Báo cáo khoa học: Structural origins for selectivity and specificity in an engineered bacterial repressor–inducer pair pdf

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Structural origins for selectivity and specificity in anengineered bacterial repressor–inducer pairMichael A. Klieber1, Oliver Scholz2,*, Susanne Lochner3, Peter Gmeiner3, Wolfgang Hillen2and Yves A. Muller11 Lehrstuhl fu¨r Biotechnik, Department of Biology, Friedrich-Alexander University, Erlangen-Nuremberg, Germany2 Lehrstuhl fu¨r Mikrobiologie, Department of Biology, Friedrich-Alexander University, Erlangen-Nuremberg, Germany3 Lehrstuhl fu¨r Pharmazeutische Chemie, Department of Chemistry and Pharmacy, Friedrich-Alexander University, Erlangen-Nuremberg,GermanyIntroductionThe bacterial repression system consisting of the effec-tor molecule tetracycline, the tetracycline-induciblerepressor protein tetracycline repressor (TetR) and thetet operator (tetO) has proven itself to comprise avaluable tool for studying gene expression not only inprokaryotes, but also in eukaryotes [1–3]. The repres-sor protein TetR, and first and foremost its ability toadopt different conformational states upon effectorKeywordsaltered inducer selectivity; altered inducerspecificity; bacterial transcription regulation;crystal structures; tetracycline repressorCorrespondenceY. A. Muller, Lehrstuhl fu¨r Biotechnik,Department of Biology, Friedrich-AlexanderUniversity Erlangen-Nuremberg, Im IZMP,Henkestrasse 91, D-91052 Erlangen,GermanyFax: +49 0 9131 8523080Tel: +49 0 9131 8523081E-mail: ymuller@biologie.uni-erlangen.de*Present addressDepartment of Biochemistry, University ofZurich, SwitzerlandDatabaseStructural data are available from the ProteinData Bank under the accession numbers3FK6 for TetR(K64L135I138) alone and 3FK7for the 4-ddma-atc complex(Received 10 March 2009, revised 9 July2009, accepted 31 July 2009)doi:10.1111/j.1742-4658.2009.07254.xThe bacterial tetracycline transcription regulation system mediated by thetetracycline repressor (TetR) is widely used to study gene expression inprokaryotes and eukaryotes. To study multiple genes in parallel, a triplemutant TetR(K64L135I138) has been engineered that is selectively inducedby the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracy-cline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-typeTetR. In the present study, we report the crystal structure ofTetR(K64L135I138) in the absence and in complex with 4-ddma-atc at reso-lutions of 2.1 A˚. Analysis of the structures in light of the available bindingdata and previously reported TetR complexes allows for a dissection of theorigins of selectivity and specificity. In all crystal structures solved to date,the ligand-binding position, as well as the positioning of the residues liningthe binding site, is extremely well conserved, irrespective of the chemicalnature of the ligand. Selective recognition of 4-ddma-atc is achievedthrough fine-tuned hydrogen-bonding constraints introduced by theHis64 fi Lys substitution, as well as a combination of hydrophobic effectand the removal of unfavorable electrostatic interactions through the intro-duction of Leu135 and Ile138.Abbreviationsatc, anhydrotetracycline; 4-ddma-atc, 4-de-dimethylamino-anhydrotetracycline; dox, 6-deoxy-5-hydoxy-tetracycline; PDB, Protein Data Bank;tc, tetracycline; tetO, tet operator; TetR, tetracycline repressor; TetR(K64L135I138), TetR-BD triple mutant H64K, S135L and S138I.5610 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBSbinding, plays a key role in this system. In the absenceof tetracycline, TetR binds with high affinity to the tetoperator tetO and thereby blocks the transcription ofany downstream genes. Upon binding of the inducertetracycline, the repressor TetR switches conformationsand dissociates from the operator DNA. As a result ofnumerous functional and structural studies, the atomicmechanism that underlies the functional switch inTetR is now understood in significant detail [4–7].To be able to control the expression of several genesin parallel, TetR mutants have been isolated in elabo-rate screens that respond to novel synthetic tetracyclineanalogs [8,9]. One of these variants is the TetR triplemutant TetR(K64L135I138) in which residues His64,Ser135 and Ser138 of TetR have been replaced by Lys,Leu and Ile, respectively [9]. This mutant is selectivelyinduced by the synthetic inducer 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc) and slightly by atc,but no longer by tetracycline. To better understand theswitch in selectivity and the acquired novel specificityof TetR(K64L135I138) for 4-ddma-atc, we solved thecrystal structure of TetR(K64L135I138) in the presenceand absence of 4-ddma-atc at resolutions of 2.06 and2.1 A˚, respectively. We show that the effects of thethree mutations on ligand binding can be generallythermodynamically dissected into individual contribu-tions and that they originate from a favorable inter-play of different physico-chemical properties, such assolvation effects, constrained hydrogen-bonding geom-etries and electrostatic discrimination.ResultsComparison of the 4-ddma-atc-bound and freeoverall TetR(K64L135I138) structureAs for all TetRs, TetR(K64L135I138) forms a dimerand, with respect to the monomer architecture, eachchain contains an N-terminal DNA-binding domain(residues 1–48) and a C-terminal effector-bindingdomain (residues 49–205) [10] (Fig. 1A). The latteralso comprises the dimer interface. The two effector-binding sites present in the dimer are identical and,because the binding sites are located within the proteininterface, each binding site is lined