Computational Design of a Chain-Specific Tetracycline Repressor Heterodimer

doi:10.1016/j.jmb.2010.07.055

J. Mol. Biol. (2010) 403, 371–385 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Computational Design of a Chain-Specific Tetracycline Repressor Heterodimer Martin T. Stiebritz, Stefanie Wengrzik, Doris L. Klein, Jan Paul Richter, Anna Srebrzynski, Sigrid Weiler and Yves A. Muller⁎ Lehrstuhl für Biotechnik, Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Im IZMP, Henkestr. 91, D-91052 Erlangen, Germany Received 3 June 2010; received in revised form 14 July 2010; accepted 27 July 2010 Available online 15 September 2010 Edited by F. Schmid Keywords: computational de novo design; crystal structure; bacterial repressor; protein–protein interaction specificity; interface remodeling

The specificity and selectivity of protein–protein interactions are of central importance for many biological processes, including signal transduction and transcription control. We used the in-house side-chain packing program MUMBO to computationally design a chain-specific heterodimeric variant of the bacterial transcription regulator tetracycline repressor (TetR), called T-AAB. Our goal was to engineer two different TetR chain variants, A and B, that no longer interact as AA or BB homodimers but selectively recombine to form heterodimers. Although 56 residues from each chain contribute to a dimer interface as large as 2200 Å2 in wild-type TetR, the substitution of only three residues in one chain and two residues in a second chain sufficed for generating specificity in a T-AAB heterodimer variant. The design was corroborated in vivo by a cell-based transcription assay, and in vitro by CD spectroscopy and X-ray crystallography. Crystal structure analyses showed that while selectivity in the B chain is achieved entirely through van der Waals repulsion, the best selectivity in the A chain is obtained for the variant with the lowest number of atoms in the interface, thus possibly leading to underpacking of the dimer interface. This results in a marked decrease in thermal stability and a drastic reduction in the solubility of the T-AAAA homodimer in comparison to the designed T-AAB heterodimer variant. © 2010 Elsevier Ltd. All rights reserved.

Introduction Knowledge validation through reengineering has proven extremely valuable in many disciplines such as physics and chemistry but has so far remained scarce in structural biology. Reengineering is hampered by a fundamental property of proteins, namely the low overall stability of their threedimensional structure in comparison to the unfolded polypeptide chain.1 As a consequence, small sequence changes can easily abrogate the structural integrity— *Corresponding author. E-mail address: [email protected]. Abbreviations used: TetR, tetracycline repressor; T-WT, wild-type TetR; PDB, Protein Data Bank.

and hence the function—of a protein. In recent years, computational protein design algorithms pursuing an ‘inverted folding’ approach2,3 yielded novel opportunities for the successful reengineering of proteins. Given a specified protein backbone conformation, these algorithms aim at identifying the energetically most favorable sequence of amino acids that will not only produce a stably folded protein but also provide the protein with novel biological activities. Pathbreaking successes in computational protein design include the redesign of a zinc finger domain,4 the design of oligomerization-specific left-handed and right-handed helix bundles,5 the design of a protein with a fold topology not observed so far in nature,6 and, lately, de novo enzyme design.7,8 Although additional examples have been reported, the overall number of experimentally validated computational

0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

372 design studies is small, and this also extends to the computer programs available to perform the underlying calculations. Only very few studies have so far aimed at redesigning protein–protein interaction specificities (for a recent review, see Mandell and Kortemme9). Whereas the goal in one study was to improve the affinity of an antibody–antigen interaction,10 the interaction of the bacterial DNase colicin E7 with the inhibitor Im7 was targeted in another study.11,12 The authors succeeded in engineering a specific protein– protein interaction pair with the added constraint that the individual proteins no longer recognize their natural partners. In an additional study, the homodimeric protein SspB was mutated, and two different chains were produced. 13 These chains were no longer able to associate to form homodimers but gave rise to a dimerization-specific heterodimer instead. Here, we report a protein design study that targeted the bacterial gene transcription regulator tetracycline repressor (TetR). TetR is an all-α-helical homodimeric protein that regulates the expression of genes that provide resistance against members of the tetracycline family of antibiotics in Gramnegative bacteria. 14,15 TetR binds to operator sequences located in between the tetA gene and the tetR gene and thereby blocks the expression of these genes. Upon interaction with the effector tetracycline, TetR undergoes a conformational change that causes its dissociation from the operator and thereby induces the transcription of the tetA and tetR genes encoding for a tetracycline antiporter and for TetR itself, respectively.14 Similar to SspB, the aim of the current study was to transform wild-type TetR (T-WT) into two nonidentical protein chains that can recombine specifically to form heterodimers, while at the same time excluding the formation of homodimers. For the desired specificity to be achieved, it was therefore not sufficient to identify the most favorable combination of amino acids that gives rise to a stable interface packing; rather, it required both stabilization of the heterodimer and destabilization of putative homodimers. In contrast to colicin E7 and SspB, where the size of the interface region is on the order of 750 Å2, the interface region targeted in TetR was considerably larger, namely as large as 2200 Å2.

Results Computational design of heterodimeric TetR variants TetR is an all-α-helical homodimeric protein, with each monomer comprising 10 α-helices (Fig. 1). Whereas N-terminal helices α1–α3 (from monomer 1)

