Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Hot-spot analysis to dissect the functional protein–protein interface of a tRNA-modifying enzyme Stephan Jakobi,1 Tran Xuan Phong Nguyen,1 Franc¸ois Debaene,2 Alexander Metz,1 Sarah Sanglier-Cianferani,2 Klaus Reuter,1 and Gerhard Klebe1* 1 Institut f€ ur Pharmazeutische Chemie, Philipps-Universit€at Marburg, Marbacher Weg 6, D-35032 Marburg, Germany 2 Laboratoire de Spectrometrie de Masse BioOrganique (LSMBO), IPHC-DSA, Universite de Strasbourg, CNRS UMR7178; 25 rue Becquerel, 67087 Strasbourg, France

ABSTRACT Interference with protein–protein interactions of interfaces larger than 1500 A˚2 by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. Proteins 2014; 82:2713–2732. C 2014 Wiley Periodicals, Inc. V

Key words: protein–protein interaction inhibition; interface binding hot spots; MM-GBSA computational stability analysis; site-directed mutagenesis; native nano-ESI mass spectrometry; crystal structure analysis.

INTRODUCTION A large number of processes in living organisms is controlled by proteins. In some cases, these biomolecules function on their own, but more frequently they cooperate with other proteins. For example, receptor signal transduction relies on the communication of proteins across well-defined interfaces of highly specific protein– protein complexes. The cytoskeleton of cells consists of large protein assemblies. Processes such as cell–cell recognition, immune response, or gene regulation by transcription factors all proceed via the formation of protein–protein complexes involving large commonly shared interfaces.1–10 In addition, many enzymes are only functionally active once adopting a particular quaternary structure. Because protein–protein interactions (PPIs) are that important, any misdirected or aberrant

C 2014 WILEY PERIODICALS, INC. V

recognition process may cause a disease. Conversely, selective inhibition of specific PPIs may provide a novel concept for therapeutic intervention. Therefore, major effort is presently dedicated to the understanding of how a small molecule can interfere with the formation of PPIs.11–18

Additional Supporting Information may be found in the online version of this article. Grant sponsor: Deutsche Forschungsgemeinschaft; grant numbers: FO 806; KL1204/13-1; Grant sponsor: the Alsace region, the CNRS, and the University of Strasbourg. *Correspondence to: Gerhard Klebe, Institut f€ ur Pharmazeutische Chemie, Philipps-Universit€at Marburg, Marbacher Weg 6, D-35037 Marburg, Germany. E-mail: [email protected] Received 31 January 2014; Revised 24 May 2014; Accepted 18 June 2014 Published online 28 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.24637

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Such interference via small molecule inhibitors or antagonists, however, is notoriously difficult owing to the shape of typical protein–protein interfaces.19 Usually, such interfaces span a surface area of 700–3000 A˚2 that is buried on each side of the interface. This surface is usually flat, wide, and rarely exhibits deep depressions or cavities reminiscent of small-molecule binding sites found in enzymes or G protein-coupled receptors. Some examples of inhibiting PPIs have been reported. Notably, most successfully addressed PPIs feature extensive crevices on the interface that encloses a protruding surface feature of the dimer mate, such as a helical stretch or a turn-type bulge. In such cases, it is possible to develop a small molecule surrogate for the protruding portion as a competitive inhibitor of the PPI.16,17 In the absence of a well-defined crevice, significant protein flexibility is required to achieve complementarity between the surfaces of both dimer mates. In addition, this flexibility may lead to interface conformations that allow the binding of, and may in fact be stabilized by, small molecules. Evidence shows that in most protein–protein interfaces only a small subset of residues that form important interactions contributes most of the interaction affinity (socalled hot spots). Therefore, small-molecule inhibitors need not cover the complete interface but may instead specifically disturb the interactions of such hot spots. To modulate the function of an enzyme, usually the strategy of active-site inhibition is followed. However, enzymatic activity is often controlled and regulated by the formation of oligomeric states. If oligomerization is required for enzyme activity, the inhibition of this oligomerization may provide an alternative strategy to activesite inhibition, even though inhibiting oligomerization appears challenging. Nevertheless, variations among oligomerization interfaces are often larger than those found in conserved active sites across protein families and different genera. Inhibiting oligomerization may thus allow specific inhibition in the presence of highly conserved active-site architectures. Recently, we reported that the bacterial tRNA-guanine transglycosylase (Tgt) is only active as a homodimer.20,21 Tgt is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q).22 In detail, Tgt replaces the genetically encoded guanine at wobble position 34 of tRNAsAsp,Asn,His,Tyr by the premodified base preQ1. As Tgt is required for efficient pathogenicity of Shigella bacteria, it is a putative drug target for selective antibiotics against bacillary dysentery.23 During catalysis one monomer of the dimeric Tgt catalyzes the nucleobase exchange while the second monomer aligns and stabilizes the bound tRNA in the required position.24 Tgt enzymes are present in all domains of life and their active sites are highly conserved.25,26 Interestingly, however, eukaryotic Tgt, which incorporates queuine at the wobble position of the above-named tRNAs, forms a heterodimer.27,28 Consequently, this