by residues fromboth monomers.The overall structures of ligand-free TetR(K64L135I138) and of TetR(K64L135I138) in complex with4-ddma-atc are very similar (for the chemical structureof 4-ddma-atc and related TetR ligands, see Fig. 2).The two crystals that have been used for structuredetermination are highly isomorphous and only smalldeviations occur in the cell axes (Table 1). They eachcontain a complete dimer in the asymmetric unit.Almost no differences exist between the monomers ineach crystal and the monomers ⁄ dimers between crys-tals. The main chain atoms of the two monomers ineach crystal can be superimposed with rmsd of 0.92and 1.34 A˚for the 4-ddma-atc-bound and effector-freeTetR(K64L135I138) structures, respectively. When direc-tly comparing the structure of 4-ddma-atc-bound TetR(K64L135I138) with that of effector-free TetR(K64L135I138), it is obvious that ligand binding does not induceany considerable changes in the tertiary structures. Thefour possible pairwise cross-superpositions betweencrystals yield an rmsd in the range 0.52–1.23 A˚for atotal of 770 common main chain atoms. This showsthat the differences between the ligand-bound andligand-free structures are not larger than the differ-ences observed between the two monomers present ineach crystal. To some extent, this is not too surprisingbecause the crystal with 4-ddma-atc bound wasobtained by soaking a ligand-free crystal with theligand. However, when considering the molecularmechanism by which TetR exerts its function, then thestructural similarity might be considered unexpected.A central function of TetR is its ability to adoptdifferent conformations. According to the conforma-tional switch model, TetR exists in two conforma-tions. In the ligand-free structure, the DNA-bindingheads are oriented such that TetR can readily bindto the operator DNA, whereas, in effector-boundTetR, the separation of the DNA-binding domain ischanged, such that TetR can no longer recognize thetetO DNA sequence (Fig. 1A) [4]. However, as notedabove, the domain orientations in the 4-ddma-atc-bound TetR(K64L135I138) and the ligand-free TetR(K64L135I138) structure are very similar and resemblethat of induced TetR more closely than that of indu-cer-free TetR (data not shown). Moreover, the mainchain of loop segment 100–105 that switches con-formations upon effector binding is in an identicalconformation in both structures and resembles thatobserved for induced TetR (Fig. 1C). In this confor-mation, segment 100–105 moves towards the ligandand thereby enables residues from the segment toparticipate in ligand binding.The observation that effector-free TetR(K64L135I138)adopts an induced-like conformation must appearunexpected. However, it can easily be rationalized ifalternative models are considered (e.g. the populationshift model) as explanations for the allosteric behaviorof TetR [11,12]. According to this model, effector-freeand DNA-free TetR is able to adopt a variety ofdivers and freely inter-converting conformations.Among these, there are also conformations that canM. A. Klieber et al. Structure of an engineered TetR-inducer pairFEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5611readily interact with DNA or that resemble the effec-tor-bound conformation as, for example, observed inthe present study in the case of the crystal structure ofthe effector-free TetR(K64L135I138). In this model,induction can be explained by a shift of the populationtowards a single DNA-binding incompetent conforma-tion. Indications that such a population shift modelmight apply to TetR have recently started to emerge[13,14].The effector-binding site of TetR(K64L135I138)inthe presence and absence of 4-ddma-atcParticularly interesting with respect to the observedspecificity and selectivity of TetR(K64L135I138) for4-ddma-atc are the interactions between the ligandand the protein in the effector-binding site (Figs 1Band 2A). Of the two binding sites that can be observedindependently in the 4-ddma-atc-complex structure,ACBFig. 1. Crystal structure of TetR(K64L135I138) in complex with 4-ddma-atc. (A) Ca-representation of the TetR(K64L135I138) dimer (shown in dif-ferent shades of magenta) in complex with 4-ddma-atc (shown in yellow) superimposed on wild-type TetR in complex with DNA (in black,PDB entry: 1QPI) [6]. According to the generally agreed induction mechanism, effector binding to TetR induces a conformational change inTetR, which alters the orientation and separation of the DNA-binding domains (indicated by two-headed arrows), so that TetR no longerbinds to the operator DNA. (B) Schematic representation of the interactions between 4-ddma-atc and TetR(K64L135I138). (C) Stereo represen-tation of the structure of TetR(K64L135I138) in complex with 4-ddma-atc (shown in magenta and yellow) superimposed onto the effector-freeTetR(K64L135I138) structure (shown in grey). Of the two binding sites present in the crystal structure, binding site I (Table 2) is shown. Thebinding sites are highly similar in the presence and absence of 4-ddma-atc. The loop segment 100–105 that switches conformations in otherligand-free and ligand-bound TetR structures is only slightly displaced in ligand-free TetR(K64L135I138) compared to the ligand-bound TetRstructure.Structure of an engineered TetR-inducer pair M. A. Klieber et al.5612 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBSboth binding sites show the density for the ligand inthe initial difference-fourier electron density maps,albeit to a different extent. The ligand is well definedin binding site I, but only poor density has beenobserved for the ligand in binding site