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

Structural origins for selectivity and specificity in an engineered bacterial repressor–inducer pair Michael A. Klieber 1 , Oliver Scholz 2, *, Susanne Lochner 3 , Peter Gmeiner 3 , Wolfgang Hillen 2 and Yves A. Muller 1 1 Lehrstuhl fu ¨ r Biotechnik, Department of Biology, Friedrich-Alexander University, Erlangen-Nuremberg, Germany 2 Lehrstuhl fu ¨ r Mikrobiologie, Department of Biology, Friedrich-Alexander University, Erlangen-Nuremberg, Germany 3 Lehrstuhl fu ¨ r Pharmazeutische Chemie, Department of Chemistry and Pharmacy, Friedrich-Alexander University, Erlangen-Nuremberg, Germany Introduction The bacterial repression system consisting of the effec- tor molecule tetracycline, the tetracycline-inducible repressor protein tetracycline repressor (TetR) and the tet operator (tetO) has proven itself to comprise a valuable tool for studying gene expression not only in prokaryotes, but also in eukaryotes [1–3]. The repres- sor protein TetR, and first and foremost its ability to adopt different conformational states upon effector Keywords altered inducer selectivity; altered inducer specificity; bacterial transcription regulation; crystal structures; tetracycline repressor Correspondence Y. A. Muller, Lehrstuhl fu ¨ r Biotechnik, Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Im IZMP, Henkestrasse 91, D-91052 Erlangen, Germany Fax: +49 0 9131 8523080 Tel: +49 0 9131 8523081 E-mail: ymuller@biologie.uni-erlangen.de *Present address Department of Biochemistry, University of Zurich, Switzerland Database Structural data are available from the Protein Data Bank under the accession numbers 3FK6 for TetR(K 64 L 135 I 138 ) alone and 3FK7 for the 4-ddma-atc complex (Received 10 March 2009, revised 9 July 2009, accepted 31 July 2009) doi:10.1111/j.1742-4658.2009.07254.x The bacterial tetracycline transcription regulation system mediated by the tetracycline repressor (TetR) is widely used to study gene expression in prokaryotes and eukaryotes. To study multiple genes in parallel, a triple mutant TetR(K 64 L 135 I 138 ) has been engineered that is selectively induced by the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracy- cline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-type TetR. In the present study, we report the crystal structure of TetR(K 64 L 135 I 138 ) 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 binding data and previously reported TetR complexes allows for a dissection of the origins of selectivity and specificity. In all crystal structures solved to date, the ligand-binding position, as well as the positioning of the residues lining the binding site, is extremely well conserved, irrespective of the chemical nature of the ligand. Selective recognition of 4-ddma-atc is achieved through fine-tuned hydrogen-bonding constraints introduced by the His64 fi Lys substitution, as well as a combination of hydrophobic effect and the removal of unfavorable electrostatic interactions through the intro- duction of Leu135 and Ile138. Abbreviations atc, 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(K 64 L 135 I 138 ), TetR-BD triple mutant H64K, S135L and S138I. 5610 FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS binding, plays a key role in this system. In the absence of tetracycline, TetR binds with high affinity to the tet operator tetO and thereby blocks the transcription of any downstream genes. Upon binding of the inducer tetracycline, the repressor TetR switches conformations and dissociates from the operator DNA. As a result of numerous functional and structural studies, the atomic mechanism that underlies the functional switch in TetR is now understood in significant detail [4–7]. To be able to control the expression of several genes in parallel, TetR mutants have been isolated in elabo- rate screens that respond to novel synthetic tetracycline analogs [8,9]. One of these variants is the TetR triple mutant TetR(K 64 L 135 I 138 ) in which residues His64, Ser135 and Ser138 of TetR have been replaced by Lys, Leu and Ile, respectively [9]. This mutant is selectively induced by the synthetic inducer 4-de-dimethylamino- anhydrotetracycline (4-ddma-atc) and slightly by atc, but no longer by tetracycline. To better understand the switch in selectivity and the acquired novel specificity of TetR(K 64 L 135 I 138 ) for 4-ddma-atc, we solved the crystal structure of TetR(K 64 L 135 I 138 ) in the presence and absence of 4-ddma-atc at resolutions of 2.06 and 2.1 A ˚ , respectively. We show that the effects of the three mutations on ligand binding can be generally thermodynamically dissected into individual contribu- tions and that they originate