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observations suggests that it may be possible to selectively inhibit the enzymatic function by inhibiting the homodimerization of bacterial Tgt. In a previous study of Zymomonas mobilis Tgt, we already concluded that homodimerization is a prerequisite for the bacterial enzyme to be functionally active. To this end, we applied native mass spectrometry (nano-ESI-MS), enzyme kinetics, and preliminary site-directed mutagenesis in order to interfere with Tgt dimerization.21 The nanoESI-MS experiments revealed an almost exclusive prevalence of dimeric Tgt and a 2:1 protein:RNA stoichiometry. These results confirmed the crystallographic observation that homodimeric bacterial Tgt interacts with a single tRNA anticodon stem loop.21,24 In order to interfere with Tgt dimerization, we created two mutants of Z. mobilis Tgt. With the first mutant Tyr330Phe, we intended to remove the H-bond formed by the tyrosine hydroxyl group of Tyr3300 and the backbone carbonyl oxygen of Ala49 located on the dimer mate (the respective dimer mate will hereinafter be indicated by an apostrophe). In the second mutant Lys52Met, we replaced Lys52 by a sterically similar but uncharged methionine in order to remove the salt bridge formed by the ammonium group of Lys52 and the carboxylate group of Glu3390 . While both mutants had little effect on KM(tRNA), kcat was reduced by factors of 10 and 50 for Tyr330Phe and Lys52Met, respectively. This supports our conclusion that dimerization is a prerequisite for the catalytic activity of Tgt.21 Furthermore, nano-ESI-MS experiments revealed an increased and concentration-dependent formation of monomers for both mutants. This suggests that the dimer interface of both mutants is destabilized but not fully disrupted. To elucidate the structural basis of this destabilization, we resolved the crystal structure of the Lys52Met variant. Despite the unchanged dimeric state in the crystal packing, the introduced mutation caused significant structural alterations.21 Although the above-mentioned mutants were merely inspired by empirical considerations with the goal to prevent Tgt dimerization, they led only to a partial monomerization. This may not come as a surprise, as effective dimer-disrupting mutants are hard to predict in the absence of detailed information about the contribution of individual interface residues to dimer stability. Our main goal in the present study was therefore to design Tgt mutants that are predominantly monomeric. To this end, we used computational analyses to identify residues that particularly contribute to dimer stability. This inspired the subsequent experimental realization of a promising double mutant. Native nano-ESI-MS confirmed that this double mutant adopts, depending on the applied concentration, a predominantly monomeric state (94–98%) in solution. Subsequent crystal structure analysis and enzyme kinetics are in agreement with this observation and provide first insights into the structural consequences of dimer destabilization.

Analysis of Tgt Homodimer Interface

MATERIALS AND METHODS Statistical analysis of distances in crystal structures

A subset of 43 Tgt structures was selected from the Protein data bank (pdb-codes: 1efz, 1enu, 1f3e, 1k4g, 1k4h, 1ozq, 1p0b, 1p0d, 1p0e, 1pud, 1pxg, 1q2r, 1q2s, 1wkd, 1wke, 1wkf, 1y5v, 1y5w, 1y5x, 2bbf, 2nqz, 2oko, 2pot, 2pwu, 2pwv, 2qii, 2qzr, 2z1v, 2z1w, 2z1x, 2z7k, 3bl3, 3c2n, 3c2y, 3c2z, 3eos, 3eou, 3gc4, 3gc5, 3ge7, 3gev, 3gfn, 3hfy). Homodimer complexes were generated by applying the crystallographic twofold symmetry axis and subsequently analyzed using the PISA server29 web interface. All parameters for interface detection were set to default. Salt bridges and H-bonds identified by PISA were classified in intervals of 0.25 A˚. Each interaction was considered only once for each homodimer. Two input structures contained a Tgt dimer in the asymmetric unit (pdb-codes: 1q2r, 1q2s), so that we considered both of the nonidentical interactions in the dimer interface. Computational analyses of interactions of interface residues

Multiple web services are available to estimate the contribution of interface residues to the stability of protein– protein complexes. In a comparative analysis we considered HotRegion,30 KFC2a and KFC2b,31 DrugScorePPI,32 Robetta,33 and the standalone version of FoldX (v3.0b6).34 These tools either perform a sequential exchange of interface residues by alanine and estimate the free energy contribution of the exchanged residues to the overall complex stability (DrugScorePPI, Robetta, and FoldX) or predict hot spots by evaluating interactions in the wild-type complex (all remaining tools). Unfortunately, the first four of these services are not prepared to handle homodimers explicitly where one single mutation contributes twice due to symmetry. Only FoldX allows testing the effect of simultaneously mutating both equivalent residues in the Tgt homodimer to alanine, using the BuildModel option of FoldX. For this purpose, we calculated the energy change upon mutating both equivalent residues in the dimer to alanine and subtracted twice the energy change upon mutating the individual residue in a monomer. All predictions used default parameters and were based on the crystal structure of the apo-Tgt dimer (pdb-code: 1pud35). If available, we used the inherent hot-spot classification of each method or a threshold of 2 kcal mol21. In an attempt to test whether this threshold exerts excessive bias on our hot-spot prediction, classifying the upper quartile of each method’s scored list of residues as hot spots did not change the overall result (data not shown). In order to consider potential effects due to flexibility and plasticity, we performed molecular dynamics (MD)

simulations using the AMBER 11 program suite.36 Starting structures were based on pdb-code: 1pud.35 Mutant structures were generated using Pymol (http://www. pymol.org/); side chain rotamers were selected based on visual inspection aiming for minimal steric overlap with the local environment. Charged residues were protonated according to standard protonation states (negative: Asp and Glu; positive: Lys and Arg). Histidine was protonated in d- or E-position based on visual inspection. The structural zinc ion was treated using the cation dummy approach by Pang;37 coordinating cysteines were set to residue type “CYM.” All crystallographically determined water molecules were included and the entire complex was placed in a box of explicit TIP3P water.38 Electroneutrality was achieved by adding sodium or chloride counter ions. For MD simulations, we used the ff99SB force field,39 periodic boundary conditions with the particle mesh Ewald method,40 a 10 A˚ non-bonded cutoff, and SHAKE.41 Temperature was regulated using Langevin dynamics42 with a frequency of 1 ps21. Pressure was adjusted by isotropic position scaling at steps of 1 ps. Before productive MD simulation each system was relaxed and equilibrated by a sequence of: (i) 100 steps of steepest descent minimization followed by 400 steps of conjugate gradient minimization with high positional restraints (500 kcalmol21A˚22), (ii) 1000 steps of steepest decent minimization followed by 1500 steps of conjugate gradient minimization without restraints, (iii) heating the system to 300 K over 100 ps of NVT simulation with 50 kcalmol21A˚22 restraints, (iv) and pressure adjustment to 1 bar over 100 ps of NPT simulation with 25 kcalmol21A˚22 restraints. Subsequently, productive NVT simulations were run for 20 ns without restraints at 300 K and 1 bar with pmemd.cuda. For all analyses, we considered only the last 10 ns of this trajectory. We used ptraj to calculate structural RMSD values and to analyze H-bonds over the trajectory. Distance and angle cutoffs for H-bonds were 3.2 A˚ and 120 , respectively. All counter ions and water molecules were discarded and 120 snapshots were selected. Following the method of Gohlke et al.,43 we performed per residue energy decompositions using the mm_pbsa.pl module of AMBER 11. Electrostatic solvation energy was calculated using Onufriev’s Generalized Born continuum model.44 Nonpolar solvation energy was computed proportional to the solvent accessible surface area determined using the ICOSA method of AMBER. Cloning and Tgt preparation