TetR Heterodimer Design

and helices α1′–α3′ (monomer 2) form the two helix–turn–helix-type DNA-binding domains, helices α4–α10 and α4′–α10′ build up the effectorbinding domains and, at the same time, the dimerization interface.19 This interface is very extensive in TetR. Fifty-six residues from each monomer contribute to the dimerization interface [calculated for Protein Data Bank (PDB) entry 1A6i using the protein assembly service PISA at the European Bioinformatics Institute].16,17,20 The central structural feature of the dimerization interface consists of a four-helix bundle (Fig. 1b and c). Helices α8 and α10 of one monomer pack at an almost 90° angle against helices α8′ and α10′ of the second monomer. The computational heterodimer design was accomplished using the side-chain packing and selection algorithms implemented in the program MUMBO.21 During design calculations, the protein backbone was considered fixed. Discriminative heterodimeric TetR variants were obtained by a combined positive design/negative design approach.13 The two TetR chains were computationally modified to introduce a knob and a hole in each chain. In the heterodimer TetR variants, these knobs and holes are juxtaposed and complementary in shape (positive design). At the same time, the formation of homodimer variants is rendered unfavorable as a result of steric repulsion between knob positions and also potentially through the occurrence of extensive voids introduced by unmatched hole residues (negative design) (Fig. 2). In a first step, the most suitable positions to place knob residues within the four-helix bundle region of the interface were identified. All residues in the interface were mutated in silico to alanine and then mutated one by one to either phenylalanine or tryptophan. The positions were ranked according to the absence of steric clashes between the side chains and the protein backbone, as well as the Cβ atoms of surrounding residues. Two positions were identified as promising for the introduction of knob residues, namely positions 136 and 192. In the next step, and as part of the positive design, the program MUMBO was used to place tryptophan or phenylalanine residues at these positions on either chain and to optimize the side-chain packing of the surrounding residues by allowing neighboring residues (eight in total) to be substituted to either Ala, Val, Leu, Ile, or Phe. Because of the largely hydrophobic nature of the TetR interface, only hydrophobic residues were considered. At 20 additional surrounding positions, the wild-type residues were allowed to adopt alternative side-chain orientations. In a subsequent step, the two resulting homodimer scenarios were evaluated. Here, the emphasis was to make sure that formation of homodimers is rendered impossible as a result of extensive repulsion between identical chains (negative design).

TetR Heterodimer Design

373

Fig. 1. Dimerization interface in T-WT. (a) Cartoon representation of the two chains of the all-helical homodimeric T-WT (light and dark gray; PDB entry 1A6I).16,17 (b) A four-helix bundle constitutes the salient feature of the dimer interface. Residues displayed from the helices were targeted during the computational design. The viewpoint in (b) equals that in (a) after a 90° rotation around the vertical 2-fold axis defining the C2 point group of T-WT. (c) Stereographic representation of side-chain packing at the center of the four-helix bundle in T-WT. Here, the vertical 2fold symmetry is again apparent. This figure and all other structure illustrations were drawn with the program PyMOL.18

Three different TetR heterodimer variants were designed using three different A chains and one single B chain (Table 1). Position 136 was chosen for the introduction of a knob residue in chain A. In T-WT, this residue corresponds to an alanine, and the Cβ methyl group packs against Leu193 and Phe197 from the same chain and against Phe140′ and Gly189′ from the second chain (Fig. 1c). In the designed heterodimer variants, position 136 now displays a tryptophan in chain A (Fig. 2). Neighboring residue Phe140 is replaced by

Ala in chain B, and residue Phe197 is replaced by Ile in chain A. In addition, residue 193 from chain A was either retained as Leu (variant T-ALB) or replaced by Ala (T-AAB) or Ile (T-AIB) (Table 1). In chain B, residue 192 was targeted as a knob residue. In homodimeric T-WT, this residue is a serine and is surrounded by His188 from the same monomer and by Phe197′ from the second monomer (Fig. 1c). In the designed heterodimer variants, Ser192 is replaced by Trp in chain B, and space for the accommodation of the side chain is

374

TetR Heterodimer Design

Fig. 2. Design of heterodimeric TetR variants. (a) Stereographic representation of the computationally predicted favorable side-chain packing in T-AAB (positive design; chain AA in magenta and chain B in green). (b) Predicted unfavorable packing in T-AAAA and (c) T-BB (negative design; see Table 1 for the mutations introduced). Because of the C2 point group symmetry of T-WT, T-AAAA, and T-BB, two identical clash regions and void regions are present in each homodimer variant.

generated through the replacement of Phe197 by Ile in chain A (Table 1, Fig. 2). A remarkable feature of the designed variants is the low number

of substitutions introduced for selective heterodimer formation, namely three residues in chain A and two residues in chain B.

375

TetR Heterodimer Design Table 1. Designed TetR chain variants Sequence of targeted residuesa Chain

136

140

192

193

197

Sum of all side-chain atomsb

Wild type AA AI AL B

Ala Trp Trp Trp Ala

Phe Phe Phe Phe Ala

Ser Ser Ser Ser Trp

Leu Ala Ile Leu Leu

Phe Ile Ile Ile Phe

21 24 27 27 23

a

Mutated residues are highlighted in boldface. Total number of side-chain atoms present in the five positions (hydrogen atoms omitted). b

According to the interaction energies calculated, the heterodimers were expected to be highly discriminative, namely strongly favoring heterodimer formation and disfavoring homodimer formation (Table 2). In terms of stabilizing interactions, T-AAB appeared to be the most favorable variant, since the predicted heterodimer stability was almost identical with that of T-WT (−197.7 versus −212.3 kcal/mol). Unfavorable interaction energies in excess of 1000 kcal/mol for the homodimer variants originated from van der Waals repulsion. High repulsion energies were predicted to occur between Trp136 and Ala193 in the A-chain homodimer (Fig. 2b) and between Trp192 and Gln200 in the B-chain homodimer (Fig. 2c). A detailed summary of the predicted per-residue interaction energies in variants T-AAB, T-AAAA, and T-BB in comparison to T-WT is provided in Tables S1–S5. A notable feature of the design strategy and a result of the C2 point group symmetry of T-WT is that a matching wild-type interaction is still present for every interaction that is newly introduced in the heterodimers. Whereas Ser192 from T-WT is replaced by Trp in chain B, a serine is still present at this position in the A chain, where it participates in wildtype-like interactions across the dimer interface. This is not true for the negative design, which again leads to homodimeric complexes, and all destabilizing interactions are therefore repeated twice. Biological activity of heterodimeric TetR variants The biological activities of the different variants were assayed in vivo using a β-galactosidase