II. To accountfor this observation, the occupancy of the ligand inbinding site II was estimated at 50% and that in bind-ing site I at 100%. When using these values during therefinement, the temperature factors of the ligandsrefine to values similar to those of the surrounding res-idues, hinting that the estimated occupancies correctlyreflect those in the crystal. Inspection of the crystalpacking yields a plausible explanation for the differ-ences in occupancies. In the crystal, the accessibility tosite II is impaired by the packing of neighboring mole-cules, whereas site I appears to be readily accessiblethrough the solvent channels in the crystal.When superimposing the two ligand-binding sites onthe basis of the coordinates of 93 residues surroundingthe ligands, it is obvious that the ligand is slightlyshifted in site II (Table 2). This shift is not only appar-ent with respect to 4-ddma-atc bound to site I, butalso compared to various other ligand-TetR complexes(Table 2). The two 4-ddma-atc molecules differ by anrmsd of approximately 1 A˚, whereas the deviationsbetween 4-ddma-atc bound to site I and tetracyclineand 6-deoxy-5-hydoxy-tetracycline (dox) bound toTetR [15,16], as well as atc bound to revTetR [14], arein the range 0.4–0.5 A˚when considering 27 commonligand atoms. We suspect that the positional shift ofthe ligand in site II is related to the 50% occupancy.The concomitant occurrence of ligand and water mole-cules (also refined at 50% occupancy) at almost identi-cal positions might lead to increased coordinate errorsduring the refinement of the atomic positions andhence a less accurate ligand positioning in site II.Accordingly, the description of the binding of theligand in the present study is restricted to 4-ddma-atcbinding to site I in TetR(K64L135I138).4-ddma-atc binding leads to only minor rearrange-ments in the TetR(K64L135I138)-binding site (Fig. 1C).Among the most notable changes are a slight shift of theentire loop segment 100–105 in the direction of theligand, the presence of two alternative side chain confor-mations for Asn82 in the 4-ddma-atc-bound structureversus a single conformation in ligand-freeTetR(K64L135I138) and, finally, the occurrence of aslightly different rotamer for Ile138 (i.e. different posi-tioning of atom Cd) in the ligand-free and ligand-boundstructure. Overall, when considering both the mainchain fold and the conformations of the side chains, thestructures of ligand-free TetR(K64L135I138) and 4-ddma-atc-bound TetR(K64L135I138) are very similar. This simi-larity also extends to a number of water molecules thatoccupy identical positions in the two structures.Specific interactions between 4-ddma-atc andTetR(K64L135I138)TetR(K64L135I138) binds 4-ddma-atc via a number ofspecific interactions (Fig. 3A). One of the most notableones involves Lys64. Atom Nf of Lys64 interacts withtwo oxygen atoms of 4-ddma-atc, namely of the amidegroup attached to atom C2 and the OH groupABCDFig. 2. Chemical structures of (A) 4-ddma-atc, (B) atc, (C) tetracy-cline (tc) and (D) dox.M. A. Klieber et al. Structure of an engineered TetR-inducer pairFEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5613attached to atom C3 of 4-ddma-atc. Furthermore,atom Lys64-Nf is located in hydrogen-bonding dis-tance to the amide group of Asn82 and the main chaincarbonyl oxygen atom of Tyr66. It is not possible topredict the strength of the interaction between Lys64and 4-ddma-atc based on structural data only. ForTable 1. Data collection and refinement statistics.Triple mutantTetR(K64L135I138)Triple mutantTetR(K64L135I138) in complexwith 4-ddma-atcData collection statisticsSpace group C2 C2Unit cell parametersa, b, c (A˚) 126.35, 58.06, 62.62 130.22, 59.38, 63.78b (°) 96.58 97.85Molecules per asymmetric unit 2 2Resolution range (A˚)a15–2.1 (2.25–2.1) 20–2.06 (2.19–2.06)Unique reflections 24749 29597Redundancy 3.3 3.7Completeness (%) 93.3 (95.9) 98.6 (91.8)Rmerge(%) 6.4 (37.1) 6.0 (39.8)Wilson B-factor (A˚2) 15.1 26.5Refinement statisticsNumber of protein atoms, solventmolecules and ligand atoms3095, 116, 0 3180, 225, 58Rwork(%) 21.6 20.3Rfree(%) 26.2 26.1rmsd bond lengths (A˚) 0.005 0.011rmsd bond angles (°) 1.069 1.175rmsd B-factors bonded atoms: main chain,side chains (A˚2)1.51, 2.27 1.24, 2.13Percentage of residues in most favored regions,additional allowed, generously allowed anddisallowed regions of the Ramachandran plotb95.1, 4.9, 0.0, 0.0 95.5, 4.2, 0.3, 0.0Average B-factor (A˚2) 43.26 36.79aValues in parentheses refer to the highest resolution shell.bAccording to PROCHECK [27].Table 2. Superposition of the ligand-binding sites and ligand positions in selected TetR complexes.No ligandTetR(K64L135I138)IaNo ligandTetR(K64L135I138)II4-ddma-atcTetR(K64L135I138)Ia4-ddma-atcTetR(K64L135I138)IIatc revTetR(PDB code:2VKV)tc TetR(D)(PDB code:2VKE)dox TetR(D)(PDB code:2O7O)No ligandTetR(K64L135I138)I– 0.905, 1.396b0.532, 0.843 0.830, 1.360 0.854, 1.386 0.851, 1.385 0.870, 1.356No ligandTetR(K64L135I138)II– – 0.788, 1.237 0.430, 0.771 0.631 1.168 0.576, 1.170 0.603, 1.1024-ddma-atcTetR(K64L135I138)I– – – 0.633, 1.128 0.656, 1.155 0.615, 1.054 0.632, 1.0474-ddma-atcTetR(K64L135I138)II– – 1.048c– 0.489, 1.062 0.432, 1.065 0.415, 0.999Atc revTetR – – 0.431 0.896 – 0.413, 0.983 0.437, 0.918tc TetR(BD) – – 0.456 1.024 0.270 – 0.216, 0.732Dox TetR(BD) – – 0.523 0.878 0.259 0.265 –aBecause the crystals contain two molecules in the asymmetric unit, two separate binding sites (I and II) are present in each of the twoTetR(K64L135I138) crystal structures.bAbove the diagonal the rmsd (A˚) between structures of a selection of 93 residues surrounding