from a favorable inter- play of different physico-chemical properties, such as solvation effects, constrained hydrogen-bonding geom- etries and electrostatic discrimination. Results Comparison of the 4-ddma-atc-bound and free overall TetR(K 64 L 135 I 138 ) structure As for all TetRs, TetR(K 64 L 135 I 138 ) forms a dimer and, with respect to the monomer architecture, each chain contains an N-terminal DNA-binding domain (residues 1–48) and a C-terminal effector-binding domain (residues 49–205) [10] (Fig. 1A). The latter also comprises the dimer interface. The two effector- binding sites present in the dimer are identical and, because the binding sites are located within the protein interface, each binding site is lined by residues from both monomers. The overall structures of ligand-free TetR(K 64 L 135 I 138 ) and of TetR(K 64 L 135 I 138 ) in complex with 4-ddma-atc are very similar (for the chemical structure of 4-ddma-atc and related TetR ligands, see Fig. 2). The two crystals that have been used for structure determination are highly isomorphous and only small deviations occur in the cell axes (Table 1). They each contain a complete dimer in the asymmetric unit. Almost no differences exist between the monomers in each crystal and the monomers ⁄ dimers between crys- tals. The main chain atoms of the two monomers in each crystal can be superimposed with rmsd of 0.92 and 1.34 A ˚ for the 4-ddma-atc-bound and effector-free TetR(K 64 L 135 I 138 ) structures, respectively. When direc- tly comparing the structure of 4-ddma-atc-bound TetR (K 64 L 135 I 138 ) with that of effector-free TetR(K 64 L 135 I 138 ), it is obvious that ligand binding does not induce any considerable changes in the tertiary structures. The four possible pairwise cross-superpositions between crystals yield an rmsd in the range 0.52–1.23 A ˚ for a total of 770 common main chain atoms. This shows that the differences between the ligand-bound and ligand-free structures are not larger than the differ- ences observed between the two monomers present in each crystal. To some extent, this is not too surprising because the crystal with 4-ddma-atc bound was obtained by soaking a ligand-free crystal with the ligand. However, when considering the molecular mechanism by which TetR exerts its function, then the structural similarity might be considered unexpected. A central function of TetR is its ability to adopt different conformations. According to the conforma- tional switch model, TetR exists in two conforma- tions. In the ligand-free structure, the DNA-binding heads are oriented such that TetR can readily bind to the operator DNA, whereas, in effector-bound TetR, the separation of the DNA-binding domain is changed, such that TetR can no longer recognize the tetO DNA sequence (Fig. 1A) [4]. However, as noted above, the domain orientations in the 4-ddma-atc- bound TetR(K 64 L 135 I 138 ) and the ligand-free TetR (K 64 L 135 I 138 ) structure are very similar and resemble that of induced TetR more closely than that of indu- cer-free TetR (data not shown). Moreover, the main chain of loop segment 100–105 that switches con- formations upon effector binding is in an identical conformation in both structures and resembles that observed for induced TetR (Fig. 1C). In this confor- mation, segment 100–105 moves towards the ligand and thereby enables residues from the segment to participate in ligand binding. The observation that effector-free TetR(K 64 L 135 I 138 ) adopts an induced-like conformation must appear unexpected. However, it can easily be rationalized if alternative models are considered (e.g. the population shift model) as explanations for the allosteric behavior of TetR [11,12]. According to this model, effector-free and DNA-free TetR is able to adopt a variety of divers and freely inter-converting conformations. Among these, there are also conformations that can M. A. Klieber et al. Structure of an engineered TetR-inducer pair FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5611 readily interact with DNA or that resemble the effec- tor-bound conformation as, for example, observed in the present study in the case of the crystal structure of the effector-free TetR(K 64 L 135 I 138 ). In this model, induction can be explained by a shift of the population towards a single DNA-binding incompetent conforma- tion. Indications that such a population shift model might apply to TetR have recently started to emerge [13,14]. The effector-binding site of TetR(K 64 L 135 I 138 )in the presence and absence of 4-ddma-atc Particularly interesting with respect to the observed specificity and selectivity of TetR(K 64 L 135 I 138 ) 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 