reporter gene assay.22 The results show that in cases where the cells were transformed with both A chain and B chain, gene transcription is silenced in the absence of anhydrotetracycline and induced upon addition of the effector (Fig. 3). This corresponds to the activity of T-WT (positive control). Compared to T-AAB, the inducibility of the heterodimer variants T-AIB and T-ALB appears reduced. In the negative control experiment with empty plasmid vectors, 100% β-galactosidase activity can be detected in the absence and in the presence of anhydrotetracycline. When the cells are transformed with the B chain alone, then repression is significantly impeded, and a β-galactosidase activity corresponding to 63% of the negative control can be detected even in the absence of any effector. This indicates that the B chain, by itself, only poorly represses transcription. Unexpectedly, however, in all experiments where the different A chains were present alone, activities similar to those of T-WT are observed (Fig. 3). This hints that in the case of the A-chain variants, biologically functional homodimers can still be formed. At the same time, it questions the interpretation of the results obtained for the heterodimer variants, since the observed TetR-like function of the heterodimers could be readily explained by functionally active A-chain homodimers alone. Because of this ambiguity, it became imperative to purify the individual variants and to further characterize these in vitro. Secondary structure and thermal stability The heterodimeric variants T-AAB, T-ALB, and T-AIB were purified in milligram amounts using a His-tag attached to the B chain (exemplarily shown for T-AAB in Fig. S1). The A and B chains coelute in equimolar amounts in each fraction from a His-trap affinity column. The equimolar ratio indicates that the B chain, by itself, does not form any stable homodimers and always elutes as a component of a heterodimeric variant. This is supported by the observation that Escherichia coli cells transformed with the B-chain plasmid alone do not yield any soluble protein that can be immobilized on a His-trap column (data not shown).

Table 2. Predicted dimer interaction energies in homodimeric and heterodimeric TetR variants TetR variant T-WT T-AAB T-AIB T-ALB a

Dimer composition Chain I Wild type AA AI AL

Predicted interaction energies (kcal/mol)

Chain II

Homodimera I–I

Homodimera II–II

Heterodimer I–II

Wild type B B B

− 211.9 1244.4 4461.0 5666.7

− 211.9 710.1 710.1 710.1

− 211.9 − 197.5 − 171.5 − 112.8

b

In the text, the corresponding homodimers are referred to as T-AAAA, T-BB, T-AIAI, and T-ALAL. As for all protein design programs, the interaction energies calculated with MUMBO are not on an absolute scale. As a consequence, only relative energies should be considered. b

376

TetR Heterodimer Design

Fig. 3. In vivo repression and induction activity of the various TetR variants. TetR-controlled lacZ expression in the absence of the effector (repression; black bars) and upon addition of 0.4 μM anhydrotetracycline (induction; color and gray bars) monitored by measuring β-galactosidase activity in a cell-based assay. Enzyme activities were normalized against a negative control with no repressor present (NC; fully induced state). The results are not fully conclusive. Although all heterodimer variants (red, yellow, and pink) behave like T-WT as intended (PC: positive control) and the expression of the B chain, by itself, leads to a functionally impaired protein (brown bar), the expression of the A chains alone unexpectedly leads also to functional proteins (blue, green, and cyan bars). Hence, there is a possibility that the biological activity of the heterodimeric variants results from A-chain homodimers alone rather than from a successful heterodimer design. All heterodimer experiments were performed in duplicate, swapping the chains between two different plasmids (see Materials and Methods).

This is also in agreement with the impaired biological activity of T-BB in a cell-based reporter gene assay (see the text above) and in full agreement with the design goal. Unexpectedly, the homodimeric variants T-AAAA, T-AIAI, and T-ALAL could also be isolated. However, whereas T-AIAI and T-ALAL could be purified to homogeneity after cleaving off the His-tag, variant T-AAAA could only be isolated as a His-tag fusion protein. Removal of the His-tag with thrombin caused T-AAAA to fully precipitate (Fig. 4a). This is not the case for the corresponding heterodimer variant T-AAB, which remains in solution irrespective of whether or not the His-tag is present. Thus, at the level of solubility, the heterodimeric variant T-AAB displays a clear advantage over the corresponding T-AAAA homodimeric variant. Binding affinities between the different chains cannot be measured directly since TetR forms a permanent homodimer that folds and unfolds cooperatively. TetR is expected to exist in solution either as an unfolded monomer or as a folded dimer.23 As an indicator for the success of our design, we investigated instead the stability of the different variants by CD spectroscopy (Fig. 4). The CD spectra of T-WT, Histagged T-AAAA, and variants T-AAB, T-ALB, T-AIB, T-AIAI, and T-ALAL show that all proteins appear properly folded and display the typical spectra of αhelical proteins (Fig. 4b and c). Although heatinduced thermal unfolding of TetR is irreversible and therefore does not allow for derivation of thermodynamic equilibrium parameters,23 thermal

unfolding provides valuable insight into the stability of the variants when performed under identical conditions. T-WT unfolds cooperatively at 55 °C, which is close to the previously reported melting temperature.23 In contrast, T-AAB unfolds at 44 °C, while His-tagged T-AAAA unfolds at 41 °C (Fig. 4d). Hence, T-AAB seems more stable than T-AAAA. The stability of a protein is also mirrored by the cooperativity of the unfolding process, which can be evaluated from the steepness of the CD curve during the unfolding transition. As can be seen in Fig. 4d, unfolding of His-tagged T-AAAA appears to proceed less cooperatively than in the cases of T-AAB and T-WT. As mentioned before, His-tagfree T-AAAA could not be investigated due to its low solubility (see the text above). In contrast to the T-AAB versus His-tagged T-AAAA variant where a difference in stability in favor of the heterodimeric version can be observed, no such positive discrimination occurs for variants T-AIB and T-ALB. In the case of T-AIB, the heterodimeric and homodimeric variants show a highly similar unfolding behavior with a Tm of about 50 °C (Fig. 4e). In the case of T-ALB, it appears that the homodimeric variant T-ALAL (Tm = 55 °C) is as stable as T-WT and, at the same time, even more stable than heterodimeric T-ALB. Moreover, unfolding of T-ALB occurs in a twostage process: After an initial melting of T-ALB at around 47 °C, the A chains appear to reassociate to form T-ALAL homodimers, which then unfold at around 55 °C (Fig. 4e).

