 and GluR2 from Rattus norvegicus (Sobolevsky et al., 2009; Armstrong andGouaux, 2000). The SBP in these proteins is made out of two half domains, whichare separated in primary sequence. In these proteins ligand binding induces aconformational change in the transmembrane domains, which subsequently leadsto channel opening. The metabotropic glutamate receptors (mGluR) are G-proteincoupled receptors, with an N-terminal SBP, followed by a transmembrane domainand a cytosolic C-terminal domain. Upon ligand binding, conformational changeslead to signal transduction over the membrane.
Transcriptional regulators, like the lac-repressor have two main domains, a DNA-binding domain and a SBD (Lewis et al., 1996; Friedman et al., 1995; Schumacheret al., 1994). Ligand binding to the SBD of these proteins alters the affinity of theDNA-binding domain to its cognate DNA, thereby allowing, or repressing, geneexpression. This type of proteins forms either dimers (as the purine and carboncatabolite repressors (Schumacher et al., 1994, 2004)) or tetramers (as the lactose andfructose repressors (Friedman et al., 1995; Ramseier et al., 1993)), with the dimer
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General introduction to substrate-binding proteins 5
interface at the SBD and a separate small domain forming a tetramerization helix(in the case of LacR) (Bell and Lewis, 2001).
1.1.3 Structures of SBPs
The first SBP crystal structure, the L-arabinose binding protein (ABP), was solvedin 1974 (Quiocho et al., 1974). Many more have been elucidated since (Table1.2), supplying us with a wealth of structural information regarding this familyof proteins. Overall the SBPs are built of two α/β domains connected by a hinge-region, with the ligand-binding site buried in between the two domains. In theabsence of ligand, the protein is flexible with the two domains rotating aroundthe hinge (Tang et al., 2007) and exists largely in the open conformation with bothsubdomains spread more or less apart (Quiocho and Ledvina, 1996) (Fig. 1.2).Upon substrate binding, the closed conformation is stabilized, and the ligand iscompletely buried inside the protein. This process has been called the ’Venus Fly-trap’ mechanism (Mao et al., 1982).
Based on the available structures, SBPs can in principle exist in four distinctstructural states: (i) open-unliganded (ii) open-liganded (iii) closed-unliganded and(iv) closed-liganded (Fig. 1.3). As stated above, SBPs mainly exist in the open-unliganded state in the absence of substrate, and only a small fraction (
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Figure 1.2. Slice through surface representation of OppA structure from L. lactis, with the top panelshowing the closed state and the ligand-binding cavity completely buried inside the protein. Lowerpanel shows the open conformation, which exposes the ligand-binding site to the solvent.
analysis, SBPs can be grouped in three distinct classes. In class I the sheet topologyof both domains is β2β1β3β4β5 whereas class II has β2β1β3βnβ4 as topology, with nrepresenting the strand just after the first cross-over from the N-terminal domain tothe C-terminal domain, and vice versa (Fukami-Kobayashi et al., 1999). Examplesof proteins belonging to Class I are leucine / isoleucine / valine-binding protein(LIVBP) (Sack, Saper, and Quiocho, 1989) and lysine-binding protein (LBP) (Sack,Trakhanov, et al., 1989). Prominent members of Class II are the histidine-bindingprotein (HisJ) (Oh et al., 1994) and the oligopeptide binding protein (OppA) (Tameet al., 1994). Subsequently, the hinge-region is generally formed by three connectingstrands in Class I and two connecting strands in Class II SBPs. A third class of SBPswere found after this classification was made, with the first example being TroA(Y. H. Lee et al., 1999). The distinguishing feature of Class III SBPs is a single α-helix which serving as the linker between the two α/β domains. Proteins includedin Class III are the vitamin B12 binding protein BtuF (Karpowich et al., 2003) andthe Zinc-binding protein TroA (Y. H. Lee et al., 1999). On has also to stress, that thesize of SBPs does not correlate with the class to which it belongs, e.g., both OppA(65 kDa) and HisJ (27 kDa) fall in Class II.
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General introduction to substrate-binding proteins 7
Figure 1.3. The different structural states of the traditional SBPs, ie the Venus Fly-trap model. A:Open-unliganded; B: Closed-unliganded; C: Open-liganded; and D: Closed-liganded. S stands forsubstrate. Without substrate, the equilibrium is towards the open-unliganded conformation. Uponbinding of substrate, the open-liganded conformation is formed and the equilibrium is shifted to closed-liganded.
1.2 Structural classification
Traditionally and in contrast to the classification of Fukami-Kobayashi, SBPs havebeen grouped into different groups on the basis of their substrates (metal-, carbo-hydrate-, vitamin-, tetrahedral oxyanions-, compatible solutes-, amino acids- andpeptide-binding proteins), but it is clear from the many existing structures that SBPsthat have similar structure do not bind the same ligands. Sequence alignment showthat the SBPs, for which high resolution structural information and functional dataare available (Table 1.2), are very diverse in sequence. Since phylogenetic analysisbased on multiple sequence alignment did not yield a stable phylogenetic tree(the sequence identity of the proteins are often
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8 Chapter 1
SBPs available in the protein data bank (PDB), which are associated with membraneproteins and for which both functional and structural data were available, werecollected and summarized in Table 1.2. Here the protein name, organism, ligand,affinity range, isoelectric point and the molecular weight are listed. Furthermorefrom a structural point of view, Table 1.2 contains the resolution, PDB code(s),structural class and the functional state (liganded, unliganded, closed, open) inwhich the protein has been crystallized. The PDB was searched in two ways: (i)via structural homology searches, using the FFAS server (Jaroszewski et al., 2005)and (ii) via protein BLAST, searching against the PDB (Altschul et al., 1997). In bothmethods the sequences of known SBPs were used as search entries, and the processwas repeated with members of all the clusters identified in the subsequent analysis(Fig. 1.4). Proteins with 70% or higher identity to the search query were not in-cluded in the table, except for cases of proteins for which important functional datawas available. Although the majority of SBPs that were used in the structural align-ment were proteins that were both structurally and biochemically characterized,the structure of eight proteins were included which had not been functionally char-acterized, and thus had unknown ligands, or possibly wrongly annotated ligandsin the PDB. They were chosen on the basis of having low sequence homology to anyof the existing SBPs (verified via Psi-BLAST (Altschul et al., 1997)). The structures ofthe resulting 106 SBPs (102 closed structures and 4 open structures) (Table 1.2) werepairwise superpositioned with each other, and the resulting RMSDs were used toproduce a structural distance tree (Fig. 1.4), using the kitch program of the Phylippackage (http://evolution.genetics.washington.edu/phylip). As seen in the tree,the SBPs group into six defined clusters (A-F). It is worth mentioning that threeclusters (cluster A, D and F) can be even further subdivided by the actual ligand ofthe SBPs (see below). For a brief overview of the features of each (sub)cluster, seetable 1.1. The six clusters will be explained in the following part of the review. Forevery cluster we took one representative and showed the characteristic feature ofthis cluster in Figure 3.