be observed independently in the 4-ddma-atc-complex structure, A C B Fig. 1. Crystal structure of TetR(K 64 L 135 I 138 ) in complex with 4-ddma-atc. (A) Ca-representation of the TetR(K 64 L 135 I 138 ) 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 in TetR, which alters the orientation and separation of the DNA-binding domains (indicated by two-headed arrows), so that TetR no longer binds to the operator DNA. (B) Schematic representation of the interactions between 4-ddma-atc and TetR(K 64 L 135 I 138 ). (C) Stereo represen- tation of the structure of TetR(K 64 L 135 I 138 ) in complex with 4-ddma-atc (shown in magenta and yellow) superimposed onto the effector-free TetR(K 64 L 135 I 138 ) structure (shown in grey). Of the two binding sites present in the crystal structure, binding site I (Table 2) is shown. The binding sites are highly similar in the presence and absence of 4-ddma-atc. The loop segment 100–105 that switches conformations in other ligand-free and ligand-bound TetR structures is only slightly displaced in ligand-free TetR(K 64 L 135 I 138 ) compared to the ligand-bound TetR structure. 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 FEBS both binding sites show the density for the ligand in the initial difference-fourier electron density maps, albeit to a different extent. The ligand is well defined in binding site I, but only poor density has been observed for the ligand in binding site II. To account for this observation, the occupancy of the ligand in binding site II was estimated at 50% and that in bind- ing site I at 100%. When using these values during the refinement, the temperature factors of the ligands refine to values similar to those of the surrounding res- idues, hinting that the estimated occupancies correctly reflect those in the crystal. Inspection of the crystal packing yields a plausible explanation for the differ- ences in occupancies. In the crystal, the accessibility to site II is impaired by the packing of neighboring mole- cules, whereas site I appears to be readily accessible through the solvent channels in the crystal. When superimposing the two ligand-binding sites on the basis of the coordinates of 93 residues surrounding the ligands, it is obvious that the ligand is slightly shifted in site II (Table 2). This shift is not only appar- ent with respect to 4-ddma-atc bound to site I, but also compared to various other ligand-TetR complexes (Table 2). The two 4-ddma-atc molecules differ by an rmsd of approximately 1 A ˚ , whereas the deviations between 4-ddma-atc bound to site I and tetracycline and 6-deoxy-5-hydoxy-tetracycline (dox) bound to TetR [15,16], as well as atc bound to revTetR [14], are in the range 0.4–0.5 A ˚ when considering 27 common ligand atoms. We suspect that the positional shift of the 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 errors during the refinement of the atomic positions and hence a less accurate ligand positioning in site II. Accordingly, the description of the binding of the ligand in the present study is restricted to 4-ddma-atc binding to site I in TetR(K 64 L 135 I 138 ). 4-ddma-atc binding leads to only minor rearrange- ments in the TetR(K 64 L 135 I 138 )-binding site (Fig. 1C). Among the most notable changes are a slight shift of the entire loop segment 100–105 in the direction of the ligand, the presence of two alternative side chain confor- mations for Asn82 in the 4-ddma-atc-bound structure versus a single conformation in ligand-free TetR(K 64 L 135 I 138 ) and, finally, the occurrence of a slightly different rotamer for Ile138 (i.e. different posi- tioning of atom Cd) in the ligand-free and ligand-bound structure. Overall, when considering both the main chain fold and the conformations of the side chains, the structures of ligand-free TetR(K 64 L 135 I 138 ) and 4-ddma- atc-bound TetR(K 64 L 135 I 138 ) are very similar. This simi- larity also extends to a number of water molecules that occupy identical positions in the two structures. Specific interactions between 4-ddma-atc and TetR(K 64 L 135 I 138 ) TetR(K 64 L 135 I 138 ) binds 4-ddma-atc via a number of specific interactions (Fig. 3A). One of the most notable ones involves Lys64. Atom Nf of Lys64 interacts with two oxygen atoms of 4-ddma-atc, namely of the amide group attached to atom C2 and the OH group A B C D Fig. 