TetR Heterodimer Design

377

Fig. 4. In vitro protein characterization. (a) Solubility time course of T-AAAA (blue bars) and T-AAB (red bars) upon removal of His-tag with thrombin. (b) CD spectra of T-WT, T-AAB, and His-tagged T-AAAA recorded with identical protein concentrations. The overall shapes of the recorded spectra are highly similar. The amplitude of the measured CD signal is highest for T-WT and reduced for T-AAB and His-tagged T-AAAA. (c) CD spectra of T-WT, T-AIB, and T-ALB. (d) Thermal unfolding analysis of T-WT, T-AAB, and His-tagged T-AAAA monitored by CD at 222 nm. (e) Thermal unfolding of T-WT, T-AIB, T-ALB, T-AIAI, and T-ALAL. The melting temperatures are as follows: 40.7 °C (His-tagged T-AAAA), 44.2 °C (T-AAB), 55.2 °C (T-WT), 49.6 °C (T-AIB), 50.1 °C (T-AIAI), and 55.4 °C (T-ALAL). Please note that, in (b) and (d), the His-tagged version of variant T-AAAA was used since removal of the His-tag led to the precipitation of T-AAAA (see (a)).

In summary, thermal unfolding and solubility measurements reveal a positive discrimination between T-AAB and T-AAAA. This discrimination

is, however, considerably smaller than that for T-BB, which could not be isolated at all for in vitro characterization.

378

TetR Heterodimer Design

Table 3. Crystallographic data collection and refinement statistics Data collection statistics X-ray source Wavelength (Å) Space group Cell dimensions a, b, c (Å) Cell dimensions α, β, γ (°) Resolution range (Å) Number of unique reflections Redundancy Completeness (%) Mean I/σ(I) Rint Rmeas Refinement statistics Number of residues in asymmetric unit Number of refined atoms Number of solvent molecules Resolution range Rwork (%) Rfree (%) RMSD bond lengths (Å) RMSD bond angles (°) Ramachandran analysis PDB entry coordinates (structure factor amplitudes)

T-AAB

T-AIB

T-ALAL

Rotating anode 1.5418 I4122 69.1, 69.1, 183.0 90, 90, 90 20–2.10 (2.23–2.10)a 13,364 (2148) 13.6 (13.5) 99.6 (99.3) 28.6 (5.6) 0.055 (0.535) 0.057 (0.554)

BESSY Synchrotron 0.9184 I4122 69.2, 69.2, 183.4 90, 90, 90 40–2.15 (2.28–2.15) 12,563 (1972) 14.2 (12.0) 99.9 (99.9) 29.1 (4.6) 0.055 (0.632) 0.057 (0.660)

BESSY Synchrotron 0.9184 I4122 69.7, 69.7, 184.3 90, 90, 90 40–2.25 (2.39–2.25) 11,142 (1733) 19.4 (14.1) 99.3 (96.2) 28.2 (4.2) 0.063 (0.686) 0.064 (0.711)

194 1697 106 20–2.14 21.0 26.0 0.012 1.378 94.8, 4.1, 1.2, 0.0b 2xge (2xgesf)

194 1646 58 35–2.15 21.7 27.1 0.013 1.402 90.3, 7.4, 2.3, 0.0 2xgc (2xgcsf)

195 1607 56 35–2.25 21.7 25.9 0.014 1.445 90.8, 6.4, 1.7, 1.2 2xgd (2xgdsf)

a

Values for the highest-resolution shell are listed in parentheses. Listed are the percentages of residues in the core, allowed, generously allowed, and disallowed regions of the Ramachandran plot, according to the program PROCHECK.24 b

Crystal structures To validate the success of our design, we solved the crystal structures of three TetR variants to resolutions between 2.1 and 2.3 Å (Table 3). All structures were solved in space group I4122, with the particularity that the asymmetric unit in these crystals contains only one single monomer. Hence, the molecular unit in the heterodimeric variants exceeds in size the asymmetric unit of the crystals. Upon evaluation of different refinement strategies, we opted for a bimodal local disorder model in which heterodimers stochastically adopt two different orientations at each lattice position (see Materials and Methods). Such a bimodal disorder is not uncommon in crystallography. Bimodal disorder occurs, for example, in crystals that contain near-palindromic double-stranded DNA, as well as in a number of DNA–protein complexes. 25–27 Its occurrence can easily be explained in the case of the TetR heterodimers. Since all mutated positions are buried within the dimer interface, chains A and B are indistinguishable when viewed from the surface. Therefore, both chains can participate in identical crystal packing contacts. As a consequence of the local disorder, the density displayed at residue positions that differ between A and B corresponds to the averaged electron density of the amino acids present at this position in both chains (Fig. S2).

Because of this bimodal disorder, the accuracy with which the atomic positions of the mutated residues are determined has to be questioned. However, since no two side chains can simultaneously share an identical space, the placement of the side chains into the electron density maps was unambiguous in all cases and, hence, reliable models could be obtained. The crystal structures of variants T-AAB, T-AIB, and T-ALAL were refined to crystallographic R-factors ranging from 21% to 22%, with deviations from ideal bond lengths between 0.012 and 0.014 Å (Table 3). An overall comparison of the crystal structures with the starting coordinate file (PDB entry 1A6i)16 yields RMSDs between 0.39 and 0.44 Å for the main-chain atoms, indicating that no severe structural rearrangements were induced by the mutations. This is especially true for the four-helix bundle motif itself. Here, very small overall RMSDs are observed (0.27–0.45 Å) between structures. The overall agreement between the design and the experimentally determined crystal structures of T-AAB and T-AIB is quite good (Fig. 5a and b). As is apparent from Table 4, the precision with which the 30 residues that were either mutated or allowed to reorient during the calculations have been placed during the design is relatively high, with N73% correctly predicted χ1 angles and RMSDs between 0.8 and 1.6 Å.

TetR Heterodimer Design

379

Fig. 5. Interface packing in the crystal structures of (a) T-AAB, (b) T-AIB, and (c) T-ALAL (orange and yellow) in comparison to the predicted design. For the latter, the color coding (magenta/green) from Fig. 2 is used. (d) Sketch showing the various orientations of Trp136 (in orange), namely as predicted and as observed in T-AIB, as observed in the crystal structure of TAAB, and as observed in the crystal structure of T-ALAL.

