Structure and function of substrate-binding domains of ABC-transporters · 2016. 3. 9. · SBP....

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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).

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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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


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.


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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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


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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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).


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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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


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


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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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,


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


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


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


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

Structure and function of substrate-binding domains of ABC-transporters · 2016. 3. 9. · SBP....

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Transcript of Structure and function of substrate-binding domains of ABC-transporters · 2016. 3. 9. · SBP....