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 pair FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5613 attached 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 chain carbonyl oxygen atom of Tyr66. It is not possible to predict the strength of the interaction between Lys64 and 4-ddma-atc based on structural data only. For Table 1. Data collection and refinement statistics. Triple mutant TetR(K 64 L 135 I 138 ) Triple mutant TetR(K 64 L 135 I 138 ) in complex with 4-ddma-atc Data collection statistics Space group C2 C2 Unit cell parameters a, b, c (A ˚ ) 126.35, 58.06, 62.62 130.22, 59.38, 63.78 b (°) 96.58 97.85 Molecules per asymmetric unit 2 2 Resolution range (A ˚ ) a 15–2.1 (2.25–2.1) 20–2.06 (2.19–2.06) Unique reflections 24749 29597 Redundancy 3.3 3.7 Completeness (%) 93.3 (95.9) 98.6 (91.8) R merge (%) 6.4 (37.1) 6.0 (39.8) Wilson B-factor (A ˚ 2 ) 15.1 26.5 Refinement statistics Number of protein atoms, solvent molecules and ligand atoms 3095, 116, 0 3180, 225, 58 R work (%) 21.6 20.3 R free (%) 26.2 26.1 rmsd bond lengths (A ˚ ) 0.005 0.011 rmsd bond angles (°) 1.069 1.175 rmsd B-factors bonded atoms: main chain, side chains (A ˚ 2 ) 1.51, 2.27 1.24, 2.13 Percentage of residues in most favored regions, additional allowed, generously allowed and disallowed regions of the Ramachandran plot b 95.1, 4.9, 0.0, 0.0 95.5, 4.2, 0.3, 0.0 Average B-factor (A ˚ 2 ) 43.26 36.79 a Values in parentheses refer to the highest resolution shell. b According to PROCHECK [27]. Table 2. Superposition of the ligand-binding sites and ligand positions in selected TetR complexes. No ligand TetR (K 64 L 135 I 138 )I a No ligand TetR (K 64 L 135 I 138 )II 4-ddma-atc TetR (K 64 L 135 I 138 )I a 4-ddma-atc TetR (K 64 L 135 I 138 )II atc revTetR (PDB code: 2VKV) tc TetR(D) (PDB code: 2VKE) dox TetR(D) (PDB code: 2O7O) No ligand TetR(K 64 L 135 I 138 )I – 0.905, 1.396 b 0.532, 0.843 0.830, 1.360 0.854, 1.386 0.851, 1.385 0.870, 1.356 No ligand TetR(K 64 L 135 I 138 )II – – 0.788, 1.237 0.430, 0.771 0.631 1.168 0.576, 1.170 0.603, 1.102 4-ddma-atc TetR(K 64 L 135 I 138 )I – – – 0.633, 1.128 0.656, 1.155 0.615, 1.054 0.632, 1.047 4-ddma-atc TetR(K 64 L 135 I 138 )II – – 1.048 c – 0.489, 1.062 0.432, 1.065 0.415, 0.999 Atc revTetR – – 0.431 0.896 – 0.413, 0.983 0.437, 0.918 tc TetR(BD) – – 0.456 1.024 0.270 – 0.216, 0.732 Dox TetR(BD) – – 0.523 0.878 0.259 0.265 – a Because the crystals contain two molecules in the asymmetric unit, two separate binding sites (I and II) are present in each of the two TetR(K 64 L 135 I 138 ) crystal structures. b Above the diagonal the rmsd (A ˚ ) between structures of a selection of 93 residues surrounding the ligand-binding site is reported (first number, rmsd obtained upon superposition of all main chain atoms of the selection; second number, superposition of all atoms). c Below 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 FEBS example, the structure does not allow a distinction of whether the side chain of Lys64 is protonated or not. Uncertainties also arise with respect to the correct orientation of the amide group attached to atom C2 of 4-ddma-atc, as well as the amide group of amino acid Asn82, because the amide oxygen and nitrogen atoms are indistinguishable at the resolutions of the solved crystal structures. Because atom Nf of Lys64 can only participate in three hydrogen bonds, it should be noted that, of the four potential hydrogen bond acceptors ⁄ donors, atom O from the amide group of 4-ddma-atc is the furthest apart (3.1 A ˚ versus 2.5–2.8 A ˚ for the other hydrogen bond acceptors ⁄ donors). However, all four potential hydrogen bond partners are geometri- cally quite favorably placed. In all four cases, almost ideal linear hydrogen bond geometries can be antici- pated because the angle formed between atoms Lys64-Ce, Lys64-Nf and the potential acceptor ⁄ donor atoms are in the range 108–120°, in line with the tetrahedral positioning of the hydrogen atoms attached to Lys64-Nf. TetR(K 64 L 135 I 138 ) forms an extended hydrophobic contact with the ligand at the ‘backside’ of 4-ddma- atc. 4-ddma-atc directly contacts the mutated resi- dues 135 (Ser135Leu) and 138 (Ser138Ile) of TetR(K 64 L 135 I 138 ). Because of the hydrophobic nature of the Leu and Ile side chains and because of their increased size compared to the serine residues in A C B Fig. 3. (A) Close-up view on the ligand-binding site of 4-ddma-atc in complex with TetR(K 64 L 135 I 138 ) 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 residue 64, namely Lys64 in TetR(K 64 L 135 I 138 ) (A) and His64 in TetR (B), participates, is indicated by dashed lines. Water molecules present in the two 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(K 64 L 135 I 138 ) (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 in the 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 pair FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5615 wild-type TetR, a number of water molecules that bridge between the serines and the effector in other effector complexes