Close inspection of the individual structures, however, reveals slight deviations from the design. Whereas the two Trp residues in T-AIB introduced

as knob residues, namely Trp136 and Trp192, adopt the predicted orientations with only slight deviations in χ1 and χ2 values (Fig. 5b), this is not true for

380

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Table 4. Accuracy of design calculations TetR variant RMSD (Å)a Δχ1 b 20° (%) All Δχi b 20° (%)

T-AAB

T-AIB

T-ALAL

1.53/1.55b 84.6/100.0 69.5/84.6

1.33/0.78 86.7/87.5 68.9/82.1

2.57/2.91 73.3/60.0 56.2/44.4

a

Calculated for all side-chain atoms (including Cβ). The first number refers to all residues that were either allowed to be mutated or allowed to adopt alternative side-chain orientations during the computational design process. The second number refers to the mutated residues136, 140, 192, 193, and 197 only. b

T-AAB. In T-AAB, the indole ring of Trp136 from chain A is flipped over by about 180° and becomes wedged between the side chain of Ala193 and the side chain of Phe140, both from chain A (Fig. 5a). As a consequence of the change in χ2 from 70° (design) to − 85° (crystal structure), the indole ring in the crystal structure now predominantly participates in intramolecular interactions instead of intermolecular interactions. At the same time, a void is created around Ala140 from chain B. The crystal structure of the homodimeric variant T-ALAL is of significant interest for the corroboration of the design, since this variant was predicted to be highly instable and yet the Tm of the protein produced in E. coli is comparable to that of wild type. The largest deviation can be seen for the side chain of Trp136. Instead of making close contacts with Leu193, Trp136 escapes into a cleft between helix α8 and α10′ (Fig. 5c). Concomitantly, the mainchain atoms of helices α8 and α10′ are displaced, and the residues surrounding this cleft are slightly pushed outwards. This reorientation of Trp136 appears unfortunate. It now occupies a hole that we introduced upon mutation of Phe197 to isoleucine as part of the A-chain design. In all heterodimers, this hole is occupied by Trp192 from chain B; however, in the A-chain homodimers, this hole was wrongly expected to be inaccessible and to remain empty. Overall, the crystal structures show that most side-chain orientations were predicted correctly for the heterodimer variants. The crystal structure of T-AL AL, however, suggests that unpredicted readjustments in the protein backbone allow for an unanticipated reorientation of Trp136, thereby allowing for a relief of the engineered van der Waals repulsion and the occurrence of stable T-ALAL and T-AIAI variants.

Discussion

cooperatively at 44 °C. T-AAB proves to be uniquely folded, and with the exception of the orientation of the side chain of Trp136, which is flipped over by about 180°, the crystal structure of T-AAB reveals that the packing of the interface side chains is highly similar to that predicted by computational design. Variant T-AAB also shows a positive discrimination against the homodimer variants T-AAAA and T-BB. Although variant T-AAAA could be produced as a His-tag version and displays only a slightly lower Tm than T-AAB (40.7 versus 44.2 °C) in the presence of the His-tag, removal of the His-tag in T-AAAA results in complete precipitation of the protein (Fig. 4a). In contrast, T-AAB stays in solution without His-tag and is amendable to structural investigations using X-ray crystallography. In the case of T-BB, we were never able to produce any soluble protein in E. coli for in vitro characterization, leading to the conclusion that, as intended, formation of a stable T-BB homodimer is impeded. Variant T-AAB shows a reduced thermal stability when compared to T-WT. Such a behavior was also observed in a similar study on the SspB adapter protein, where the authors concluded that specificity was gained at the cost of stability.13 Although we see the same effect here, we do not see a necessary correlation between selectivity and stability. The reduced stability might just reflect a shortcoming of the design, as it is generally agreed that interaction energies calculated using computational de novo design algorithms only roughly correlate with experimentally measured stabilities. The tendency of T-AAAA to aggregate upon removal of the His-tag reflects—as intended by our design—a reduced fitness of this variant in comparison to T-AAB. At the same time, aggregation proneness represents the strongest single criterion that discriminates between T-AAB and T-AAAA. We suspect that the lowered solubility is caused by a destabilized interface and the concomitant occurrence of voids. This is not immediately obvious when only comparing variants T-AAAA, T-AAB, and T-WT. Counting the number of side-chain atoms displayed in the five positions targeted during the design yields 21 atoms for the wild-type chain, 24 atoms for chain AA, and 23 atoms for chain B (Table 1). However, in comparison to the unexpectedly stable homodimeric variants T-AIAI and T-ALAL, T-AAAA displays the lowest number of atoms within the dimer interface; hence, we suspect that an underpacking of this interface in comparison to the more stable versions T-AIAI and T-ALAL might be the origin of the lower solubility of T-AAAA.

Design success

Design limits

In the case of variant T-AAB, the computational design can be viewed as a success. T-AAB forms a stable heterodimeric protein that unfolds highly

Overall, the knobs-into-hole design, with one knob being presented from each chain, appears promising. The fixed main-chain assumption