1.2.1 Cluster A
Cluster A consists solely of Class III SBPs, and they also cluster in our structurebased alignment. This is due to the alpha helix serving as the hinge between thetwo domains (Figure 1.5A, the helix is coloured in orange). This helix ensures arigid overall structure reflecting in the small movement of both domains during
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General introduction to substrate-binding proteins 9
Figure 1.4. Structural distance tree of all SBPs that are mentioned in table 1.2. The PDB codes of theSBPs in the closed-liganded conformation were pairwise superimposed using SSM Superpose (Krissineland Henrick, 2004). Four structures out of 106 were not available in a closed form, in those cases theopen structure was used in the superimposition. The resulting RMSD values were converted, by takingthe value to the power of 2.1, to values that were empirically determined suitable for input into thekitch program of the Phylip package (http://evolution.genetics.washington.edu/phylip). The resultingstructural distance tree has 6 well-defined clusters (A-F). For a brief description of each cluster, see table1.1.
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10 Chapter 1
Table 1.1. Overview of the determined clusters of SBPs (Fig. 1.5 and 1.4), with information over theirligand specificity, class and additional features.
Cluster Types of ligands Classification byFukami-Kobayashi etal., 1999
Additionalinformation
A-I Metal ions Class IIIA-II Siderophores Class IIIB Carbohydrates,
Leu, Ile, Val,Autoinducer-2
Class I Homologous to lac-repressor
C Di- & oligopep-tides, Arg, Celllu-biose, Nickel
Class II extra large domain
D-I Carbohydrates Class II extra domainD-II Putrescine,
thiamineClass II
D-III Tetrahedraloxyanions
Class II
D-IV Iron ions Class IIE Sialic acid, 2-keto
acids, ectoine, py-roglutamic acid
Class II TRAP transporter asso-ciated SBPs
F-I Trigonal planaranions, unknownligands
Class II
F-II Methionine Class IIF-III Compatible
solutesClass II
F-IV Amino acids Class II
closing towards each other, for example in BtuF, the SBP of the vitamin B12 ABCimporter from E.coli, the open structure rotates only by 4◦, when closing uponsubstrate binding (Hvorup et al., 2007). All of these SBPs play a role in metalbinding. Inside this cluster the SBPs can be subdivided into two subgroups (A-I and A-II) by their cognate substrates. In A-I the SBPs either bind metal ions(iron, zinc, manganese) via direct interactions with the metal ion, and in A-II theybind sequestered metals, in the way of siderophores like enterobactin, catecholateand hydroxamate or heme. BtuF clusters together with this latter subgroup, sincevitamin B12 contains a porphyrin ring that binds in a similar way as the othersiderophores (Borths et al., 2002).
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General introduction to substrate-binding proteins 11
1.2.2 Cluster B
This cluster consists of Class I SBPs binding mainly carbohydrates (such as ribose,glucose and arabinose), but also branched chain amino acids and autoinducer-2(AI-2). The carbohydrate- and amino acid-binding proteins are all associated
Figure 1.5. The different clusters of SBPs are shown with their distinct structural feature colored inorange. A) Cluster A contains proteins having a single connection between the two domains in theform of a rigid helix. B) Cluster B contains SBPs with three interconnecting segments between the twodomains. C) Cluster C contains SBPs that have an extra domain and are significantly larger in sizewhen compared with the others. D) Cluster D contains SBPs with two relative short hinges E) ClusterE contains SBP associated with TRAP-transporters which all contain a large helix functioning as hingeregion. F) Cluster F contains SBPs with two hinges similar like cluster D, however this hinges havealmost double the length creating more flexibility inside the SBP. Please note that Cluster A, D and F canfurther be subdivided based on the substrate of the SBP (see text).
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12 Chapter 1
with ABC-transporters, whereas the AI-2 binding proteins bind to histidine sensorcomplexes and are involved in bacterial quorum sensing (Neiditch et al., 2006). Thiscluster contains SBPs with three hinges between the two domains (highlighted inFigure 1.5B in orange). Therefore the N- and C-terminus are not located within thesame domain.
Homologous to the proteins in cluster B are the lac-repressor type proteins, such asthe LacR and PurR from E. coli (Lewis et al., 1996; Friedman et al., 1995; Schumacheret al., 1994). If analyzed in their whole, this type of proteins would form a separatecluster with their DNA-binding domain as the distinct feature, but based solely ontheir SBD, which belong to class I, they cluster together with the other class I SBPsin cluster B, with especially close similarity to the ribose-binding protein (data notshown).
1.2.3 Cluster C
The proteins belonging to cluster C are all Class II SBPs. They bind very differentligands, such as di- and oligopeptides, arginine, nickel ions and cellubiose. Specificfor the SBP in this cluster is their large size, ranging from 55 to 70 kDa. Whencompared to other SBPs they all have an extra domain, as highlighted in figure1.5C. Only for some of these proteins the exact function of these domains havebeen clarified. For AppA from B. subtilis and OppA from L. lactis it has been shownthat the extra domain is taking part in extending the oligopeptide-binding cavity(Levdikov et al., 2005; Berntsson et al., 2009) in order to accommodate their largeligands. The fact that the nickel binding protein NikA has an extra domain issurprising, as other metal binding proteins (Table 1.2) are not as large in size anddo not contain an extra domain. Despite the difference in size inside the cluster allof these proteins align very nicely on a structural level (data not shown).