All plasmids used for DNA manipulation were prepared using the peqGOLD Plasmid Miniprep Kit II (PEQLAB, Erlangen, Germany). For site-directed mutagenesis the QuickChangeTM Lightning kit (Stratagene, CA) was used according to the vendor’s protocol. The PROTEINS

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required DNA primers were purchased from Eurofins MWG Operon (Ebersberg, Germany). In each case, sequence analyses of the entire tgt gene (performed by Eurofins MWG Operon, Ebersberg, Germany) confirmed the presence of the desired mutation(s) as well as the absence of any further unwanted mutation. The Trp326Glu and Glu339Gln mutations were successively introduced into the Z. mobilis tgt expression plasmid pET9d-ZM4. Subsequently, the mutated plasmid R (DE3)was transformed into E. coli BL21-CodonPlusV RIL cells (Stratagene, CA). The transformed cells were used for the preparation of Tgt(Trp326Glu/Glu339Gln) according to the protocol of Romier et al.45 However, the significantly altered physicochemical properties of this variant required establishing new expression and purification protocols to yield pure protein. For this purpose, the Z. mobilis tgt gene including the Trp326Glu and Glu339Gln mutations was chemically synthesized by GENEART (Regensburg, Germany) with its codons optimized for expression in Escherichia coli. Additional sequences of 17 base pairs length were attached immediately upstream of the start codon and downstream of the stop codon. Each of these sequences contained a BsaI recognition site allowing the insertion of the target gene into the expression vector pASK-IBA13plus (IBA, G€ ottingen, Germany) as shown in Supporting Information Figure S1. In the resulting plasmid, the mutated tgt gene was fused at its 50 -end to a sequence encoding an N-terR separated from the tgt start codon by minal Strep-tag IIV a spacer sequence and a sequence encoding a thrombin cleavage site. The Cys158Ser and Cys281Ser exchanges were introduced successively. For the production of Tgt(Glu339Gln), Glu326 was backmutated to tryptophan. For the purification of wild-type Tgt via affinity chromatography Gln339 was further backmutated to glutamate (for sequences of DNA primers used for mutagenesis see the Supporting Information Table S1; for complete sequence of codon-optimized wild-type tgt see the Supporting Information Sequence S1). For overexpressing tgt gene variants, the respective plasmids were transformed into E. coli BL21-CodonPlus(DE3)RIPL (Stratagene, CA). Transformed cells were grown at 37 C in 2 3 YT medium46 containing 100 mgL21 ampicillin and 10 mgL21 chloramphenicol until an OD600 0.7 was reached. Subsequently, the bacterial culture was cooled down to 15 C and gene expression was induced by addition of anhydrotetracycline to a final concentration of 0.2 mgL21. Incubation was continued for a period of 14 h at 15 C whereupon cells were harvested by centrifugation and resuspended in 50 mL of buffer W (150 mmolL21 NaCl, 100 mmolL21 TRIS pH 7.8, 1 mmolL21 EDTA). Cell lysis occurred on ice by ultrasonication (6 3 90 s). Afterwards, the sonified cell extract was centrifuged at 30,000g at 4 C for 1 h. The clear supernatant was loaded onto a streptactin superflow sepharose column (IBA, G€ ottingen) with a bed volume of 25 mL. The column was

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washed with buffer W followed by elution of the respective Tgt variant with buffer E (150 mmolL21 NaCl, 100 mmolL21 TRIS pH 7.8, 1 mmolL21 EDTA, 2.5 mmolL21 desthiobiotin) at a flow rate of 4 mLmin21. The Tgt containing fractions were concentrated to a volume of 2 mL using a Vivaspin 20 mL concentrator with a cutoff of 30 kDa (Sartorius Stedim Biotech, G€ ottingen) and loaded onto a HiLoad Superdex200 prep grade column with a diameter of 16 mm and a height of 60 cm (GE Healthcare; Chalfont St Giles, UK) for size exclusion chromatography. Elution occurred with buffer G (150 mmolL21 NaCl, 10 mmolL21 TRIS pH 7.8, 1 mmolL21 EDTA) at a flow rate of 1.5 mLmin21. All chromatographic steps were carried out at room tempera€ ture using an AKTA purifier LC system (GE Healthcare). R was proteolytically chipped from Finally, the Strep-tag IIV the target protein in buffer G for 20 h at 20 C using 1 U of biotinylated thrombin per mg target protein. Both, the bioR , were then sepatinylated thrombin and the Strep-tag IIV rated from the target protein using Streptavidin Agarose and Spin Filter columns. Biotinylated thrombin, Streptavidin Agarose, and Spin Filter columns are components of the Thrombin Kit (Novagen). The procedure was done according to the vendor’s protocol. Compared to the original Tgt, the additional amino acid sequence Gly-Ser remained attached to the N-terminal methionine after thrombin cleavage. The virtually pure recombinant Tgt was finally concentrated to about 10 gL21. The yield achieved by this protocol typically amounted to about 3 mg of mutated Tgt per L bacterial culture. Tgt(Lys52Met) was prepared as described by Ritschel et al.21 Native nano-ESI-mass spectrometry experiments

Prior to native nano-ESI-MS experiments, Tgt(Glu339Gln) and Tgt(Trp326Glu/Glu339Gln) were buffer exchanged against 1 molL21 ammonium acetate pH 8.0 (adjusted with ammonia) using microcentrifuge gel-filtration columns (Zeba 0.5 mL, Thermo Scientific, Rockford, IL). Protein concentration was further determined with Bradford assay using 2 g L21 BSA as standard. Native nano-ESI-MS experiments were performed on a hybrid electrospray quadrupole time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Manchester, UK) coupled to an automated chip-based nanoelectrospray device (Triversa Nanomate, Advion Biosciences, Ithaca, NY) operating in positive ion mode. Molecular mass, integrity, and homogeneity of the Tgt variants were checked under denaturing conditions with classical interface tuning parameters (cone voltage Vc was set to 40 V, pressure in the interface region Pi to 2.1 mbar) by diluting protein samples to a concentration of 2 lmolL21 in 1:1 (v/v) water/acetonitrile acidified with 1% (v/v) formic acid. Calibration was achieved using 2 lmolL21 horse heart myoglobin. The measured