theligand-binding site is reported (first number, rmsd obtained upon superposition of all main chain atoms of the selection; second number,superposition of all atoms).cBelow the diagonal: rmsd (A˚) calculated between the ligands (27 common ligand atoms) in the different com-plexes after optimal superposition of the structures based on the main chain atoms of 93 residues surrounding the binding site.Structure of an engineered TetR-inducer pair M. A. Klieber et al.5614 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBSexample, the structure does not allow a distinction ofwhether the side chain of Lys64 is protonated or not.Uncertainties also arise with respect to the correctorientation of the amide group attached to atom C2 of4-ddma-atc, as well as the amide group of amino acidAsn82, because the amide oxygen and nitrogen atomsare indistinguishable at the resolutions of the solvedcrystal structures. Because atom Nf of Lys64 can onlyparticipate in three hydrogen bonds, it should be notedthat, of the four potential hydrogen bond acceptors ⁄donors, atom O from the amide group of 4-ddma-atcis the furthest apart (3.1 A˚versus 2.5–2.8 A˚for theother hydrogen bond acceptors ⁄ donors). However, allfour potential hydrogen bond partners are geometri-cally quite favorably placed. In all four cases, almostideal linear hydrogen bond geometries can be antici-pated because the angle formed between atomsLys64-Ce, Lys64-Nf and the potential acceptor ⁄ donoratoms are in the range 108–120°, in line with thetetrahedral positioning of the hydrogen atoms attachedto Lys64-Nf.TetR(K64L135I138) forms an extended hydrophobiccontact with the ligand at the ‘backside’ of 4-ddma-atc. 4-ddma-atc directly contacts the mutated resi-dues 135 (Ser135Leu) and 138 (Ser138Ile) ofTetR(K64L135I138). Because of the hydrophobic natureof the Leu and Ile side chains and because of theirincreased size compared to the serine residues inACBFig. 3. (A) Close-up view on the ligand-binding site of 4-ddma-atc in complex with TetR(K64L135I138) and (B) tetracycline in complex with TetR[15] (PDB entry: 2VKE). The residues that differ between the two structures are underlined. The hydrogen-bonding network in which residue64, namely Lys64 in TetR(K64L135I138) (A) and His64 in TetR (B), participates, is indicated by dashed lines. Water molecules present in thetwo structures between residue 135 and the ‘backside’ of the ligand are depicted; all other water molecules are omitted. (C) Stereo repre-sentation of the superimposed structures of 4-ddma-atc bound to TetR(K64L135I138) (shown in yellow and magenta), atc bound to revTetR[14] (PDB entry: 2VKV; shown in cyan) and tetracycline bound to TetR [15] (PDB entry: 2VKE; shown in grey and blue). The stereo represen-tation shows that the binding position of the ligands and the orientations of the side chains lining the binding sites are highly conserved inthe different complexes. In wild-type TetR, this also extends to water molecules surrounding residue 135.M. A. Klieber et al. Structure of an engineered TetR-inducer pairFEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5615wild-type TetR, a number of water molecules thatbridge between the serines and the effector in othereffector complexes are absent in the 4-ddma-atcTetR(K64L135I138) complex (Fig. 3).Although the TetR(K64L135I138) crystals were soakedwith 4-ddma-atc without the addition of any extramagnesium, a partially occupied magnesium ion can beobserved at a position identical to that observed in otherTetR effector complexes. With the exception of theLys64 interaction and the extended hydrophobic inter-face introduced by Leu135 and Ile138, all other ligandprotein interactions are highly similar to those observedin other TetR-ligand complexes (see also below).Discussion4-ddma-atc binding to TetR(K64L135I138) comparedto tetracycline, atc and dox binding to TetRVarious crystal structures of TetR in complex with tet-racycline, dox and atc have already been solved tohigh resolution. Comparing these structures amongthemselves and to TetR(K64L135I138) promises to pro-vide insight into why TetR(K64L135I138) specificallyrecognizes 4-ddma-atc and why wild-type TetR isselectively induced by tetracycline, dox and atc andnot by 4-ddma-atc [9,17]. Upon superposition of thesestructures, it is immediately obvious that, in all thesestructures, the effector-binding sites are highly similarboth with respect to the positioning of the ligands andthe conformations of the residues lining the bindingsite (Table 2). In an initial comparison of the ligandpositions of tetracycline, dox and atc in TetR, theseligands superimpose with an average rmsd of 0.26 A˚for 27 common ligand atoms. In the case of the4-ddma-atc complex, the ligand appears to be slightlydisplaced with respect to the other ligands (4-ddma-atcbound to site I, average rmsd of 0.47 A˚compared tothe other ligands; Table 2). However the difference issmall and only slightly exceeds the estimates for thecoordinate errors in the different crystal structures.A major difference between TetR(K64L135I138)incomplex with 4-ddma-atc and all other effector TetRcomplexes is seen for the interaction between residue64 and the various effectors. As noted above, inTetR(K64L135I138) Lys64 is involved in a number ofspecific interactions with 4-ddma-atc and it appearsthat, in the wild-type TetR complexes, histidine is ableto participate in similar interactions because the posi-tion of atom Ne of His64 coincides almost exactly withthat of atom Nf of Lys64 (atom displacement of0.9 A˚; Fig. 3C). Compared to Lys64, however, a histi-dine residue can participate in