1.2.4 Cluster D
This cluster contains exclusively SBPs belonging to Class II. They are recognizedby two hinge region connecting domain I with domain II, thereby the N- and C-terminus are located within the same domain (Fig. 1.5D). This large group of SBPsbinds a large variety of substrates; carbohydrates, putrescine, thiamin, tetrahedraloxyanion as well as irons. Interestingly the subclasses found in this cluster resemble
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General introduction to substrate-binding proteins 13
exactly the bound ligand inside the proteins. A structural difference between thesesubgroups however was not seen. Likely, these subgroups are present due to thespecific orientation and composition of the binding sites in these SBPs.
The first subgroup (cluster D-I) contains a rather narrow substrate spectrum con-taining only carbohydrates such as maltose, glucose and galacturonide. Inspectionof the structural distance tree reveals that these proteins have a larger similarityto the proteins in Clusters C to F, than to the proteins in Cluster B, althoughthese proteins bind similar ligands. However this subgroup has two additionaldistinguished features when compared to the proteins of Cluster B, namely sizeand domain organization. In subgroup D-I the SBPs are all slightly larger, withmolecular weights above 40 kDa compared to 35 kDa of Cluster B SBPs, and theyall seem to have one small extra subdomain, as described for the maltose bindingprotein (MBP) being the best characterized member of this subgroup (Spurlino etal., 1991).
A second subgroup (cluster D-II) contains polyamine-binding proteins, as well asthe thiamine-binding protein TbpA. The ligand affinities vary greatly, with bothTbpA and PotD from T. pallidum having KD values in the low nM range (Sorianoet al., 2008; Sugiyama et al., 1996), while the other two proteins in the cluster haveKD values in the µM range (Machius et al., 2007; Vassylyev et al., 1998). The threepolyamine-binding proteins are clearly related, but why TbpA cluster with them isunknown.
The third subgroup (cluster D-III) is a very well-defined structural cluster, withproteins that bind tetrahedral oxyanions, and consists of molybdate-, sulfate-,and phophate-binding proteins, like the molybdate binding protein ModA formA. fulgifus (Hollenstein et al., 2007). Tetrahedral oxyanion-binding proteins bindtheir ligands with dissociation constants in the (sub)µM range (Wang et al., 1997;Jacobson and Quiocho, 1988). The last subgroup (cluster D-IV) contains only iron-binding proteins (FBPs). They all bind either ferrous or ferric iron, usually via directinteractions with protein side-chains. A subset of the proteins (e.g. hFBP) alsochelates the iron via an exogenous anion, whereas others do not require a secondanion (e.g. SfuA). The coordination of the iron ion, especially those that chelatethe metal via a second anion, is remarkably similar to mammalian transferrins, andthese proteins have also been referred to as bacterial transferrins (Dhungana et al.,2003). However, the similarities in the metal coordination between the FBPs andthe mammalian transferrins are believed to have arisen by convergent evolution(Bruns et al., 1997).
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1.2.5 Cluster E
In this cluster all substrates binding protein represent the known structures of theTRAP-Ts (tripartite ATP-independent periplasmic transporters) family. In contrastto ABC transporters these TRAP-transporter use an electrochemical gradient to(H+ or Na+) fuel the uphill transport of substrates. The remarkable feature ofTRAP-SBPs is a large single β-strand that belongs to both five-stranded antiparallelβ-sheet of the two subdomains. Interestingly, not only the strand order alsoobserved in SBPs of ABC transporters but also the additional β-strand connectingboth subdomains and the number and positioning of the flanking α-helices areconserved in all TRAP-dependent ESRs structurally characterized so far. Quitestriking is the long helix (residues 225-260) of UehA lying on top of the proteinspanning both domains of UehA. Such a long helix is found in all other crystalstructures of SBP proteins reported for TRAP-Ts, although in some structures thishelix is interrupted by a kink. (Fig. 1.5E). Please note that SBPs of TRAP-T arealso referred to as ESR (extracellular receptors) to distinguish these proteins fromthe SBPs of ABC transporters. To date, only a few structure of TRAP transporterSBPs are known therefore the substrates in this cluster are limited to ectoine,pyroglutamic acid, lactate, 2-keto acids and sialic acid. Recently, a selectivity helixhas been described, explaining how these SBPs discriminate their substrates basedon their size (Lecher et al., 2009).
One member of this cluster, TakP, is one of the few prokaryotic SBPs that arebelieved to function as a dimer. The ligand-binding domains of mammalianglutamate receptors are known to act as dimers, and some SBPs associated withABC transporters have been proposed to form dimers (Richarme, 1982, 1983). Theonly other known example of a prokaryotic dimeric SBP that we are aware of is theiron-binding protein FitE (Shi et al., 2009), belonging to cluster D. It is clear thatthese proteins dimerize in solution, but whether these dimers are of physiologicalrelevance remains unclear and is not further discussed here.
1.2.6 Cluster F
Like SBPs in cluster D these proteins possess two hinges connecting domain Iand domain II. In striking contrast however these hinges are significantly longer,8-10 amino acids (Fig. 3F) compared to 4-5 amino acids normally observed inSBPs. Thereby more flexibility between the open and closed conformation of these
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General introduction to substrate-binding proteins 15
binding protein is likely to be possible. They bind a large variety of substratesranging from trigonal planar anions, nitrate, bicarbonates to amino acid as well ascompatible solutes. The overall structure of the proteins within cluster F remainssimilar, but they can be subdivided based on their substrates.