Figure 1 Influence of point mutations on Tgt dimer stability. Native mass spectra obtained for wild-type Tgt or Tgt mutant variants at either 10 lM (A–D) or 1 lM (E–H) protein concentration in 1 molL21 ammonium acetate buffer pH 7.5. Vc 5 80 V, Pi 5 6.0 mbar. (*Tgt monomer, **Tgt dimer).

molecular masses were found in good agreement with those calculated from the amino acid sequence (Tgtwt: MWobs 5 43,013.8 6 0.2 Da, MWth 5 43,013.2 Da; Tgt(Glu339Gln): MWobs 5 43,012.6 6 0.4 Da, MWth 5 43,012.2 Da; Tgt (Trp326Glu/Glu339Gln): MWobs 5 42,956.2 6 0.4 Da, MWth 5 42,955.7 Da). For native nano-ESI-MS experiments, tuning parameters were carefully optimized to improve desolvation and ion transfer as well as to maintain weak interactions (Vc 5 80 V, Pi 5 6 mbar). Calibration was performed using monovalent ions produced by a 2 mg mL21 solution of Cesium iodide in 1:1 2-propanol/water (v/v). Native MS data interpretation was performed using MassLynx 4.1 (Waters). Relative species quantification presented in Figure 1 was calculated from average peak intensities of major charge states (111 to 131 for monomeric forms and 181 to 201 for dimeric forms). Kinetic characterization of the mutated variants

KM(tRNA) and kcat were determined for Tgt(Trp326Glu/ Glu339Gln), Tgt(Glu339Gln), and wild-type Tgt (prepared R affinity chromatography) as described by via Strep-tag IIV Biela et al.26 using 150 nmolL21 Tgt (monomer), 10 lmolL21 3H-guanine (A 5 12 Ci mmol21, Hartmann Analytic, Braunschweig, Germany), and various concentrations (0.5–15 lmolL21) of in vitro-transcribed tRNATyr.

Preparation of unmodified tRNATyr (ECY2)47 via in vitro transcription was done using the RiboMAXTM Large-Scale RNA Production System-T7 (Promega, Madison, WI) according to the vendor’s protocol. The concentration of tRNA was determined via UV photometry (k 5 260 nm).

Crystallization

All Tgt variants were crystallized using the sitting-drop vapor diffusion method at 18 C. For crystallization of Tgt(Lys52Met), a 1-mL droplet of protein solution (12 gL21 in 10 mmolL21 TRIS pH 7.8, 1 mmolL21 EDTA, 2 molL21 NaCl) was mixed with 2 mL of reservoir solution (100 mmolL21 TRIS pH 8.5, 10% (v/v) DMSO, 12% (w/v) PEG8000). For crystallization of Tgt(Glu339Gln), a 2-mL droplet of protein solution (10 gL21 in 10 mmolL21 TRIS pH 7.8, 150 mmolL21 NaCl, 1 mmolL21 EDTA) was mixed with 2 mL of reservoir solution (100 mmolL21 TRIS pH 8.5, 10% (v/v) DMSO, 7% (w/v) PEG8000). Crystals grew after a few days and were selected for data collection after transferring them into cryo buffer [reservoir solution plus glycerol at a ratio of 4:1 (v/v)] for 1 min and flash-frozen in liquid nitrogen for data collection. Tgt(Cys158Ser/Cys281Ser/Trp326Glu/Glu339Gln) failed to crystallize under customary conditions, thus required PROTEINS

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Table I Crystallographic Parameters

PDB code A. Data collection and processing Wavelength () Space group Unit cell parameters a, b, c () b ( ) B. Diffraction data Resolution range () Unique reflections R(I)sym (%) Completeness (%) Redundancy I/r(I) C. Refinement Resolution range () Reflections used in refinement Rfree (%) Rwork (%) Number of atoms Protein Waters Ligands RMS deviation from ideality Bond angles ( ) Bond length () Ramachandran plot Favored (%) Additionally allowed (%) Generously allowed (%) Mean B-factors (2) Protein Water

Tgt(Lys52Met)

Tgt(Glu339Gln)

Tgt(Cys158Ser/Cys281Ser/ Trp326Glu/Glu339Gln)

4dxx

3unt

3uvi

0.91841 C2

0.91841 C2

0.91841 C2

91.2, 64.9, 70.1 96.0

91.3, 64.9, 70.2 96.0

91.0, 65.0, 70.5 95.6

50.0–1.66 (1.69–1.66) 47,912 (2330) 4.9 (28.5) 99.6 (96.6) 2.6 (2.5) 18.1 (3.8)

30.0–1.80 (1.83–1.80) 36,234 (1618) 8.9 (28.3) 95.7 (87.5) 2.7 (2.0) 10.9 (2.8)

50.0–1.55 (1.58–1.55) 58,805 (2729) 6.0 (27.4) 99.0 (89.7) 3.0 (2.2) 16.1 (2.8)

34.9–1.66 (1.69–1.66) 47,886 (2768) 19.9 (25.2) 16.6 (22.5)

18.9–1.80 (1.85–1.80) 36,210 (2421) 18.5 (24.6) 15.4 (20.3)

35.0–1.55 (1.58–1.55) 58,746 (2416) 19.1 (28.3) 16.5 (22.9)

2989 421 –

2899 399 23

3029 479 41

1.084 0.006

1.036 0.007

1.054 0.006

95.3 4.4 0.3

95.0 4.7 0.3

94.7 4.7 0.3

15.62 28.55

15.66 27.57

15.04 29.69

group C2 and contained one monomer per asymmetric unit. Unit cell dimensions are given in Table I. The data were processed and scaled with the HKL2000 package (for statistics see Table I).48 For phasing and initial

establishing new conditions. In total, 1248 conditions from commercially available crystallization screens (AMSO4 Suite, Anions Suite, Classics Suite, Classics Lite Suite, Cryos Suite, JCSG1 Suite, JCSG Core I Suite, JCSG Core II Suite, JCSG Core III Suite, JCSG Core IV Suite, PACT Suite, MbClass Suite, and MbClass II Suite from Qiagen (Hilden, Germany)) were screened automatically using a Cartesian pipetting robot (Tecan, Switzerland). Thereby, 0.3 mL of the protein solution (9 gL21 in 10 mmolL21 TRIS pH 7.8, 150 mmolL21 NaCl, 1 mmolL21 EDTA) were mixed with 0.3 mL of the respective screening solution. Droplets were inspected every other day. After 1 week, crystals appeared under various conditions. After 4 weeks, a crystal from solution 51 of the PACT suite (200 mmolL21 NaI, 20% (w/v) PEG3350) was selected, transferred into cyro buffer (reservoir solution plus glycerol at a ratio of 4:1) for 1 min, and flash-frozen in liquid nitrogen for data collection.