fewer hydrogen bonds.In wild-type TetR, it appears that a putative hydrogenatom attached to Ne of His64 is poised to interactwith the oxygen atom attached to atom C3 present inall tetracycline derivatives. This oxygen is positioned inplane with the imidazole ring, and a linear almost idealhydrogen bond can be anticipated for this interaction.In comparison, an additional interaction often dis-cussed as being important for ligand binding [16],namely the interaction between Ne of His64 and theamide group attached to atom C2 of tetracycline,appears less favorable because the amide group is con-siderably displaced out of the plane of the imidazolering.The presence of a histidine at position 64 comparedto a lysine appears to affect a neighboring asparagineresidue. As noted above, Asn82 is hydrodrogen-bonded to Lys64 in TetR(K64L135I138). In all otherTetR structures with a histidine at position 64, nosuch interaction exists because the amide group ofAsn82 participates in a bidental interaction with alltetracycline derivatives possessing a 4-dimethyl-amino-group, namely with the nitrogen of the dimethyl-amino-group and the oxygen attached to atom C3(Figs 2 and 3).A further notable difference between 4-ddma-atc-bound TetR(K64L135I138) and other complexes com-prises the number of water molecules in the interfacebetween the ligand and the protein. By contrast toTetR(K64L135I138), where Ser135 and Ser138 arereplaced by Leu and Ile, water molecules are attachedto the serines in all other TetR structures and fill acleft between the ligand and the protein. Close inspec-tion of these water molecules shows that their posi-tions are largely conserved in the complexes formedbetween TetR and tetracycline, dox or atc (Fig. 3C).The specificity and selectivity of the 4-ddma-atcTetR(K64L135I138) interactionThe structural investigations reported in the presentstudy aimed to gain insight into the mechanismby which effector selectivity is switched inTetR(K64L135I138) compared to wild-type TetR. Previ-ously published data on induction efficiencies andinducer affinities of TetR(K64L135I138), as well as forall corresponding single and double mutant variants,are highly valuable for discussing the origins of speci-ficity and selectivity [8,9] (Table 3). When analyzingthe changes in the free binding energies of the mutantsfor the different ligands, it is apparent that thesechanges are often additive. For example, the sum ofthe changes in the binding affinities (DDG) observed inthe three single site mutants His64Lys, Ser135Leu andStructure of an engineered TetR-inducer pair M. A. Klieber et al.5616 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBSSer138Leu for the ligand dox corresponds almostexactly to the change observed in the triple mutantTetR(K64L135I138) (6.09 versus 5.86 kcalÆmol)1) com-pared to wild-type TetR (Table 3). In the case of theligands atc and 4-ddma-atc, only near additivity isachieved in the mutants (7.93 versus 6.61 kcalÆmol)1for atc and )5.71 versus )3.60 kcalÆmol)1for 4-ddma-atc binding).In many cases, it is also possible to formulate almostperfect thermodynamic cycles. For example, the freebinding energy difference observed for the binding ofthe ligands atc and 4-ddma-atc to wild-type TetR(DDG = 7.63 kcalÆmol)1) corresponds exactly to thesum of the changes observed for atc binding to themutant His64Lys (4.7 kcalÆmol)1), the difference inbinding energies for the ligands atc and 4-ddma-atc tothe same mutant (1.28 kcalÆmol)1) and the differ-ence in binding energies observed for 4-ddma-atc bind-ing to wild-type TetR and to the His64Lys mutant(1.65 kcalÆmol)1).Juxtaposition of the binding affinities to the induc-tion efficiencies suggests that free binding energies inexcess of approximately )12 kcalÆmol)1are requiredfor efficient induction (Table 3). If this is indeed thecase, then the switch in induction specificity inTetR(K64L135I138) can be explained as the result of anincrease in the free binding energies (DG) above)12 kcalÆmol)1for the ligands dox or atc and the con-comitant lowering of the free binding energy to)12.44 kcalÆmol)1for 4-ddma-atc. This appears tohold true for all the variants, with the exception of theligand dox in combination with the mutant Ser138Ile.Only very little induction is observed for this mutantwith the ligand dox [9] (Table 3), although the freebinding energy is lower than )12 kcalÆmol)1. It shouldbe noted, however, that the binding affinity data fromwhich the free energies have been calculated are notfree of errors. The standard deviations have beenestimated to be in the range 10–40% of the reportedvalues [9]. When translated to DG, this corresponds toapproximately 0.25 kcalÆmol)1(Table 3).The structures that we have determined in the pres-ent study and the comparison of these structures withpreviously solved crystal structures are in agreementwith the proposed additivity or near additivity of thefree binding energies. In all the structures, the effectormolecule binds at almost exactly the same position,and the introduction of mutations and ⁄ or changes inthe ligand does not lead to any notable changes in theside chain or backbone conformations of the residueslining the binding site. Although the structures of eachsingle and double mutant have not been solved, it isreasonable to assume that structure conservation alsoextends to these mutants. As a result of the structuralTable 3. Induction efficiencies and free binding energies of TetR and mutants for various tetracycline analogs. Data are compiled fromHenssler et al. [9].dox atc 4-ddma-atcInduction efficienciesTetR wild-type ++++a++++ )H64K )))S138I ) ++ )S135L ++++ +++ +H64K S138I )))S135L S138I +++ +++ )H64K S135L ++ +++ +++H64K S135L S138I [TetR(K64L135I138)] ))++Free binding energies (kcalÆmol)1)b,cTetR wild-type )15.31 )16.47 )8.84H64K )11.29 (+4.02) )11.77 (+4.70) )10.49 ()1.65)S138I )13.32 (+1.99) )12.82 (+3.65) )9.14 ()0.30)S135L )15.23 (+0.08) )16.89 ()0.42) )12.10 () 3.26)H64K S138I )9.34 (+5.97) )9.34 (+7.13) )10.63 ()1.79)S135L S138I )15.20 (+0.11) )13.63 (+2.84) )9.83 ()0.99)H64K S135L )12.01 (+3.30) )12.84 (+3.63) )12.75 ()3.91)H64K S135L S138I [TetR(K64L135I138)] )9.45 (+5.86) )9.86 (+6.61) )12.44 ()3.60)aInduction efficiencies: Less than 20% induction efficiency in a b-galactosidase reporter assay. +, ++, +++, ++++: between 20–40%,40–60%, 60–80%, and 80–100% induction efficiency, respectively.bFree binding energies derived from the experimentally determinedbinding affinities reported in Henssler et al.[9] and calculated according to DG=)RT lnK (t = 298.15 °K). In parentheses: DDG=DG(mutant)) DG(wild-type).cThe standard deviations of the affinities reported in Henssler et al. [9] have been estimated to be in the range 10–40%.Assuming a standard error propagation model with dDG=)RT (dK ⁄ K), this translates into 0.05–0.25 kcalÆmol)1as an error estimate for DG.M. A. Klieber et al. Structure of an engineered TetR-inducer pairFEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5617conservation and the near additivity of the DG values,it is possible to discuss the observed selectivity in lightof individual changes introduced in the binding site bythe various mutations.With respect to single changes, the most drastic dif-ferences in the free binding energies are observed forthe His64Lys mutation and for the removal of thedimethyl-amino-group attached to atom C4 of thetetracycline derivatives in wild-type TetR or in mutantsin which His64 is retained. Replacing the histidine bya lysine causes all tetracycline derivatives to be recog-nized with almost identical binding affinities (Table 3).This is the result of a drastic decrease in the affinitiesfor dox and atc, and a significant increase in affinity for4-ddma-atc. Because the free binding energies exceed)12 kcalÆmol)1, the His64Lys mutation is not inducedby any of the four ligands anymore. Accordingly, it isapparent that histidine is particular well suited to recog-nize the dimethyl-amino-group attached to atom C4.Inspection of the crystal structures shows that recogni-tion occurs indirectly and that Asn82 most likely makesa key contribution to this discrimination. In all com-plexes with ligands containing a dimethyl-amino-groupattached to C4, the amide group of Asn82 partici-pates in a bidental interaction with the tcs, namelywith the dimethyl-amino-group and the oxygen atomattached at position C3 (Fig. 3). The amide group ofAsn82 does not directly interact with His64 but onlyindirectly because both His64 and Asn82 interact withthe oxygen attached at C3. By contrast, when His64is replaced by a lysine residue, a direct interactionoccurs between Nf of Lys64 and the carbonyl groupof Asn82. Because all ligands are now recognizedwith similar affinities, it is apparent that the Lys64-Asn82 interaction disrupts any favorable interactionbetween the dimethyl-amino-group and Asn82. Thisdisruption could, for example, be caused by a flip inthe orientation of the amide group, which leads to anexchange of the positions of the nitrogen and oxygenatoms. As a result, a less favorable interaction couldoccur between the amide-NH2group and thedimethyl-amino-group of dox and atc that is assumedto be protonated in the TetR complex [16]. Theimportance of Asn82 for ligand discrimination is fur-ther highlighted by the fact that randomization ofposition 82 does not allow the identification of anyadditional residue with 4-ddma-atc specificity [9].Upon mutation of residue Ser135 to leucine, theaffinity of TetR for almost all ligands increases(Table 3). This holds true for all the variants intowhich this mutation is introduced. The only exceptionappears to be the binding of dox to the single mutantSer135Leu for which a small decrease in affinity canbe observed compared to wild-type TetR(DDG = +0.08 kcalÆmol)1). Introducing the mutationSer135Leu to any other variant also enhances thebinding affinity of dox. The amounts by which theaffinities increase differ for the various mutants andthe ligands. The most significant increase is observedfor 4-ddma-atc binding. Inspection of the crystal struc-tures suggests that this increase in affinity is a directconsequence of increased hydrophilic interactions andthe associated hydrophobic effect. As noted above, res-idue 135 interacts with the largely hydrophobic D ring.Whereas, in most crystal structures, a number ofhighly conserved water molecules bridges between theserine at position 135 and the tetracycline derivative,the water molecules are expelled from this interface inTetR(K64L135I138) in complex with 4-ddma-atc, in linewith an acquired entropic advantage for this complex.Because of the removal of the dimethyl-amino-group,4-ddma-atc represents the most hydrophobic com-pound of all the derivatives discussed in the presentstudy. Consequently, we expect the hydrophobic effectto be the largest for this tetracycline derivative andtherefore the largest increase in affinity should beobserved for 4-ddma-atc binding to any mutant inwhich Ser135 is mutated to leucine.The main role played by the Ser138Ile mutationappears to be that of a negative selection filter because,in most variants, the introduction of this mutationcauses a significant reduction in affinity for dox andatc and, at the same time, only slightly