Subgroup F-I consists of for example NrtA and CmpA that both bind trigonalplanar anions such as nitrate and bicarbonate, respectively. SsuA, has not beenfunctionally characterized, but is annotated in the PDB as a nitrate binding protein.The other three proteins have not been functionally characterized and are also notannotated in the PDB. One of the proteins however, TTHA1568, has a tartaric acidmolecule bound inside the protein, at a position where one would expect a ligandto bind. It is thus possible that these latter proteins bind different sort of ligands.
Subgroup F-II is a small cluster of Class II SBPs, which all, with the exception of theuncharacterized YhfZ, bind methionine. The outlier in the group is GmpC sinceit binds a dipeptide, glycylmethionine, but the residues involved in binding of thedipeptide are rather conserved and similar to those of the methionine SBPs, thusmaking this protein fall into this cluster.
Subgroup F-III is a small well-defined cluster with Class II SBPs, binding thecompatible solutes glycine betaine, proline betaine and choline. These substratesare imported into the cell upon an osmotic upshift, to maintain volume andintracellular ionic strength within certain limits. All the proteins within this clusterhave both a similar overall structure, but also very similar ligand binding sites.They coordinate the quaternary ammonium group of their substrates via cation-π interactions. These cation-π interactions are usually formed via a tryptophanprism, as in ProX from E. coli and OpuAC from B. subtilis (Horn et al., 2006; Smitset al., 2008). The binding site can also be made out of tyrosines as in ProX from A.fulgidus (Schiefner et al., 2004). Noteworthy is the difference in primary sequencebetween OpuAC from B. subtilis and the rest of the members in the cluster. Analysisof the sequence has revealed that a domain swap has taken place, with OpuACfrom B. subtilis having swapped domain I and II when compared to the otherproteins in this group.
Subgroup F-IV is a large subgroup consisting of Class II amino acid-binding pro-teins, with the only exception being the ectoine-binding protein EhuB. Amino acid-binding proteins usually exhibit high to moderate affinity binding. Typical affinitiesfor their substrates are in the nano- to (sub)micromolar range, e.g. a KD of 40 nM ofhistidine for binding to the histidine-binding protein, HisJ (Oh et al., 1994), a KD of
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16 Chapter 1
about 15 nM for lysine and arginine binding to LAO (lysine, arginine, ornithine-binding protein) (Nikaido and Ames, 1992) and a KD of 500 nM for glutaminebinding to GlnBP (Sun et al., 1998). Even though these proteins often have thepossibility to bind other amino acids than their primary substrate, they generallydo so with an order of magnitude lower affinity. In all structurally examined aminoacid-binding proteins, the amine and carboxyl groups are charge-neutralized andstabilized by a couple of conserved residues. These are Asp161 and Arg77 in HisJ(Oh et al., 1994), which interact with the amine and carboxyl group, respectively.Hydrogen bonds are usually formed to the side-chains of the amino acids, but dueto their different sizes, polarity and structures, there is no conserved binding motifexcept for the charge neutralization of the termini.
1.3 Discussion
SBPs have relatively low sequence similarity, but they usually share an overallsimilar tertiary structure as previously described (Quiocho and Ledvina, 1996).This is also evident based on the analysis of the structure based on the phylogenetictree presented here (Fig. 1.4). All proteins within a cluster have defined similaritiesin their structure, although it does not always mean that they bind similar ligands.A number of clusters contain proteins with similar types of ligands, but some donot. An example of each would be cluster C and F. Cluster C contains proteinswhich all have the same scaffold, containing a third domain, but with very differentligand specificities (arginine, di- and oligopeptides, nickel and cellubiose). ClusterF on the other hand is a large structural cluster, which can further be subdividedinto subgroups. In cluster F-IV, for example, all SBPs bind amino acids (exceptEhuB, which binds ectoine). These different clusters are clear-cut examples that incertain cases ligand specificity has co-evolved with structure, while in other casesevolution seems to have used certain structural scaffolds and evolved the ligandspecificity afterwards.
That SBPs are associated with such a large range of different protein complexesillustrates the flexibility and modularity of their fold. In both pro- and eukaryotes,SBPs act mechanistically in an analogous manner, they bind ligands which shiftsthe intrinsic equilibrium between an open and closed structure towards the closedconformation. In prokaryotic systems, with SBPs associated with ABC or TRAPtransporters. Here, the substrate is delivered to the TMD after lignad binding,
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General introduction to substrate-binding proteins 17
which initiates the transmembrane translocation event. In the lac-type repressorproteins ligand binding is associated with a conformational changes that causes theprotein to alter its affinity towards DNA, changing the gene expression. Anothermechanism is evident in histidine kinase sensors, where a SBP, in the presence ofligand, interacts with the membrane protein complex and a signal is transducedacross the membrane. In eukaryotic systems like the glutamate receptors, the clo-sure of the SBDs leads to a conformational change in the transmembrane domain,which triggers a channel opening, in the case of iGluRs, or a signaling event, as inthe case of mGluRs.
Comparing ABC-transporters with mGluRs, it is clear that the precise structuraldetails are different, but the overall steps are similiar. In both cases, the process isinitiated by the ligand binding to the SBP. Second, a signal is transduced acrossthe membrane to domains on the cytosolic side of the membrane protein. InmGluR this signal is triggered by ligand binding, whereas in ABC transporters thissignal occurs when the SBP docks to the TMD. The binding event is followed by asignaling event, as in mGluR; in ABC transporters the signaling event is followedby events in the NBDs, which ultimately drives the translocation process. Theconservation of the SBP structures and mechanism of action is obviously a clearcase of divergent evolution; the successful protein fold is used over and over againand only the substrate-binding site evolved, see Felder et al., 1999 for a review onthis topic.
Acknowledgments
The authors would like to thank Tejas Gandhi for providing the scripts for thesuperimposition and conversion of the output files. We apologize to all authorsof the overwhelming amount of publication concerning SBPs, which we could notall cite in this review. This work was supported by grants from Marie Curie EarlyStage Training (EST, to RB), The Netherlands Organisation for Scientific research(NWO, Vidi grant to DJS, Top-subsidy grant 700.56.302 to BP) and EU (EDICTprogram).