are absent in the 4-ddma-atc TetR(K 64 L 135 I 138 ) complex (Fig. 3). Although the TetR(K 64 L 135 I 138 ) crystals were soaked with 4-ddma-atc without the addition of any extra magnesium, a partially occupied magnesium ion can be observed at a position identical to that observed in other TetR effector complexes. With the exception of the Lys64 interaction and the extended hydrophobic inter- face introduced by Leu135 and Ile138, all other ligand protein interactions are highly similar to those observed in other TetR-ligand complexes (see also below). Discussion 4-ddma-atc binding to TetR(K 64 L 135 I 138 ) compared to tetracycline, atc and dox binding to TetR Various crystal structures of TetR in complex with tet- racycline, dox and atc have already been solved to high resolution. Comparing these structures among themselves and to TetR(K 64 L 135 I 138 ) promises to pro- vide insight into why TetR(K 64 L 135 I 138 ) specifically recognizes 4-ddma-atc and why wild-type TetR is selectively induced by tetracycline, dox and atc and not by 4-ddma-atc [9,17]. Upon superposition of these structures, it is immediately obvious that, in all these structures, the effector-binding sites are highly similar both with respect to the positioning of the ligands and the conformations of the residues lining the binding site (Table 2). In an initial comparison of the ligand positions of tetracycline, dox and atc in TetR, these ligands superimpose with an average rmsd of 0.26 A ˚ for 27 common ligand atoms. In the case of the 4-ddma-atc complex, the ligand appears to be slightly displaced with respect to the other ligands (4-ddma-atc bound to site I, average rmsd of 0.47 A ˚ compared to the other ligands; Table 2). However the difference is small and only slightly exceeds the estimates for the coordinate errors in the different crystal structures. A major difference between TetR(K 64 L 135 I 138 )in complex with 4-ddma-atc and all other effector TetR complexes is seen for the interaction between residue 64 and the various effectors. As noted above, in TetR(K 64 L 135 I 138 ) Lys64 is involved in a number of specific interactions with 4-ddma-atc and it appears that, in the wild-type TetR complexes, histidine is able to participate in similar interactions because the posi- tion of atom Ne of His64 coincides almost exactly with that of atom Nf of Lys64 (atom displacement of 0.9 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 hydrogen atom attached to Ne of His64 is poised to interact with the oxygen atom attached to atom C3 present in all tetracycline derivatives. This oxygen is positioned in plane with the imidazole ring, and a linear almost ideal hydrogen 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 the amide group attached to atom C2 of tetracycline, appears less favorable because the amide group is con- siderably displaced out of the plane of the imidazole ring. The presence of a histidine at position 64 compared to a lysine appears to affect a neighboring asparagine residue. As noted above, Asn82 is hydrodrogen- bonded to Lys64 in TetR(K 64 L 135 I 138 ). In all other TetR structures with a histidine at position 64, no such interaction exists because the amide group of Asn82 participates in a bidental interaction with all tetracycline 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(K 64 L 135 I 138 ) and other complexes com- prises the number of water molecules in the interface between the ligand and the protein. By contrast to TetR(K 64 L 135 I 138 ), where Ser135 and Ser138 are replaced by Leu and Ile, water molecules are attached to the serines in all other TetR structures and fill a cleft between the ligand and the protein. Close inspec- tion of these water molecules shows that their posi- tions are largely conserved in the complexes formed between TetR and tetracycline, dox or atc (Fig. 3C). The specificity and selectivity of the 4-ddma-atc TetR(K 64 L 135 I 138 ) interaction The structural investigations reported in the present study aimed to gain insight into the mechanism by which effector selectivity is switched in TetR(K 64 L 135 I 138 ) compared to wild-type TetR. Previ- ously published data on induction efficiencies and inducer affinities of TetR(K 64 L 135 I 138 ), as well as for all corresponding single and double mutant variants, are highly valuable for discussing the origins of speci- ficity and selectivity [8,9] (Table 3). When analyzing the changes in the free binding energies of the mutants for the different ligands, it is apparent that these changes are often additive. For example, the sum of the changes in the binding affinities (DDG) observed in the three single site mutants His64Lys, Ser135Leu and