modeling of the structures, coordinates of the apo-Tgt crystal structure (pdb-code: 1pud) were used in the program phaser49 of the ccp4 program suite.50 For this purpose, residues 46–62 and side chains of 158, 281, 326, and 339 were not considered, depending on the studied variant. Further refinement cycles were made with the program phenix.51 Randomly selected 5% of all data were used for the calculation of Rfree. Amino acids were fit to |Fo| – |Fc| and 2 |Fo| – |Fc| electron density maps using coot.52 Subsequently, water and other ligand molecules were located in the difference electron density and included in further refinement cycles. The coordinates of the Tgt structures were deposited in the RCSD Protein Data Bank (pdb-codes given in Table I).

Structure determination

Protein data bank accession codes

Diffraction data were collected at BESSY beamline 14.2. All crystals exhibited monoclinic symmetry in space

Coordinates and structure factors have been deposited under the following accession codes:

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Figure 2 (A) The Z. mobilis Tgt dimer is shown in ribbon representation, structural Zn21 ions are shown as orange spheres. Helix a1(0 ) as well as the preceding b1(0 )a1(0 ) loop are colored red, the extended helix aE(0 )— turn—helix aF(0 ) motif is colored blue. (B) The dimer is folded apart showing the symmetrical halves side-by-side. (C) Surface representation of the dimer halves in the same orientation as in (B). The individual contributions of surface residues to dimer stability according to Supporting Information Table S2 are mapped onto the solvent accessible surface in terms of a gray scale (the darker the region is shown the more it contributes to the dimer stability).

4dxx: Tgt(Lys52Met), 3unt: Tgt(Glu339Gln), and 3uvi : Tgt(Cys158Ser/Cys281Ser/Trp326Glu/Glu339Gln).

RESULTS Structural analysis of the dimer interface

In the apo-Tgt crystal structure (pdb-code: 1pud),35 the Tgt homodimer covers an interface area of 1660 A˚2; each monomer contributes 43 interface residues.21

The dimer contact is established by multiple directional interactions comprising 10 salt bridges and eight H-bonds (see below). Most of these interactions involve residues that reside on helix a1 (Lys55 to Gly63) or on the preceding ß1a1-loop (Val45 to Leu54) (Fig. 2). In the following, this stretch (Val45 to Gly63) will be referred to as “loop-helix motif.” As in the apo-Tgt crystal structure, the subunits of the homodimer are related to each other by a crystallographic twofold axis, any interaction contributes twice to the interface. Accordingly, each of the two b1a1-loops provides one residue to form the Lys52Glu3390 salt bridges and two to form the Ala48His3330 and Ala49Tyr3300 H-bonds [Fig. 3(A)]. Notably, the Lys52Glu3390 salt bridges are observed in only about two thirds of all Tgt crystal structures, while in the remaining structures Lys52 adopts a conformation allowing an H-bond to the backbone carbonyl of Thr285. The two a1 helices add two further salt bridges, Lys55Glu3480 and Glu57Lys3250 . In some structures, the latter salt bridge is mediated by a water molecule [Fig. 3(B)]. The complementary binding region on the dimer mate is located on an extensive helix-turnhelix motif formed by helices aE0 (Ser3270 to Arg3360 ) and aF0 (Ile3400 to Glu3670 ; Fig. 2). The two helices are connected via a three-residue turn comprising Ala3370 , Gly3380 , and Glu3390 with the glutamate exposed at the apex between both helices. Helix aE0 exhibits a patch of aromatic residues (Trp3260 , Tyr3300 , and His3330 ) that form H-bonds and van-der-Waals interactions to the dimer mate [Fig. 3(C)]. While His3330 interacts with Leu74 and Pro78, Tyr3300 forms hydrophobic interactions to Phe92 and Met93. The backbone carbonyl oxygen of the latter residue forms an H-bond to the indole NH of Trp3260 , which additionally interacts with the adjacent Pro56 at the C-terminus of helix a1. This hydrophobic interface region is bordered by Leu86 and Leu3110 on one side and by the ß1a1-loop on the opposite side. Altogether, nonpolar carbon and sulfur atoms contribute 60.7% (982 A˚2) of the interface area, while polar oxygen and nitrogen atoms contribute 39.3% (636 A˚2). Accordingly, the Tgt interface is predominantly hydrophobic similar to most protein–protein interfaces.

Consistency of directional interactions across the Tgt interface in crystal structures and molecular dynamics simulations

To assess the geometric variation of directional interactions formed across the Tgt interface, we analyzed 43 crystal structures of Tgt. In most Tgt structures, the rotational symmetry of the dimer is imposed by the crystallographic twofold axis in space group C2. One Tgt/ inhibitor complex exhibits a reduced P2 symmetry20 and the stem-loop bound tRNA-Tgt dimer lacks this imposed symmetry constraint.24 While the distances of some of PROTEINS

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Figure 3 Directional interface contacts of the Tgt dimer. The color code of the subfigures is the same as in Figure 2. Individual residues are shown in stick representation with nitrogen atoms in blue, oxygen atoms in red, and sulfur atoms in orange. Water molecules are shown as red spheres. H-bonds and salt bridges are indicated by dashed gray lines. (A) The Lys52Glu3390 salt bridge as well as the Ala48His3330 and Ala49Tyr3300 H-bonds as present in the high-resolution crystal structure of wild-type Tgt in complex with a competitive inhibitor (pdb-code: 2z7k). The Lys52Glu3390 salt bridge is observed in only about two thirds of all deposited Tgt crystal structures while in the remaining ones Lys52 adopts an alternative conformation allowing an H-bond to the backbone carbonyl of Thr285. (B) The Lys55Glu3480 salt bridge and a water mediated interaction between the side chain carboxylate of Glu57 and the side chain ammonium of Lys3250 are observed in the crystal structure of wild-type apo-Tgt (pdb-code: 1pud). While in some crystal structures of Tgt the latter residues directly form a salt bridge via their side chain functional groups, in some structures no interaction between these residues is formed. (C) Aromatic hot-spot region formed by residues Phe92, Trp3260 , Tyr3300 , and His3330 (pdb-code ???: 1pud). This hydrophobic interface contact region is shielded from water access by Leu86 and Leu3110 on one side and by the b1a1loop (omitted for the sake of clarity) on the opposite side. (D) Same detail as in (C) but from Tgt(Cys158Ser/Cys281Ser/Trp326Glu/Glu339Gln) (pdb-code: 3uvi). The empty space generated by the Trp3260 Glu exchange is filled by six water molecules making H-bonds to the Glu3260 side chain carboxylate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