-
18 Chapter 1
Tabl
e1.
2.O
verv
iew
ofsu
bstr
ate-
bind
ing
prot
eins
avai
labl
ein
the
prot
ein
data
bank
(PD
B).T
hePD
Bw
asse
arch
edin
two
way
s,i:
via
stru
ctur
alho
mol
ogy
sear
ches
,us
ing
the
FFA
Sse
rver
(Jar
osze
wsk
ieta
l.,20
05)a
ndii:
via
prot
ein
BLA
ST,s
earc
hing
agai
nsth
ePD
B.In
both
met
hods
know
nSB
Psw
ere
used
asse
arch
entr
ies.
Prot
eins
with
70%
orhi
gher
iden
tity
toth
ese
arch
quer
yw
ere
noti
nclu
ded
inth
eTa
ble,
exce
ptfo
rcas
esof
prot
eins
forw
hich
impo
rtan
tfun
ctio
nald
ata
isav
aila
ble.
Onl
ypr
otei
ns,f
unct
iona
llych
arac
teri
zed
and
corr
ectly
anno
tate
d,ha
vebe
enin
clud
edin
the
tabl
e,w
ithth
eex
cept
ion
ofei
ghtp
rote
ins
from
stru
ctur
alge
nom
ics
cons
ortia
.Tho
seei
ghtp
rote
ins
did
noth
ave
any
clos
eho
mol
ogue
sba
sed
onth
eir
sequ
ence
,and
was
incl
uded
inth
eta
ble
and
subs
eque
ntan
alys
is.
Con
form
atio
nsav
aila
ble
Prot
ein
Org
anis
mLi
gand
(s)
Cla
ssO
pen-
unlig
ande
dO
pen-
ligan
ded
Clo
sed-
unlig
ande
dC
lose
d-lig
ande
dH
ighe
stre
solu
tion
(Å)
PDB
Cod
e(s)
Max
affin
itypI
MW
(kD
a)
3C9H
Agr
obac
teri
umtu
mef
acie
nsn.
d.II
--
??
1.9
3C9H
n.d.
5.9
39.9
3CV
GC
occi
dioi
des
imm
i-tis
n.d.
II-
-?
?2
3CV
Gn.
d.6.
731
.7
3HN
0Pa
raba
cter
oide
sdi
stas
onis
nitr
ate
II-
-?
?1.
73H
N0
n.d.
8.4
33.6
ABP
Esch
eric
hia
coli
L-ar
abin
ose
I-
--
Y1.
71A
BE,1
ABF
0.1
mM
6.2
35.6
Adc
AII
Stre
ptoc
occu
spn
eum
onia
ezi
ncII
I-
--
Y2.
43C
X3
n.d.
5.3
34.7
AF1
704
Arc
haeo
glub
usfu
lgid
usn.
d.II
--
?-
2.3
1ZBM
n.d.
4.8
30.8
ALB
PEs
cher
ichi
aco
liD
-allo
seI
Y-
-Y
1.7
1GU
B,1G
UD
,1R
PJ0.
33µ
M6.
732
.9A
lgQ
1Sp
hing
omon
assp
.A
1al
gina
teI
Y-
-Y
1.6
1Y3N
,1Y
3P,1
Y3Q
0.23
µM
8.8
60.3
Alg
Q2
Sphi
ngom
onas
sp.
A1
algi
nate
IY
--
Y1.
61J
1N,1
KW
H0.
15µ
M8.
859
.6
App
ABa
cillu
ssub
tilis
olig
opep
tide
II-
--
Y1.
61X
OC
n.d.
661
.9A
rtJ
Geo
baci
llus
stea
roth
erm
ophi
lus
L-ar
gini
ne,
L-ly
sine
,L-
hist
idin
e
II-
--
Y1.
82P
VU
,2Q
2A,2
Q2C
39nM
5.2
29.8
BtuF
Esch
eric
hia
coli
vita
min
B12
III
Y-
-Y
21N
2Z,1
N4A
,1N
4D,2
QI9
15nM
8.8
29.4
Ceu
EC
ampy
loba
cter
jeju
nien
tero
bact
inII
I-
--
Y2.
42C
HU
n.d.
8.4
32.6
cFbp
AC
ampy
loba
cter
jeju
niir
onII
--
-Y
1.4
1Y4T
,1Y
9Un.
d.8.
837
.4
Cho
XSi
norh
izob
ium
mel
iloti
chol
ine
IIY
-Y
Y1.
82R
EG,2
REJ
,2R
F1,2
RIN
,3H
CQ
2.7
µM
4.6
34
Cja
AC
ampy
loba
cter
jeju
niL-
cyst
eine
II-
--
Y2
1XT8
0.1
µM
6.2
30.9
Cm
pASy
nech
ocys
tissp
.PC
C68
03bi
carb
onat
eII
Y-
-Y
1.35
2I48
,2I4
9,2I
4B,2
I4C
5µ
M5.
549
.5
Con
tinue
don
next
page
-
General introduction to substrate-binding proteins 19
Con
form
atio
nsav
aila
ble
Prot
ein
Org
anis
mLi
gand
(s)
Cla
ssO
pen-
unlig
ande
dO
pen-
ligan
ded
Clo
sed-
unlig
ande
dC
lose
d-lig
ande
dH
ighe
stre
solu
tion
(Å)
PDB
Cod
e(s)
Max
affin
itypI
MW
(kD
a)
Dct
P6Bo
rdet
ella
pert
ussi
spy
rogl
utam
icac
idII
--
-Y
1.8
2PFZ
n.d.
9.1
35.7
Dct
P7Bo
rdet
ella
pert
ussi
spy
rogl
utam
icac
idII
--
-Y
2.2
2PFY
0.3
µM
9.1
34.7
Dpp
AEs
cher
ichi
aco
lidi
pept
ide
IIY
--
Y2
1DPP
,1D
PE1
µM
6.2
60.3
EhuB
Sino
rhiz
obiu
mm
elilo
tiec
toin
eII
--
-Y
1.9
2Q88
,2Q
890.