improves theaffinity for 4-ddma-atc (Table 3). This behavior can beeasily explained by considering that the dimethyl-amino group present in dox and atc, which interactswith Ser138 in wild-type TetR, is assumed to be posi-tively charged in the TetR-bound effector [16]. Becauseof the polar nature of its side chain, a serine is bettersuited to stabilize an adjacent positive charge than anisoleucine. Vice versa, the increased hydrophobicity ofthe isoleucine residue in the Ser138Ile mutants matchesthe increased hydrophobicity of 4-ddma-atc comparedto atc, possibly explaining the slight improvement inaffinity for 4-ddma-atc in variants containing this sub-stitution. However, it has been observed that spacerequirements are equally important for achieving selec-tivity at this position because only a serine to isoleu-cine substitution leads to the observed shift inspecificities and no other hydrophobic residue is toler-ated at this position [9]. The structure hints that anisoleucine fits perfectly between the protein and thetetracycline A and B rings.The results obtained in the present study showthat the observed specificity and selectivity inTetR(K64L135I138) for 4-ddma-atc can be explainedStructure of an engineered TetR-inducer pair M. A. Klieber et al.5618 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBSthrough a defined set of contributions. Whereas theHis64Lys mutation abolishes the selectivity present inwild-type TetR, and the Ser135Leu mutation improvesthe binding of all three effectors, the Ser138Ile muta-tion selectively disfavors effector molecules containinga dimethyl-amino group and, at the same time, onlyslightly improves 4-ddma-atc binding. The physico-chemical contributions of the individual residuesappear to be finely balanced and include geometricallyconstrained hydrogen-bonding networks, electrostaticinteractions and solvation and dissolvation effects.TetR(K64L135I138) was identified using extensive muta-tional screens and, in light of the structures presentedhere, it is obvious that it would have been difficult toconstruct such a highly specific repressor–inducer pairemploying a rational design and using structural infor-mation only. Conversely, however, the structures pre-sented here, when taken together with the previouslyavailable biochemical characterization, represent achallenging benchmark data set for testing and validat-ing computational models aimed at predicting anddesigning the specificity and selectivity of protein–ligand complexes.Experimental proceduresProtein expression, purification andcrystallizationFor protein production, Escherichia coli strain RB791 wastransformed with the plasmid pWH610, which encodes forthe triple mutant TetR(K64L135I138) [9]. In this construct,the chimeric TetR variant TetR(BD) [8], which contains theDNA-binding domain (residues 1–50) from TetR variant Band the effector-binding domain (residues 51–208) fromTetR variant D, is further modified through the introduc-tion of three single site mutations, namely His64 fi Lys,Ser135 fi Leu and Ser138 fi Ile. Transformed E. coli cellswere grown in LB medium at 28 °C and induced with1mm isopropyl thio-b-d-galactoside after an A600of 0.8was reached in the cell cultures. After further incubationfor 4 h, cells were harvested by centrifugation, and the pel-let dissolved in 30 mL of 20 mm sodium phosphate buffer(pH 6.8) containing 5 mm EDTA and 1 mm Pefabloc(Roche Diagnostics, Mannheim, Germany) before the cellwalls were disrupted by sonication. After centrifugationfor 1 h at 100 000 g, the supernatant was purified employ-ing a four-step chromatographic protocol. The supernatantwas first applied onto a weak cation-exchange column(SP-Sepharose FF; GE Healthcare Bio-Sciences, Uppsala,Sweden) and subsequently onto two strong anion-exchangecolumns (Resource Q and Mono Q; GE HealthcareBio-Sciences). Whereas, in the case of the cation-exchangecolumn, the buffer was identical to the buffer used duringthe sonication step, the two anion-exchange columns wereequilibrated with 50 mm NaCl, 20 mm Tris (pH 8.0). Theproteins were eluted from all three columns using a stan-dard NaCl gradient (20 mm to 1 m). As a final chromato-graphic step, a gel filtration chromatography run wasperformed (Superdex 75; GE Healthcare Bio-Sciences)using a buffer consisting of 200 mm NaCl and 50 m m Tris(pH 8.0).Initial crystallization conditions for TetR(K64L135I138)without 4-ddma-atc were identified using standard factorialscreens (Hampton Research, Aliso Viejo, CA, USA) and aprotein solution containing 10 mgÆmL)1protein in 200 mmNaCl and 50 mm Tris (pH 8.0) buffer. X-ray quality crys-tals were grown using the hanging drop method and mixing1 lL of protein solution with 1 lL of reservoir solution(1 m K2HPO4, 200 mm NaCl, 50 mm Tris, pH 8.0). Thedroplets were equilibrated over 1 mL of reservoir solutionuntil the crystals reached their final sizes of approximately150 · 70 · 50 lm after 10 days of incubation at 19 °C. Forcompleteness, it should be noted that, in addition, tetracy-cline at a concentration of 1 mm was present in the crystal-lization droplets, although it was known from biochemicalexperiments that TetR(K64L135I138) is not induced by tetra-cycline [9]. Careful inspection of the initial and final elec-tron density maps of the 4-ddma-atc-free TetR(K64L135I138)structure did not provide any hints for the density of tetra-cycline bound to the effector-binding site.Crystals of TetR(K64L135I138) with 4-ddma-atc wereobtained upon soaking the previously grown ligand-freecrystals with a saturated solution of the poorly soluble4-ddma-atc compound. The yellowish coloring of the