Figure 4 Statistical analysis of directed dimer interface contacts (salt bridges and H-bonds, BB: interactions to backbone functional groups) of a subset of 43 Tgt structures deposited with the protein data bank. The counts of each contact as a function of distance (0.25 A˚ bins) are given as blue bars. Some of the contacts populate a broad range of distances suggesting an intrinsic flexibility of the involved residues. In contrast, other contacts exhibit nearly the same distance in all considered crystal structures indicating an important role in dimer stabilization. Some contacts, which are likely of minor importance, are present in only a few structures. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

the H-bonds and salt bridges traversing the interface vary only slightly among the evaluated crystal structures (Fig. 4), others differ more widely or are missing in some complexes. This suggests a substantial flexibility and adaptability of the Tgt interface. For a better understanding of the influence and stability of individual hydrogen bonds formed across the interface, we performed explicit solvent MD simulations starting from the apo-Tgt crystal structure (pdb-code: 1pud). All MD simulations were performed according to standard protocols using AMBER30 (see Materials and Methods section). During 10 ns of productive MD simulation, all secondary structure elements remained virtually unchanged and the backbone hardly moved. This suggests that the MD trajectory correctly reproduced the properties of wild-type Tgt protein under equilibrium conditions. Nevertheless, solvent exposed side chains and individual water molecules were mobile. We calculated the frequency of occurrence (hereinafter denoted “occupancy”) of particular intersubunit H-bonds and salt bridges (Fig. 5), using distance and angle cutoffs (for H-bonds only) of 3.2 A˚ and 120 , respectively. A successively high occupancy (red) indicates strong interactions while a successively low occupancy (white) indicates weak interactions. Interactions that did not reach 10% occupancy were omitted in this analysis. Notably, some occupancies of equivalent interaction pairs in the homodimer differ significantly. This is due to small differences in the geometry of the Tgt subunits,

such as the concomitance of Ala48Lys3250 and Ala480 His333. This observation shows that some of the studied interactions are temporarily stable throughout the 10 ns trajectory. In principal, longer simulations will reproduce an equivalent behavior for equivalent interactions but, however, may require rather long simulation periods. A careful interpretation of the interaction statistics (Fig. 5) may provide some information about the contributions of individual residues to dimer stability. For instance, the persistent H-bond Met93Trp3260 , formed between indole NH and backbone carbonyl, indicates a strong contribution to dimer stability by these residues. In contrast, the incomplete or temporary formation of the H-bond network involving Glu3090 , Arg82, and Lys85 (Arg82/Lys85Glu3090 ) is clearly less consistent presumably reflecting a minor contribution of these interactions to dimer stability. Most convincingly, the frequencies of interactions in the trajectory agree well the respective occurrences across multiple crystal structures, shown in Figure 4. A single charge-assisted H-bond from the simulation trajectory, Gly94Lys3250 , misses in the crystal structure, where Ala48Lys3250 is formed instead. Computational analysis of the contributions of individual residues to dimer stability

To quantify contributions of individual residues to dimer stability, we calculated per-residue contributions PROTEINS

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Figure 5 Occupancies of particular H-bonds and salt bridges during 10 ns of MD simulation of wild-type Tgt. The occupancies, calculated for time intervals of 400 ps, are visualized using a linear color scale between high (red) and low (white) occupancy. A successively high occupancy (red) indicates strong interactions, for example, for Trp326Met930 . A successively low occupancy (white) indicates weak interactions, for example, for Glu309Arg820 /Lys850 . [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

to the binding effective energy by MM-GBSA, implemented into AMBER, as described by Gohlke et al.43 In addition, we used several web services to predict hot spots of Tgt dimerization (see below). Methodically, MM-GBSA calculates the contribution of each residue as the sum of gas phase energies and desolvation terms. Supporting Information Table S2 lists energetic contributions to Tgt dimerization of all involved residues. Generally, contributions by the side chains are higher than those of the backbone functional groups, except for significant contributions of the backbone carbonyl groups of Ala49, Met93, and Gly94. As expected for charged amino acids residues, strong compensations of electrostatic and desolvation energies are observed, that is, the attraction to an inversely charged residue on the dimer mate is virtually nullified by the high cost of desolvation. For charged residues that are not complemented upon dimerization, in a salt bridge to a residue on the dimer mate, their functional groups remain solvated in a local water environment, for example, as found for Glu81, Asp96, Arg303, and Arg336. Significant predicted energy contributions to Tgt dimerization (60.5 kcalmol21) are depicted in Figure 6. Particularly strong contributions (23.0 kcalmol21) are found for Leu86, Arg82, Trp326, Leu341, Lys55, and Tyr330. Notably, aromatic residues have an exceptionally strong contribution to Tgt dimerization. This is even more striking considering that there are only five aromatic interface residues compared to 20 aliphatic, six

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polar, and 12 charged ones. In addition, the average contributions of aromatic interface residues are also stronger (22.6 kcalmol21) than those of aliphatic (21.3 kcalmol21), polar (20.2 kcalmol21), or charged interface residues (21.1 kcalmol21). Importantly, residues that contribute significantly to Tgt dimerization are not scattered across the interface but are evidently clustered in hot-spot regions [Fig. 2(B,C)]. Due to the twofold symmetry of the dimer, each hot spot occurs twice in the interface. Most of the crucial residues are located on a-helices, except for Trp326 and Phe92, which are located in loops immediately after ahelices. For comparison, we also consulted several webservices30–34 to estimate the stability contribution of the contacting residues. Even though these approaches provide a fast ranking of important residues, they mostly do not allow to consider the twofold symmetry of a homodimers such as Tgt. Only in case of FoldX,34 we succeeded to take this symmetry into account. In total the web-based analyses suggested 24 residues to be hot spots by at least one of the applied methods (Fig. 7). In agreement with MM-GBSA, the aromatic interface residues stand out and are predicted to be hot spots by five (His333), six (Trp326), seven (Phe92), or all methods (Tyr330) including MM-GBSA. Nevertheless, several hot spots are predicted by individual methods only, in these cases mostly including MM-GBSA (Ala49, Lys55, Leu86, Ile340, and Ile341). Notably, MM-GBSA is the only method that predicts Lys52 and Arg82 as hot spots,