5µ
M5
29.6
FbpA
Man
nhei
mia
haem
olyt
ica
iron
IIY
Y-
Y1.
21S
I0,1
SI1,
1Q35
n.d.
8.2
38
FcsS
BPSt
rept
ococ
cus
pneu
mon
iae
olig
osac
hari
deII
--
-Y
2.35
2W7Y
1µ
M5.
746
.5
FeuA
Baci
lluss
ubtil
isca
tech
olat
eII
IY
--
Y1.
62W
HY,
2WI8
n.d.
7.8
35.1
FhuD
Esch
eric
hia
coli
hydr
oxam
ate
III
--
-Y
21E
SZ,1
K2V
,1K
7S0.
3µ
M6
33Fi
tEEs
cher
ichi
aco
lisi
dero
phor
esII
IY
--
Y1.
823B
E5,3
BE6
n.d.
6.4
34.5
FutA
1Sy
nech
ocys
tissp
.PC
C68
03ir
onII
Y-
-Y
1.7
2PT1
,2PT
2,3F
11n.
d.4.
939
.4
FutA
2Sy
nech
ocys
tissp
.PC
C68
03ir
onII
Y-
-Y
202V
OZ
,2V
P1n.
d.5.
838
.2
GG
BPEs
cher
ichi
aco
liD
-glu
cose
,D
-ga
lact
ose
IY
--
Y0.
922F
VY,
2FW
0,2G
BP,
2HPH
,2Q
W1,
3GA
50.
2µ
M5.
735
.7
GG
BPSa
lmon
ella
typh
imur
ium
D-g
luco
se,
D-
gala
ctos
eI
Y-
YY
1.9
1GC
G,2
FVY,
2FW
0,3G
A5
0.5
µM
5.8
35.8
GG
BPTh
erm
usth
erm
ophi
lus
D-g
luco
se,
D-
gala
ctos
eI
--
-Y
1.56
2B3B
,2B3
F0.
08µ
M9.
245
.4
GL-
BPBi
fidob
acte
rium
long
umla
cto-
N-b
iose
,ga
lact
o-N
-bi
ose
I-
--
Y1.
652Z
8D,2
Z8E
,2Z
8F10
nM4.
646
.4
Gln
HEs
cher
ichi
aco
liL-
glut
amin
eII
Y-
-Y
1.94
1WD
N,1
GG
G0.
5µ
M8.
527
.2G
luR
0N
osto
cpu
nctif
orm
eL-
glut
amat
eII
--
-Y
2.1
2PY
Y25
µM
5.4
25.1
Glu
R2
Rat
tusn
orve
gicu
sL-
glut
amat
eII
--
-Y
1.5
1GR
2,1M
5B,1
M5C
,1M
5E,1
M5F
12nM
8.2
30.7
Glu
R3
Rat
tusn
orve
gicu
sL-
glut
amat
eII
--
-Y
1.9
3DLN
,3D
P440
µM
9.1
30.9
Glu
R4
Rat
tusn
orve
gicu
sL-
glut
amat
eII
--
-Y
1.4
3FA
S,3F
AT
26nM
929
Glu
R5
Rat
tusn
orve
gicu
sL-
glut
amat
eII
--
-Y
1.8
2F34
,2F3
5,2F
3657
nM8.
329
.2G
luR
6ra
ttus
norv
egic
usL-
glut
amat
e,ki
anat
eII
--
-Y
1.7
1S50
,1S7
Y,1S
9T,1
SD3,
1TT1
,1TX
F35
nM5.
929
.3
Gm
pCSt
aphy
loco
ccus
au-
reus
dipe
ptid
e(G
lyM
et)
II-
--
Y1.
71P
99n.
d.9
30.5
Gna
1946
Nei
sser
iam
enin
gi-
tidis
L-m
ethi
onin
eII
--
-Y
2.1
3GX
A,3
IR1
n.d.
5.2
31.3
hFBP
Hae
mop
hilu
sin
fluen
zae
iron
II-
--
Y1.
61M
RP,
1D9V
n.d.
8.7
36.2
His
JEs
cher
ichi
aco
liL-
hist
idin
eII
--
-Y
1.9
1HPB
,1H
SL40
nM6.
128
.4
Con
tinue
don
next
page
-
20 Chapter 1C
onfo
rmat
ions
avai
labl
ePr
otei
nO
rgan
ism
Liga
nd(s
)C
lass
Ope
n-un
ligan
ded
Ope
n-lig
ande
dC
lose
d-un
ligan
ded
Clo
sed-
ligan
ded
Hig
hest
reso
lutio
n(Å
)PD
BC
ode(
s)M
axaf
finity
pIM
W(k
Da)
Hts
ASt
aphy
loco
ccus
au-
reus
stap
hylo
ferr
inII
IY
--
-1.
353E
IW,3
EIX
n.d.
9.4
36.6
IsdE
Stap
hylo
cocc
usau
-re
ushe
me
III
--
-Y
1.95
2Q8P
,2Q
8Qn.
d.9.
433
.3
LAO
BPSa
lmon
ella
typh
imur
ium
L-ly
sine
,L-
argi
nine
,L-
orni
thin
e
IIY
--
Y1.
81L
AF,
1LA
G,1
LAH
,1LS
T,2L
AO
14nM
628
.2
LBP
Esch
eric
hia
coli
L-le
ucin
eI
--
-Y
1.5
1USG
,1U
SI,1
USK
,2LB
P0.
4µ
M5.
539
.4Lb
pSt
rept
ococ
cus
pyo-
gene
szi
ncII
I-
--
Y2.
453G
I110
µM
7.9
34.2
LivJ
(LIV
BP)
Esch
eric
hia
coli
L-le
ucin
e,L-
isol
euci
ne,
L-va
line
IY
Y-
Y1.
71Z
15,1
Z16
,1Z
17,1
Z18
,2LI
V0.
1µ
M5.