381

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proved highly appropriate for the positive design. In the two heterodimeric versions that we investigated using X-ray crystallography, no significant differences were observed between the design and the experimentally determined structure. The most pronounced deviation was observed for the orientation of Trp136 in T-AAB, which unexpectedly becomes sandwiched in between the side chain of Ala193 and the side chain of Phe140. MUMBO failed to identify this rotamer (χ1 = 180°, χ2 = 91°) as part of the global energy minimum. Inspection of the backbone-dependent rotamer library used during the calculations reveals the existence of a closely related rotamer with dihedral angles χ1 = − 180.7° and χ2 = 85.5°,28 which was however not recognized as the energetically most favorable rotamer by MUMBO. The reason for this is that a small difference in χ2 leads to a severe van der Waals clash with the Cβ atom of Ala193. This is a generally acknowledged problem in computational de novo design.29 The packing algorithms enforce idealized rotameric states and select the best combination of side-chain orientations based on the interaction of these idealized rotamers. However, in proteins, and as seen here for T-AAB, small side-chain readjustments are able to relieve high van der Waals repulsion energies. A number of remedies have been proposed to circumvent this problem, namely reducing van der Waals radii to about 95%, expanding rotamer diversity by adding angular increments to χ dihedral values, or using a softened van der Waals repulsion energy term.29 However, even when applying these retrospectively, we still failed to predict the correct rotamer (data not shown). With respect to the negative design, namely the discrimination against homodimers, our design approach shows clear limitations. Because of a slight readjustment of helices α8 and α10 in variant T-ALAL, Trp136 is able to escape van der Waals clashes and points into an interhelical cleft (Fig. 5d). It is obvious to us that the fixed backbone approach is responsible for this discrepancy between the design and the experimental crystal structure. When the altered main-chain conformation from the T-ALAL crystal structure is used as starting model, the side-chain packing in the crystal structure can readily be reproduced with MUMBO (data not shown). During the design of the TetR variants, we estimated the probability for the occurrence of backbone rearrangements to be small, since the targeted residues are displayed from defined secondary structure elements, namely α-helices, which pack against each other to form a defined four-helix bundle supersecondary structure motif (Fig. 1b). The crystal structure of T-ALAL, however, shows that this did not prevent small main-chain shifts. Interestingly, however, the fixed backbone assumption appears justified in the case of the B chain. Here,

a similar knob design was used to preclude homodimer formation, and a significant discrimination against homodimer formation was observed. Heterodimer design in comparison to homodimer specificity in natural TetR variants Position 192 had already been identified in a previous study as an important determinant for the dimerization specificity in TetR.30 The naturally occurring variants TetR(B) and TetR(D) share 63% identical amino acids and do not form heterodimers in vivo. Schnappinger et al. showed that upon mutation of a single residue, namely Leu192 in TetR(B), to serine (as present in TetR (D)), the dimerization specificity became significantly relaxed. Mutated TetR(B) now interacts with both wild-type TetR(D) and TetR(B), as observed in a pull-down experiment. Through the introduction of three further substitutions, namely additionally mutating positions 188, 193, and 197, to the corresponding residues of TetR(D), a further shift in specificity was obtained. This mutant TetR(B) variant now only interacted with TetR(D) and did not interact anymore with unmodified TetR(B). However, all mutants remained fully inducible and behaved like wild type in in vivo expression tests, hinting that all these mutants were still able to form homodimers. Our design clearly followed a different goal, namely to generate a TetR heterodimer variant while precluding homodimer formation. Interestingly, we also identified residues around position 192 as showing considerable promise towards this goal. In contrast to the mutations discussed by Schnappinger et al., our B-chain design, which introduces a Trp residue at position 192, is homodimerization deficient and also significantly hampered in an in vivo transcription assay (Fig. 3).30 This successful replacement could not have been derived from a comparative genomics analysis, since none of the known natural TetR variants contains a Trp at position 192 (Christian Berens, personal communication). However, it clearly appears that position 192 is outstanding and might therefore qualify as a dimerization specificity hot spot. We therefore propose that the concept of functional hot spots— which was initially derived from studying the interaction specificity in transient complexes such as the specific binding of growth hormone to growth hormone receptor—also extends to large interfaces such as those observed in permanent oligomeric assemblies in TetR.31

Conclusions Our design shows that as little as two mutations in one chain and three mutations in a second chain are

382 able to considerably shift the interaction specificity in a protein interface as large as 2200 Å2. This is surprising since, in total, 2 × 56 residues participate in the dimerization interface in T-WT. The residues targeted during the design only contribute 103 Å2 (chain A) and 123 Å2 (chain B) to the interface region in T-WT. This amounts to only 4–6% of the total interaction area. Our computational design study differs from previous studies in several aspects. In comparison to SspB and colicin E7, the targeted interface is considerably larger (2200 versus 750 Å2). In addition, the number of mutations that we introduced was also lower than that in SspB (5 versus 7 in SspB). In contrast to colicin E7, the interface in TetR is almost entirely hydrophobic. The TetR interface can be considered a permanent interface;32 no TetR monomers have been reported so far, and TetR is considered to unfold according to a two-state model in which folded dimers are in equilibrium with unfolded monomer chains.33 Highly hydrophobic interfaces are often seen in permanent interaction surfaces and may present a number of advantages for computational de novo design. Van der Waals interaction energies can be calculated reliably, and the resulting interfaces are also devoid of any bridging water molecules, which are very difficult to predict using side-chain packing algorithms. Nevertheless, we did run into typical protein design problems, with the unanticipated shift in the main-chain conformation being the most severe. Our results clearly show that even such large interfaces are amendable to computational de novo design. The question on whether our study has any biological implications arises. For example, do our results help to understand adaptive changes in molecular recognition during protein evolution? The fact that such a limited number of mutations are able to considerably shift protein interaction specificities makes such an evolutionary route—in which a homodimeric protein, upon gene duplication, evolves into a more specialized heterodimeric protein—quite plausible.

Materials and Methods Computational design The coordinates from PDB entry 1A6I16,17 describing the homodimeric variant TetR(BD) were used as starting point for all design calculations. In this entry, TetR is neither bound to DNA nor in complex with a small-molecule effector molecule. In TetR(BD), the DNA-binding domain (residues 1–50) of TetR(B) (Swiss-Prot accession number P04483) is fused to the effector-binding domain of TetR(D) (P0ACT4).30,34 This TetR(BD) variant was used throughout the present study as a synonym for T-WT. All designed variants were derived from this protein.