Structure 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 FEBS Ser138Leu for the ligand dox corresponds almost exactly to the change observed in the triple mutant TetR(K 64 L 135 I 138 ) (6.09 versus 5.86 kcalÆmol )1 ) com- pared to wild-type TetR (Table 3). In the case of the ligands atc and 4-ddma-atc, only near additivity is achieved in the mutants (7.93 versus 6.61 kcalÆmol )1 for atc and )5.71 versus )3.60 kcalÆmol )1 for 4-ddma- atc binding). In many cases, it is also possible to formulate almost perfect thermodynamic cycles. For example, the free binding energy difference observed for the binding of the ligands atc and 4-ddma-atc to wild-type TetR (DDG = 7.63 kcalÆmol )1 ) corresponds exactly to the sum of the changes observed for atc binding to the mutant His64Lys (4.7 kcalÆmol )1 ), the difference in binding energies for the ligands atc and 4-ddma-atc to the 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 in excess of approximately )12 kcalÆmol )1 are required for efficient induction (Table 3). If this is indeed the case, then the switch in induction specificity in TetR(K 64 L 135 I 138 ) can be explained as the result of an increase in the free binding energies (DG) above )12 kcalÆmol )1 for the ligands dox or atc and the con- comitant lowering of the free binding energy to )12.44 kcalÆmol )1 for 4-ddma-atc. This appears to hold true for all the variants, with the exception of the ligand dox in combination with the mutant Ser138Ile. Only very little induction is observed for this mutant with the ligand dox [9] (Table 3), although the free binding energy is lower than )12 kcalÆmol )1 . It should be noted, however, that the binding affinity data from which the free energies have been calculated are not free of errors. The standard deviations have been estimated to be in the range 10–40% of the reported values [9]. When translated to DG, this corresponds to approximately 0.25 kcalÆmol )1 (Table 3). The structures that we have determined in the pres- ent study and the comparison of these structures with previously solved crystal structures are in agreement with the proposed additivity or near additivity of the free binding energies. In all the structures, the effector molecule binds at almost exactly the same position, and the introduction of mutations and ⁄ or changes in the ligand does not lead to any notable changes in the side chain or backbone conformations of the residues lining the binding site. Although the structures of each single and double mutant have not been solved, it is reasonable to assume that structure conservation also extends to these mutants. As a result of the structural Table 3. Induction efficiencies and free binding energies of TetR and mutants for various tetracycline analogs. Data are compiled from Henssler et al. [9]. dox atc 4-ddma-atc Induction efficiencies TetR wild-type ++++ a ++++ ) H64K ))) S138I ) ++ ) S135L ++++ +++ + H64K S138I ))) S135L S138I +++ +++ ) H64K S135L ++ +++ +++ H64K S135L S138I [TetR(K 64 L 135 I 138 )] ))++ Free binding energies (kcalÆmol )1 ) b,c TetR wild-type )15.31 )16.47 )8.84 H64K )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(K 64 L 135 I 138 )] )9.45 (+5.86) )9.86 (+6.61) )12.44 ()3.60) a Induction 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. b Free binding energies derived from the experimentally determined binding 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). c The 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 )1 as an error estimate for DG. M. A. Klieber et al. Structure of an engineered TetR-inducer pair FEBS Journal 276 (2009) 5610–5621 ª 2009 The Authors Journal compilation ª 2009 FEBS 5617 conservation and the near additivity of the DG values, it is possible to discuss the observed selectivity in light of individual changes introduced in the binding site by the various mutations. With respect to single changes, the most drastic dif- ferences in the free binding energies are observed for the His64Lys mutation and for the removal of the dimethyl-amino-group attached to atom C4 of the tetracycline derivatives in wild-type TetR or in mutants in which His64 is retained. Replacing the histidine by a 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 affinities for dox and atc, and a significant increase in affinity for 4-ddma-atc. Because the free binding energies exceed )12 kcalÆmol )1 , the His64Lys mutation is not induced by any of the four ligands anymore. Accordingly, it is apparent 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 makes a key contribution to this discrimination. In all com- plexes with ligands containing a dimethyl-amino-group attached to C4, the amide group of Asn82 partici- pates in a