539
.1
LsrB
Salm
onel
laty
phim
uriu
mau
toin
duce
r-2
IY
--
Y1.
31T
JY,1
TM2
n.d.
6.5
36.8
LsrB
Sino
rhiz
obiu
mm
elilo
tiau
toin
duce
r-2
I-
--
Y1.
83E
JWn.
d.5.
136
.5
LuxP
Vibr
ioha
rvey
iau
toin
duce
r-2
IY
--
Y1.
51J
X6,
1ZH
H,2
HJ9
n.d.
5.6
41.5
MBP
Esch
eric
hia
coli
olig
osac
hari
deI
YY
-Y
1.67
1AN
F,1D
MB,
1EZ
9,1E
ZO
,1E
ZP,
1FQ
A,
1FQ
B,1F
QC
,1F
QD
,1M
DP,
1MD
Q,1
OM
P,4M
BP
0.16
µM
5.2
40.7
MBP
Terh
oact
inom
yces
vulg
aris
olig
osac
hari
deI
--
-Y
2.3
2DFZ
,2Z
YK
0.2
µM
945
.8
Mnt
CSy
nech
ocys
tissp
.PC
C68
03m
anga
nese
III
--
-Y
2.9
1XV
Ln.
d.4.
436
.1
Mod
AA
rcha
eogl
obus
fulg
idus
mol
ybda
te,
tung
sten
II-
--
Y1.
552O
NR
,2O
NS
n.d.
5.6
38.6
Mod
AA
zoto
bact
ervi
nela
ndii
mol
ybda
te,
tung
sten
II-
--
Y1.
21A
TGn.
d.9
24.4
Mod
AEs
cher
ichi
aco
lim
olyb
date
,tu
ngst
enII
--
-Y
1.7
1AM
F,1W
OD
3µ
M8
27.4
Mts
ASt
rept
ococ
cus
pyo-
gene
sir
onII
I-
--
Y1.
93H
H8
4.3
µM
6.4
34.4
nFBP
Nei
sser
iago
norr
hoea
eir
onII
--
-Y
1.7
1O7T
n.d.
9.6
35.9
Nik
AEs
cher
ichi
aco
lini
ckel
IIY
--
Y1.
851U
IU,1
UIV
11µ
M5.
858
.7N
R1
Rat
tusn
orve
gicu
sgl
ycin
e,se
rine
II-
Y-
Y1.
41P
B7,1
PB8,
1PB9
,1PB
Q4
nM8.
133
.3
NR
2AR
attu
snor
vegi
cus
L-gl
utam
ate,
glyc
ine
II-
--
Y1.
72A
5S,2
A5T
n.d.
7.7
31.8
NR
3AR
attu
snor
vegi
cus
glyc
ine,
seri
neII
--
-Y
1.5
2RC
7,2R
C8,
2RC
95
µM
5.3
32.9
Con
tinue
don
next
page
-
General introduction to substrate-binding proteins 21
Con
form
atio
nsav
aila
ble
Prot
ein
Org
anis
mLi
gand
(s)
Cla
ssO
pen-
unlig
ande
dO
pen-
ligan
ded
Clo
sed-
unlig
ande
dC
lose
d-lig
ande
dH
ighe
stre
solu
tion
(Å)
PDB
Cod
e(s)
Max
affin
itypI
MW
(kD
a)
Nrt
ASy
nech
ocys
tissp
.PC
C68
03ni
trat
eII
--
-Y
1.5
2G29
0.3
mM
5.2
49
Opp
ALa
ctoc
occu
slac
tisol
igop
eptid
e(5
-35
a.a.
)II
YY
-Y
1.3
3DR
F,3D
RG
,3D
RH
,3D
RI,
3DR
J,3D
RK
,3FT
O0.
1µ
M8.
965
.9
Opp
ASa
lmon
ella
typh
imur
ium
olig
opep
tide
(3-5
a.a.
)II
Y-
-Y
1.2
1B05
,1B
0H,1
B1H
,1B2
H,1
B32,
1B3F
,1B
3G,1
B3H
,1B3
L,1B
40,1
B46,
1B4H
,1B
4Z,
1B51
,1B
52,
1B58
,1B
5H,
1B5I
,1B
5J,
1B6H
,1B
7H,
1B9J
,1J
ET,
1JEU
,1J
EV,
1OLA
,1O
LC,
1QK
A,
1QK
B,1R
KM
,2O
LB,2
RK
M
1µ
M6.
161
.3
Opp
AYe
rsin
iape
stis
olig
opep
tide
(3-5
a.a.
)II
--
-Y
1.8
2OLB
n.d.
5.8
61.7
Opp
A2
Stre
ptom
yces
clav
ulig
erus
argi
nine
,ol
igop
eptid
esII
Y-
-Y
1.45
2WO
K,2
WO
L,2W
OP
n.d.
5.4
62
Opu
AC
Baci
lluss
ubtil
isgl
ycin
ebe
tain
e,pr
olin
ebe
tain
e
II-
--
Y2
2B4L
,2B4
M,3
CH
G40
µM
7.8
32.2
Opu
AC
Lact
ococ
cusl
actis
glyc
ine
beta
ine,
prol
ine
beta
ine
IIY
--
Y1.
93L
6G,3
L6H
4µ
M7.
929
PBP
Esch
eric
hia
coli
phos
phat
eII
--
-Y
0.98
1IX
H,2
ABH
3µ
M8.
437
Peb1
aC
ampy
loba
cter
jeju
niL-
aspa
rtat
e,L-
glut
amat
eII
--
-Y
1.5
2V25
1.9
µM
928
.2
PEB3
Cam
pylo
bact
erje
juni
citr
ate
II-
--
Y1.
62H
XW
n.d.
9.4
25.6
PhuT
Shig
ella
dyse
nter
iae
hem
eII
I-
--
Y2.
42R
79n.
d.7.
131
.1
PnrA
Trep
onem
apa
llidu
min
osin
eI
--
-Y
1.7
2FQ
X,2
FQY,
2FQ
W0.