TetR Heterodimer Design

All computational design calculations were performed with the computer program MUMBO using a fixed backbone approach.21 Residue diversity and conformational flexibility were introduced using a backbonedependent rotamer library.28 Selection of the best combination of side chains and rotamers for a given set of residues was accomplished using the dead-end elimination algorithm and variations thereof.35,36 A Metropolis Monte Carlo simulated annealing search was performed on the remaining residues/rotamers in cases where the remaining diversity needed to be further reduced after dead-end elimination. The force field included solvation, hydrogen bonding, and rotamerprobabilities-derived energy terms, as well as van der Waals and electrostatic interactions using CHARMM19derived parameterization.21,37 Detailed lists of predicted per-residue interaction energies for the designed variants are provided in Tables S1–S5. Mutagenesis and in vivo protein function assay A plasmid encoding for T-WT (TetR(BD))30,34 was mutated according to the design predictions using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), the QuikChange multi-site-directed mutagenesis kit, or a two-stage PCR protocol.38 The biological activity of the designed variants was characterized with a reporter gene assay in accordance with Wissmann et al.22 The experimental setup consisted of two compatible expression plasmids, termed pWH1411 and pWH1925, that constitutively expressed the individual TetR chains. These plasmids were cotransformed into E. coli strain WH207(λtet50), which carries a chromosomal tetA–lacZ fusion under the control of the tetO operator. Thus, binding of TetR to tetO regulates lacZ transcription, and the repression activity and inducibility of the TetR variants can be studied directly by quantifying the β-galactosidase activity in the absence and in the presence of tetracyclines.22 Induction was studied upon addition of 0.4 μM anhydrotetracycline. Experiments with the heterodimeric variants were performed in duplicate. First, the chain A variants were cloned into pWH1411 and the chain B variant was cloned into pWH1925, and then the chains were swapped between plasmids. This was performed to monitor potential artifacts that could arise from differences in the protein expression levels of the two constitutively expressing plasmids. In assays where the activity of the homodimer variants was investigated, the plasmids pWH1411 and pWH1925 encoded for identical chains. As a negative control experiment, the E. coli reporter strain WH207(λtet50) was transformed with insert-free pWH1411 and pWH1925 plasmids. The plasmid for T-WT and plasmids pWH1411 and pWH1925, as well as E. coli strain WH207(λtet50), were all kindly provided by W. Hillen of University Erlangen-Nuremberg. Protein expression and purification For recombinant protein production, the A chains were cloned into the plasmid pET-43.1b carrying an ampicillin resistance gene, and the B chain was cloned into pET-28b carrying a kanamycin resistance gene (Novagen, Darmstadt, Germany). In the latter, the protein coding region is fused to a hexa-His-tag via a thrombin cleavage site. In the

383

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case of the pET-43.1b plasmid, the TetR sequences were inserted such that the Nus-tag was eliminated. To produce the homodimeric T-AAAA variant as a His-tag fusion protein, we also cloned the corresponding AA chain (Table 1) into pET-28b. Overproduction of the different variants was achieved in E. coli strain Rosetta (DE3) (Novagen) transformed either with two plasmids (heterodimeric variants) or with a single plasmid (homodimeric variants). Cells were grown in 8 × 1 L of LB medium to an OD600 of 0.6 at 37 °C. The temperature was then lowered to 22 °C, and the cells were induced with 0.5 mM IPTG. Upon further incubation for an additional 4–6 h, the cells were harvested through centrifugation, resuspended in 20 ml of sodium phosphate buffer [20 mM sodium phosphate, 0.5 M NaCl, and 2 mM Pefabloc (pH 7.4)], and disrupted by sonication. The variants that carried a His-tag (T-AAB, T-AVB, T-AIB, and T-AAAA) were purified by a two-step protocol, namely affinity chromatography (HisTrap FF crude column; GE Healthcare, Waukesha, WI) followed by size-exclusion chromatography (HiLoad 26/60 Superdex75 prep grade column; GE Healthcare). Whereas a phosphate buffer [loading buffer: 50 mM sodium phosphate buffer, 30 mM imidazole, and 0.5 M NaCl (pH 7.4); elution buffer: 50 mM sodium phosphate buffer, 500 mM imidazole, and 0.5 M NaCl (pH 8.0)] was used for affinity chromatography, the buffer for size-exclusion chromatography consisted of 50 mM Tris–HCl (pH 8.0) and 200 mM NaCl. In all cases, with the exception of variant T-AAAA, the His-tag was removed upon incubation with thrombin (20 °C for 1.5 h) between the affinity chromatography step and the size-exclusion chromatography step. Protein variants T-WT, T-ALAL, and T-AIAI were produced without His-tag and purified using a weak cation-exchange chromatography column (SP Sepharose FF media; GE Healthcare) and then an anion-exchange column (Resource Q media; GE Healthcare). The buffer used for cation-exchange chromatography consisted of a sodium phosphate [50 mM sodium phosphate, 5 mM ethylenediaminetetraacetic acid, and 50 mM NaCl (pH 6.8)]. In the case of anion-exchange chromatography, a buffer consisting of 20 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid, and 50 mM NaCl (pH 8.0) was used. In both cases, the proteins were eluted using a NaCl gradient from 50 mM to 1 M. The ion-exchange chromatography steps were followed by size-exclusion chromatography under identical conditions, as described for the His-tagged versions. All purified proteins were stored at −80 °C before further characterization. Protein solubility assays To quantify the solubility of the protein variants upon removal of the His-tag, we incubated the purified His-tag variants of T-AAB and T-AAAA with thrombin (5 NIH units thrombin/mg protein) at 25 °C. At defined time points, samples were drawn and centrifuged for 15 min at 21,000g and 4 °C. The protein concentrations in the supernatants were determined using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA). Each experiment was performed in triplicate using a protein concentration of 1 mg/ml in two experiments and a protein concentration of 1.5 mg/ml in a third experiment.