Figure 6 Energy contributions of selected amino acid residues to the stabilization or destabilization of the Tgt dimer interface as calculated by the MMGBSA43 method. The total stabilization energies are shown as red bars and refer to the scaling on the left ordinate. The stabilization energies, decomposed into gas-phase and solvation contributions, are indicated by blue and green dots, respectively. They refer to the scaling on the right ordinate which is rammed by a factor of 10. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

suggesting a potential benefit of considering MD in an explicit solvent environment. Computational analysis and design of mutant variants preventing Tgt dimerization

Based on the above-described hot-spot predictions, we conceived various amino acid exchanges, or combinations thereof, with the goal to interfere with Tgt dimeri-

zation. For this purpose, we focused on altering side chain interactions, also because affecting backbone interactions is hard and may affect local protein folding as well. We analyzed several mutations computationally, using MM-GBSA to estimate their effect on dimer stability. This elaborate MD-based approach offers clear advantages over most web services, as it is able to consider multiple and non-alanine mutations. We considered the following mutations:

Figure 7 Depicted are the rescaled values of the hot spot indicator variables as provided by the webservices HotRegion,30 KFC2a and KFC2b,31 DrugScorePPI,32 Robetta,33 FoldX (v3.0b6),34 and by MM-GBSA. Non-hot spots are indicated by a dummy value of 20.1. In total 24 residues (indicated as three-letter code) are suggested as hot spots; however, only 11 of them are considered by at least three methods. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The Lys52Met mutation, previously experimentally realized by Ritschel et al., led to a concentrationdependent interference with Tgt dimerization.21 The crystal structure of Tgt(Lys52Met) is markedly disordered in a stretch comprising most of the loop-helix motif (residues 50 to 62), suggesting an enhanced mobility or adaptability in this region. In wild-type Tgt, the Lys52Glu3390 salt bridge is located at the apex between helices aE0 and aF0. The distance of this salt bridge varies

significantly among Tgt crystal structures (Fig. 4). In fact, it is missing in about a third of all crystal structures, in which case Lys52 adopts an alternative conformation with its side chain ammonium group forming an intramolecular charge-assisted H-bond to the backbone carbonyl group of Thr285 on the same dimer mate [Fig. 8(A)]. During MD simulations, however, the Lys52Glu3390 salt bridge remained rather stable (Fig. 5). Remarkably, MM-GBSA is the only method that suggested Lys52 as a hot spot (Fig. 7). In order to elucidate the role of Lys52 for Tgt dimerization, we decided to reinvestigate the Lys52Met mutation. Due to the charged state of Glu339, a high cost of desolvation accompanies the monomer-dimer transition, which can only be compensated if a salt bridge is formed with Lys52. In order to elucidate the role of Glu339, we considered the exchange of Glu339 by an uncharged Gln. To further evaluate the contributions of salt bridges across the interface we also exchanged for glutamine: Glu3090 , which forms a salt bridge to Arg82, as well as Lys85 and Glu3480 , which form salt bridges to Lys55. As aromatic amino acids seem to contribute particularly to the interface (see Fig. 7), we decided to study Tyr330 and Trp326 more closely. Tyr330 had already been exchanged to phenylalanine by Ritschel et al. and a measurable amount of monomerization has been detected.21 We decided to reinvestigate this mutation. Our MD simulations strongly suggested that Trp326 and Met930 form an H-bond between the indole NH and the backbone carbonyl group of Met930 [Figs. 3(C) and 5]. Finally, the arrangement formed by Trp326 with Tyr330 Figure 8 (A) The Lys52 side chain as present in the crystal structure of wild-type apo-Tgt (pdb-code: 1pud) forms, via its ammonium group, an H-bond to the backbone carbonyl of Thr285. The crystal structure was superimposed with that of 2z7k where Lys52 adopts an alternative conformation leading to a salt bridge with Glu3390 . For the sake of clarity only the Lys52 side chain of 2z7k is shown (carbon atoms in bright yellow). The course of the remaining part of the detail as well as of the conformations of Thr285 and Glu3390 are identical in both structures. (B) Superimposition of wild-type apo-Tgt (pdb-code: 1pud) with Tgt(Lys52Met) crystallized at pH 8.5 (pdb-code: 4dxx). Loop b1a1 containing Met52 is present in two different conformations. Only the conformation deviating in its course from 1pud is shown in mint green. The side chain of Met52 is shown in stick representation (carbon atoms in mint green) in both conformations. (C) Superimposition of wildtype apo-Tgt (1pud) with Tgt(Cys158Ser/Cys281Ser/Trp326Glu/ Glu339Gln) (3uvi). Loop b1a1 and the Lys52 side chain of 3uvi are shown in green. The remaining part of the protein adopts identical geometries in 1pud and 3uvi and is, for the sake of clarity, only shown for 1pud. The dimer mate including Gln3390 is shown for 3uvi. The side chain conformation of Gln3390 is nearly identical to that of the original Glu3390 . The altered course of loop b1a1 enables the side chain carboxamide of Gln3390 to form, via its oxygen, H-bonds to the backbone amides of Thr500 and Lys520 and, via its nitrogen, to the backbone carbonyl of Ala480 . Furthermore, it allows the Lys520 backbone carbonyl to form a strong H-bond to the adjacent Leu341 backbone amide (for the sake of clarity, these interactions are not shown in the present figure). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 9 Changes in the contributions of particular amino acid residues to the dimer stability upon mutation of different interface residues. The underlying mutations, or rather combinations thereof, are color coded as indicated. The values are derived from MD simulations and MM-GBSA43 calculations in comparison to wild-type Tgt. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