1µ
M4.
837
.8
PotD
Esch
eric
hia
coli
putr
esci
ne,
sper
mid
ine
II-
--
Y1.
81P
OT,
1PO
Y3.
2µ
M5.
238
.9
PotD
Trep
onem
apa
llidu
mpu
tres
cine
,sp
erm
idin
eII
--
-Y
1.8
2V84
10nM
6.4
39.8
PotF
Esch
eric
hia
coli
putr
esci
neII
--
-Y
2.3
1A99
2.0
µM
5.9
40.8
ProX
Arc
heog
lobu
sfu
lgid
usgl
ycin
ebe
tain
e,pr
olin
ebe
tain
e
IIY
--
Y1.
81S
W1,
1SW
2,1S
W4,
1SW
550
nM4.
733
Con
tinue
don
next
page
-
22 Chapter 1C
onfo
rmat
ions
avai
labl
ePr
otei
nO
rgan
ism
Liga
nd(s
)C
lass
Ope
n-un
ligan
ded
Ope
n-lig
ande
dC
lose
d-un
ligan
ded
Clo
sed-
ligan
ded
Hig
hest
reso
lutio
n(Å
)PD
BC
ode(
s)M
axaf
finity
pIM
W(k
Da)
ProX
Esch
eric
hia
coli
glyc
ine
beta
ine,
prol
ine
beta
ine
II-
--
Y1.
61R
9L,1
R9Q
1µ
M5.
936
PsaA
Stre
ptoc
occu
spn
eum
onia
ezi
ncII
I-
--
Y2
1PSZ
n.d.
5.3
34.6
PstS
Yers
inia
pest
isph
osph
ate
II-
--
Y2
2Z22
n.d.
8.7
36.7
PstS
-1M
ycob
acte
rium
tu-
berc
ulos
isph
osph
ate
II-
--
Y2.
161P
C3
3µ
M5.
138
.3
RBP
Esch
eric
hia
coli
D-r
ibos
eI
Y-
-Y
1.6
1BA
2,1D
BP,1
DR
J,1D
RK
,1U
RP,
2DR
I0.
13µ
M7
31R
BPTh
erm
otog
am
ariti
ma
ribo
seI
Y-
-Y
1.4
2FN
8,2F
N9
n.d.
5.1
35.9
Sco4
506
Stre
ptom
yces
coel
i-co
lor
n.d.
II-
-?
?2
2NX
On.
d.4.
831
.4
SfuA
Yers
inia
ente
roco
l-iti
cair
onII
--
-Y
1.8
1XV
Yn.
d.9
36.2
ShuT
Shig
ella
dyse
nter
iae
hem
eII
IY
--
-2.
052R
G7
n.d.
9.4
32.8
SiaP
Hae
mop
hilu
sin
fluen
zae
sial
icac
idII
Y-
-Y
1.7
2CEY
,2C
EX58
nM6.
436
.5
SsuA
Xan
thom
onas
axon
opod
isal
kane
sulfo
nate
II-
-?
?2
3E4R
n.d.
10.3
36.2
Sulfa
teBP
Salm
onel
laty
phim
uriu
msu
lfate
II-
--
Y1.
71S
BP0.
12µ
M7.
236
.6
TakP
Rho
doba
cter
spha
eroi
des
2-ke
toac
ids
IIY
--
Y1.
42H
ZK
,2H
ZL
18nm
5.6
40
TbpA
Esch
eric
hia
coli
thia
min
II-
--
Y2.
252Q
RY2.
3nM
6.9
36.2
TeaA
Hal
omon
asel
onga
taec
toin
eII
--
-Y
1.55
2VPO
0.19
µM
4.2
38.3
TM03
22Th
erm
otog
am
ariti
ma
n.d.
IIY
--
-1.
92H
PGn.
d.6
38.2
tmC
BPTh
erm
otog
am
ariti
ma
cellu
bios
eII
--
-Y
1.5
2O7I
,2O
7J,3
I5O
0.8
µM
570
TogB
Yers
inia
ente
roco
l-iti
caol
igog
alac
turo
nide
IY
--
Y1.
82U
VG
,2U
VH
,2U
VI,
2UV
Jn.
d.6
46.3
Tp32
Trep
onem
apa
llidu
mL-
met
hion
ine
II-
--
Y1.
851X
S5n.
d.6.
729
.1
TroA
Trep
onem
apa
llidu
mzi
nc,
man
gane
seII
IY
--
Y1.
81K
0F,1
TOA
7nM
6.2
33.6
TTH
A07
66Th
erm
usth
erm
ophi
lus
Ca+
-lact
ate
II-
--
Y1.
42Z
ZV,
2ZZ
W,2
ZZ
Xn.
d.9.
540
.8
Con
tinue
don
next
page
-
General introduction to substrate-binding proteins 23
Con
form
atio
nsav
aila
ble
Prot
ein
Org
anis
mLi
gand
(s)
Cla
ssO
pen-
unlig
ande
dO
pen-
ligan
ded
Clo
sed-
unlig
ande
dC
lose
d-lig
ande
dH
ighe
stre
solu
tion
(Å)
PDB
Cod
e(s)
Max
affin
itypI
MW
(kD
a)
TTH
A15
68Th
erm
usth
erm
ophi
lus
n.d.
II-
-?
?1.
552C
ZL,
2DBP
n.d.
5.6
30
Ueh
ASi
licib
acte
rpo
mer
oyi
ecto
ine
II-
--
Y2.
93F
XB
1.1
µM
4.3
37.3
Yhf
ZSh
igel
lafle
xner
in.
d.II
Y-
--
2.3
2OZ
Zn.
d.4.
925
.4Y
tfQ
Esch
eric
hia
coli
olig
osac
hari
deI
--
-Y
1.2
2VK
21.
3µ
M6.
934
.4Z
nuA
Esch
eric
hia
coli
zinc
III
Y-
-Y
1.7
2PR
S,2P
S0,2
PS3,
2PS9
20nM
5.6
33.8
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