crys-tals indicated the successful incorporation of the ligand.X-ray structure analysis and validationDiffraction data sets of TetR(K64L135I138) in complex with4-ddma-atc and without ligand were collected at BESSYSynchrotron at the beamlines of the Protein Structure Fac-tory of Free University Berlin. Before cryo-cooling, crystalswere soaked for a few seconds in a cryo-protectant solutionconsisting of 20% ethylenglycol and 80% reservoir solu-tion. All data sets were reduced using the software xds andscaled with xscale [18]. The crystal parameters and thedata collection statistics are reported in Table 1. The struc-tures were solved by molecular replacement using amore[19,20]. As a search model for the TetR(K64L135I138) struc-ture without ligand, the Protein Data Bank (PDB) entry2TCT was used [5,21]. The solution indicated the presenceof two monomers in the asymmetric unit, and after rigidbody refinement (8 to 3 A˚resolution) the patterson correla-tion coefficient increased to 48.9%. During refinement, themodel was manually inspected with the software o [22] andcoot [23] and automatically refined with refmac [24]and cns [25] until the refinement converged at an Rworkof 21.6% and an Rfreeof 26.2% at 2.1 A˚(Table 1). TheM. A. Klieber et al. Structure of an engineered TetR-inducer pairFEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5619[...]...Structure of an engineered TetR-inducer pair M A Klieber et al determination of the ligand-bound TetR(K64L135I138) structure started with the coordinates of the ligand-free structure Refinement of the complex converged at an Rwork of 20.7% and an Rfree value of 26.1% The ligand 4-ddma-atc and ligand restraints parameters were generated using corina [26] The individual models were validated using the software... prepared using pymol [28] Structure comparisons To investigate the origins of specificity and selectivity, the structures of TetR(K64L135I138) were superimposed onto previously solved TetR structures in complex with the ligands tetracycline, dox and atc using lsqkab [20] As a model for tetracycline-bound TetR, PDB entry 2VKE was used because the structure has been solved to a resolution ˚ of 1.6 A and the... to thank Madhumati Sevvana for help during the crystallographic refinement and Benedikt Schmid for preparation of the figures and critically reading of the manuscript We would also like to acknowledge the help of Uwe Muller from the Bessy ¨ synchrotron Berlin during data collection, as well as the anonymous reviewers for their valuable comments and discussions This work was supported through funding from... suite: programs for protein crystallography Acta Crystallogr D Biol Crystallogr 50, 760–763 21 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN & Bourne PE (2000) The Protein Data Bank Nucleic Acids Res 28, 235–242 22 Jones TA, Zou J-Y, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models... Hillen W & Hinrichs W (2000) The tetracycline repressor – a paradigm for a biological switch Angew Chem Int Ed Engl 39, 2042– 2052 5620 5 Kisker C, Hinrichs W, Tovar K, Hillen W & Saenger W (1995) The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance J Mol Biol 247, 260–280 6 Orth P, Schnappinger D, Hillen W, Saenger W & Hinrichs W (2000) Structural. .. & Hinrichs W (2008) Specific binding of divalent metal ions to tetracycline and to the Tet repressor ⁄ tetracycline complex J Biol Inorg Chem 13, 1097–1110 16 Aleksandrov A, Proft J, Hinrichs W & Simonson T (2007) Protonation patterns in tetracycline:tet repressor recognition: simulations and experiments Chembiochem 8, 675–685 17 Henssler EM, Bertram R, Wisshak S & Hillen W (2005) Tet repressor mutants... affect the geometry of the binding site [15] For TetR in complex with dox, PDB entry 2O7O was used [16] ˚ (1.9 A resolution) and, as a model for atc-bound TetR, the structure of revTetR in complex with atc was selected [14] ˚ (PDB entry: 2VKV; 1.7 A resolution) In the revTetR variant, Leu17 is mutated to glycine, and, again, this substitution does not appear to affect the atc-binding site [14] Acknowledgements... foundation and DFGSFB473 References 1 Berens C & Hillen W (2003) Gene regulation by tetracyclines Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes Eur J Biochem 270, 3109–3121 2 Gossen M & Bujard H (2002) Studying gene function in eukaryotes by conditional gene inactivation Annu Rev Genet 36, 153–173 3 Berens C & Hillen W (2004) Gene regulation by tetracyclines Genet... Gmeiner P & Hillen W (2004) Structure-based design of Tet repressor to optimize a new inducer specificity Biochemistry 43, 9512–9518 10 Hinrichs W, Kisker C, Duvel M, Muller A, Tovar K, Hillen W & Saenger W (1994) Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance Science 264, 418–420 11 Cui Q & Karplus M (2008) Allostery and cooperativity revisited Protein Sci... (2009) The structural basis of allosteric regulation in proteins FEBS Lett 583, 1692–1698 13 Reichheld SE & Davidson AR (2006) Two-way interdomain signal transduction in tetracycline repressor J Mol Biol 361, 382–389 14 Resch M, Striegl H, Henssler EM, Sevvana M, EgererSieber C, Schiltz E, Hillen W & Muller YA (2008) A protein functional leap: how a single mutation reverses the function of the transcription . Structural origins for selectivity and specificity in an engineered bacterial repressor–inducer pair Michael A. Klieber1, Oliver Scholz2,*, Susanne. TetR(K64L135I138) for 4-ddma-atc are the interactions between the ligand and the protein in the effector-binding site (Figs 1B and 2A). Of the two binding sites that can
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