CD spectroscopy and thermal denaturation studies For CD spectroscopy, samples of the TetR variants were adjusted to a concentration of approximately 5 μM, and 2 ml of this solution was transferred to a potassium phosphate buffer [10 mM potassium phosphate (pH 7.5)] using a NAP-25 salt-exchange column (GE Healthcare). The samples were then diluted to obtain a solution with a concentration of 4.5 μM. After centrifugation at 20,000g for 40 min, CD spectra were measured over a wavelength range from 185 to 260 nm at a scan rate of 20 nm/min at 25 °C using a Jasco J-600 spectropolarimeter (Jasco, Tokyo, Japan) and a PTC-348 WI Peltier element. Each spectrum was averaged over eight measurements and corrected for buffer absorbance. To study thermal unfolding, we heated protein samples at a concentration of 1 μM from 20 to 75 °C at a linear scan rate of 1 K/min. Unfolding was monitored by recording ellipticity at a wavelength of 222 nm. Protein crystallization and data collection Crystallization of the different TetR variants was accomplished upon repeated microseeding and macroseeding with T-WT crystals using hanging-drop vapor diffusion. A 1-μl protein solution of T-WT (10 mg/ml) was mixed with 1 μl of reservoir solution [1 M K2HPO4, 50 mM Tris–HCl (pH 8.0), and 200 mM NaCl], and crystals grew within 1–2 days at 19 °C. For macroseeding, fragments of these crystals were washed three times in reservoir solution and transferred to a fresh droplet prepared from 1 μl of protein solution of the respective TetR variant (10 mg/ml) and 1 μl of reservoir solution. Crystals that generally appeared in the vicinity of the seeds after 1 day of incubation at 19 °C were then used as new seeds in another round of crystallization. This procedure was repeated three times in order to avoid contamination with T-WT. Alternatively, for microseeding, crystals that appeared after the initial seeding were reduced to small pieces, and the suspension was then used to inoculate new droplets. This was achieved by drawing a needle through the suspension and consecutively through fresh droplets of the respective variant. Diffraction data sets of the TetR variants were collected in-house using a MicroMax-007 HF X-ray generator (Rigaku, Tokyo, Japan) with a Mar345dtb image plate (MarResearch, Norderstedt, Germany) and at the Protein Structure Factory beamline BL14.2 of Free University Berlin at BESSY Synchrotron Berlin. Before cryocooling, crystals were immersed in a cryoprotectant solution consisting of 20% (vol/vol) ethylene glycol or 20% (vol/ vol) glycerol and 80% (vol/vol) reservoir solution for approximately 20 s. All data sets were reduced with the program XDS and scaled together with XSCALE.39 Eight percent of the reflections were flagged for later calculations of Rfree. Since all variants crystallized isomorphously in space group I4122, an identical selection of reflections was marked for the monitoring of Rfree in all variants. X-ray structure analysis Since all variants crystallized isomorphously in space group I4122 (Table 3) and since the space group and unit

384 cell dimensions are identical with those of PDB entry 1A6I,16,17 all variants could be readily solved by difference Fourier techniques. However, the I4122 space group comes with a caveat. The asymmetric unit in this space group contains one TetR monomer, and the T-WT dimer representing the biologically functional molecule is generated upon application of a crystallographic 2-fold symmetry axis that runs parallel with the c axis. Such an arrangement, namely where the point group symmetry of the biological oligomer includes symmetry elements from the space group, is of course very common in protein crystallography. However, in the case of the designed heterodimeric variants T-AAB and T-AIB, the point group symmetry is reduced to C1. Hence, the biological unit is double the content of the asymmetric unit; therefore, the presence of a crystallographic dyad that passes across the middle of the heterodimer in space group I4122 has to be questioned. We considered three alternative models for the packing of the heterodimers within the observed unit cell. We considered the possibility that the true space group of the crystals corresponds to a subgroup of I4122 and that an entire dimer is present in the asymmetric unit. The subgroup obtained upon the removal of the appropriate dyad is P43212, as described before in a crystal structure of TetR with asymmetrically bound ligands.40 However, close inspection of the diffraction patterns did not reveal the presence of any reflection up to 2.1 Å resolution for any of the dimer variants, with Miller indices corresponding to h + k + l = 2n + 1. Data processed in P43212 only yielded symmetric densities for the two chains in the dimer. We further considered the possibility that crystallographic twinning might be present and that the crystals are formed by two twin domains related by a twin law that mirrors the pseudo-dyad axis in the heterodimers. However, also in this case, the space group of the individual twin domains would be P43212, and reflections with indices h + k + l = 2n + 1 should be observable. We opted for the third possibility, namely that the heterodimers are stochastically oriented at each lattice position. The scattering unit displays space group I4122, and the random orientation of the heterodimers is reflected in an asymmetric unit that contains copies of the A chain and the B chain with a 50% occupancy. The refinement of the models in space group I4122 was started with PDB entry 1A6I.16 After several rounds of rigid-body refinement, we gradually extended the resolution to the maximum resolution of each variant while refining atom positions and atomic displacement factors. Automated refinement was carried out with REFMAC5,41 and model inspection/building was carried out with Coot.42 In the heterodimers, only a single chain was modeled in the asymmetric unit, with an occupancy of 1.0 for all amino acids, except for those amino acids that differ between the A chain and the B chain. At these positions, alternative amino acids and orientations were modeled with occupancies of 0.5 and attached to the main chain using the LINK statement. TLS refinement was used in the final stage of refinement by defining four identical TLS groups in each structure comprising residues 6–45, 46–91, 92–151, and 166–205. Validation of the crystal structures was performed using PROCHECK,24 and RMSDs between different structures were calculated with LSQMAN.43 χ angle deviations describing differences in side-chain orientations between

TetR Heterodimer Design

the experimentally determined crystal structures and the computational designs were calculated with a Perl script that internally invokes LSQMAN. All model illustrations were drawn with the program PyMOL.18 PDB accession numbers Structural data (coordinates and observed structure factor amplitudes) for the final models have been deposited with the PDB under accession codes 2xge (T-AAB), 2xgc (T-AIB), and 2xgd (T-ALAL). Supplementary materials related to this article can be found online at, at doi:10.1016/j.jmb.2010.07.055.

Acknowledgements We would like to thank Roman Jakob, Barbara Eckert, and Franz-Xaver Schmid of the University of Bayreuth for access to and help with CD measurements, and Cornelius Wimmer and Wolfgang Hillen of University of Erlangen-Nuremberg for the original plasmids and for help with the cell-based reporter assay. We would like to also thank Uwe Müller of BESSY Synchrotron Berlin for support during data collection, Madhumati Sevvana for help with crystallographic refinement, and Benedikt Schmid for help with colored illustrations.

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