bidental interaction with the tcs, namely with the dimethyl-amino-group and the oxygen atom attached at position C3 (Fig. 3). The amide group of Asn82 does not directly interact with His64 but only indirectly because both His64 and Asn82 interact with the oxygen attached at C3. By contrast, when His64 is replaced by a lysine residue, a direct interaction occurs between Nf of Lys64 and the carbonyl group of Asn82. Because all ligands are now recognized with similar affinities, it is apparent that the Lys64- Asn82 interaction disrupts any favorable interaction between the dimethyl-amino-group and Asn82. This disruption could, for example, be caused by a flip in the orientation of the amide group, which leads to an exchange of the positions of the nitrogen and oxygen atoms. As a result, a less favorable interaction could occur between the amide-NH 2 group and the dimethyl-amino-group of dox and atc that is assumed to be protonated in the TetR complex [16]. The importance of Asn82 for ligand discrimination is fur- ther highlighted by the fact that randomization of position 82 does not allow the identification of any additional residue with 4-ddma-atc specificity [9]. Upon mutation of residue Ser135 to leucine, the affinity of TetR for almost all ligands increases (Table 3). This holds true for all the variants into which this mutation is introduced. The only exception appears to be the binding of dox to the single mutant Ser135Leu for which a small decrease in affinity can be observed compared to wild-type TetR (DDG = +0.08 kcalÆmol )1 ). Introducing the mutation Ser135Leu to any other variant also enhances the binding affinity of dox. The amounts by which the affinities increase differ for the various mutants and the ligands. The most significant increase is observed for 4-ddma-atc binding. Inspection of the crystal struc- tures suggests that this increase in affinity is a direct consequence of increased hydrophilic interactions and the associated hydrophobic effect. As noted above, res- idue 135 interacts with the largely hydrophobic D ring. Whereas, in most crystal structures, a number of highly conserved water molecules bridges between the serine at position 135 and the tetracycline derivative, the water molecules are expelled from this interface in TetR(K 64 L 135 I 138 ) in complex with 4-ddma-atc, in line with 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 present study. Consequently, we expect the hydrophobic effect to be the largest for this tetracycline derivative and therefore the largest increase in affinity should be observed for 4-ddma-atc binding to any mutant in which Ser135 is mutated to leucine. The main role played by the Ser138Ile mutation appears to be that of a negative selection filter because, in most variants, the introduction of this mutation causes a significant reduction in affinity for dox and atc and, at the same time, only slightly improves the affinity for 4-ddma-atc (Table 3). This behavior can be easily explained by considering that the dimethyl- amino group present in dox and atc, which interacts with Ser138 in wild-type TetR, is assumed to be posi- tively charged in the TetR-bound effector [16]. Because of the polar nature of its side chain, a serine is better suited to stabilize an adjacent positive charge than an isoleucine. Vice versa, the increased hydrophobicity of the isoleucine residue in the Ser138Ile mutants matches the increased hydrophobicity of 4-ddma-atc compared to atc, possibly explaining the slight improvement in affinity for 4-ddma-atc in variants containing this sub- stitution. However, it has been observed that space requirements are equally important for achieving selec- tivity at this position because only a serine to isoleu- cine substitution leads to the observed shift in specificities and no other hydrophobic residue is toler- ated at this position [9]. The structure hints that an isoleucine fits perfectly between the protein and the tetracycline A and B rings. The results obtained in the present study show that the observed specificity and selectivity in TetR(K 64 L 135 I 138 ) for 4-ddma-atc can be explained Structure 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 FEBS through a defined set of contributions. Whereas the His64Lys mutation abolishes the selectivity present in wild-type TetR, and the Ser135Leu mutation improves the binding of all three effectors, the Ser138Ile muta- tion selectively disfavors effector molecules containing a dimethyl-amino group and, at the same time, only slightly improves 4-ddma-atc binding. The physico- chemical contributions of the individual residues appear to be finely balanced and include geometrically constrained hydrogen-bonding networks, electrostatic interactions and solvation and dissolvation effects. TetR(K 64 L 135 I 138 ) was identified using extensive muta- tional screens and, in light of the structures presented here, it is obvious that it would have been difficult to construct such a highly specific repressor–inducer pair employing a rational design and using structural infor- mation only. Conversely, however, the structures pre- sented here, when taken together with the previously available biochemical characterization, represent a challenging benchmark data set for testing and validat- ing computational models aimed at predicting and designing the specificity and selectivity of protein– ligand complexes. Experimental procedures Protein expression, purification and crystallization For protein production, Escherichia coli strain RB791 was transformed with the plasmid pWH610, which encodes for the triple mutant TetR(K 64 L 135 I 138 ) [9]. In this construct, the chimeric TetR variant TetR(BD) [8], which contains the DNA-binding domain (residues 1–50) from TetR variant B and the effector-binding domain (residues 51–208) from TetR 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 cells were grown in LB medium at 28 °C and induced with 1mm isopropyl thio-b-d-galactoside after an A 600 of 0.8 was reached in the cell cultures. After further incubation for 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 cell walls were disrupted by sonication. After centrifugation for 1 h at 100 000 g, the supernatant was purified employ- ing a four-step chromatographic protocol. The supernatant was first applied onto a weak cation-exchange column (SP-Sepharose FF; GE Healthcare Bio-Sciences, Uppsala, Sweden) and subsequently onto two strong anion-exchange columns (Resource Q and Mono Q; GE Healthcare Bio-Sciences). Whereas, in the case of the cation-exchange column, the buffer was identical to the buffer used during the sonication step, the two anion-exchange columns were equilibrated with 50 mm NaCl, 20 mm Tris (pH 8.0). The proteins 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 was performed (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(K 64 L 135 I 138 ) without 4-ddma-atc were identified using standard factorial screens (Hampton Research, Aliso Viejo, CA, USA) and a protein solution containing 10 mgÆmL )1 protein in 200 mm NaCl and 50 mm Tris (pH 8.0) buffer. X-ray quality crys- tals were grown using the hanging drop method and mixing 1 lL of protein solution with 1 lL of reservoir solution (1 m K 2 HPO 4 , 200 mm NaCl, 50 mm Tris, pH 8.0). The droplets were equilibrated over 1 mL of reservoir solution until the crystals reached their final sizes of approximately 150 · 70 · 50 lm after 10 days of incubation at 19 °C. For completeness, 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 biochemical experiments that TetR(K 64 L 135 I 138 ) 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(K 64 L 135 I 138 ) structure did not provide any hints for the density of tetra- cycline bound to the effector-binding site. Crystals of TetR(K 64 L 135 I 138 ) with 4-ddma-atc were obtained upon soaking the previously grown ligand-free crystals with a saturated solution of the poorly soluble 4-ddma-atc compound. The yellowish coloring of the crys- tals indicated the successful incorporation of the ligand. X-ray structure analysis and validation Diffraction data sets of TetR(K 64 L 135 I 138 ) in complex with 4-ddma-atc and without ligand were collected at BESSY Synchrotron at the beamlines of the Protein Structure Fac- tory of Free University Berlin. Before cryo-cooling, crystals were soaked for a few seconds in a cryo-protectant solution consisting of 20% ethylenglycol and 80% reservoir solu- tion. All data sets were reduced using the software xds and scaled with xscale [18]. The crystal parameters and the data 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(K 64 L 135 I 138 ) struc- ture without ligand, the Protein Data Bank (PDB) entry 2TCT was used [5,21]. The solution indicated the presence of two monomers in the asymmetric unit, and after rigid body refinement (8 to 3 A ˚ resolution) the patterson correla- tion coefficient increased to 48.9%. During refinement, the model was manually inspected with the software o [22] and coot [23] and automatically refined with refmac [24] and cns [25] until the refinement converged at an R work of 21.6% and an R free of 26.2% at 2.1 A ˚ (Table 1). The M. A. Klieber et al. Structure of an engineered TetR-inducer pair FEBS 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. Klieber 1 , Oliver Scholz 2, *, Susanne. TetR(K 64 L 135 I 138 ) 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