and Leu341 contributes to a hot-spot region in the dimer interface. To study a challenging and more perturbing exchange, we decided to investigate the mutation of Trp326 to glutamate. This exchange was expected to abandon the residue’s H-bond donor properties, to suspend aromatic contacts to the neighboring residues, and to increase the cost of desolvation. Furthermore, as a favorable effect with respect to monomer stabilization, we expected this exchange to enhance the solubility of the monomer by increasing the hydrophilic character of the interface region, which is exposed in the monomer. To predict the effect of these mutations and combinations thereof, we performed MD simulations and MMGBSA analyses of the respective in silico generated mutant variants, using the same procedure as for wildtype Tgt. Overall, we investigated four mutant variants: (i) Tgt(Lys52Met), (ii) Tgt(Glu309Gln/Glu339Gln/ Glu348Gln), (iii) Tgt(Tyr330Phe/Glu339Gln), and (iv) Tgt(Trp326Glu/Glu339Gln). No structure dissociated, was disrupted, or exhibited massive rearrangements of local motifs in our MD simulation. Such changes are unlikely to occur within the limited time duration considered in our simulations. Nevertheless, the qualitative conclusions drawn from these analyses provided the rational basis for the subsequent experimental realization of putative mutant variants. In the following, we describe mutation-associated changes in the effective energy of Tgt dimerization (Fig. 9). Compared to wild-type Tgt, our MM-GBSA calculations show that the Tgt(Lys52Met) mutant variant is significantly destabilized at the mutated position (2.7 kcalmol21) while the former interaction partner Glu339 is hardly affected.

All multimutant variants featuring a Glu339Gln exchange stabilize this mutated position slightly (>20.5 kcalmol21), likely due to a more favorable desolvation. However, this effect is over-compensated by the destabilization of Lys52 due to the loss of the Lys52Glu3390 salt bridge. Similarly, Glu309Gln and Glu349Gln have little effect at the mutated positions but destabilize the positions of their interaction partners in the former Arg82/ Lys85Glu3090 and Lys55Glu3480 salt bridges, respectively. Remarkably, the mutant variant featuring a Tyr330Phe mutation destabilizes Lys52 even stronger (3.9 kcalmol21) than the Tgt(Lys52Met) mutant variant. This is in good agreement with the experimentally confirmed reduction of dimer stability for the Tgt(Tyr330Phe) mutant variant reported by Ritschel et al.21 The strongest destabilization (> 5 kcalmol21) was predicted for the mutant variants featuring the Glu348Gln and Trp326Glu mutations, respectively. Glu348Gln lacks the Lys55Glu3480 salt bridge whereas Trp326Glu compromises the Met93Trp3260 H-bond and the aromatic interaction with Tyr3300 . In addition, the newly introduced carboxylate of Glu326 becomes solvated by a five-membered water cluster throughout the MD simulation, just as one would expect for monomeric Tgt or for the initiation of a dimer-dissociation process. In summary, the replacement of Trp326 and the destabilization of the salt bridge to Glu339 are suggested as most promising candidates to achieve the desired transition to monomeric Tgt. We therefore planned to realize a double-mutant variant involving these two positions in our experimental work. PROTEINS

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Figure 10 Influence of point mutations on Tgt dimer stability as obtained by native nano-ESI-MS analyses. Tgt and four mutant variants were analyzed at concentrations ranging from 0.5 to 10 mM. The relative population of monomeric Tgt was deduced from native mass spectra based on the intensities of the three main charge states (111 to 131). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Recombinant production and characterization of Tgt(Trp326Glu/Glu339Gln)

Considering the above MM-GBSA analyses, we conducted in vitro mutagenesis experiments for the most promising double-mutant variant Tgt(Trp326Glu/ Glu339Gln). Apart from our MM-GBSA calculations the complementary Fold-X calculations34 indicated that Tgt(Trp326Glu/Glu339Gln) destabilizes the dimer interface even stronger (13.6 kcal mol21) than the corresponding double-alanine mutant variant (9.7 kcal mol21). In addition, we intended to improve our understanding of the importance of the Lys52Glu3390 salt bridge, complementing our previous in vitro characterization of Tgt(Lys52Met).21 Purification attempts according to the well-established protocol of Romier et al.45 failed owing to the altered physicochemical properties of Tgt(Trp326Glu/ Glu339Gln). In particular, the hydrophobic interaction chromatography step indicated an increased hydrophobicity of this mutant variant. Although it appeared likely that this increased hydrophobicity arises from the exposure of the formerly buried dimerization interface, this conclusion remained speculative at this stage of the study. In addition, further purification steps revealed an altered behavior of this mutant variant, such as the absence of the pronounced salting-in effect of wild-type

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Tgt. This ultimately necessitated establishing a new purification protocol. To this end, we created a recombinant R Tgt construct containing an N-terminal Strep-tag IIV separated from the protein by a thrombin cleavage site. A sequence of affinity chromatography using streptactin sepharose, preparative size exclusion chromatography, R yielded and proteolytical removal of the Strep-tag IIV enzymatically active and virtually pure Tgt(Trp326Glu/ Glu339Gln) (for experimental details see Materials and Methods section). Native nano-ESI-MS (Fig. 1) revealed that purified Tgt(Trp326Glu/Glu339Gln) is predominantly monomeric (94%) with only a small homodimeric proportion (6%) at a concentration of 10 mmolL21 (monomer, see Fig. 10). At similar conditions, hardly any monomer (2%) was observed for wild-type Tgt. Previously, we had shown that Tgt(Lys52Met) is predominantly homodimeric (96%) at equal concentration.21 Consistent with the assumption that Tgt dimerization in required for its catalytic activity, kcat of Tgt(Trp326Glu/Glu339Gln) was reduced by a factor of 20 with respect to wild-type Tgt (Table II). In contrast, KM(tRNA) of this mutant variant remained virtually unchanged, indicating that the small proportion of dimeric enzyme is able to bind the tRNA substrate with the same affinity as the wild type. Crystallographic analysis of Tgt(Trp326Glu/Glu339Gln) mutant variant

To investigate the structural consequences of the introduced mutations in comparison to our computational predictions, we crystallized Tgt(Trp326Glu/Glu339Gln). Notably, we introduced two additional mutations (Cys158Ser/Cys281Ser) into the crystallized protein in order to facilitate site specific tethering experiments (which will be described elsewhere). However, these additional mutations did not affect the structural and functional properties of the enzyme as demonstrated by unchanged Michaelis-Menten parameters for tRNA and guanine as substrates of Tgt(Cys158Ser/Cys281Ser) compared to wild-type Tgt (data not shown). The well-established crystallization conditions for wild-type and variant Tgt45 failed for Tgt(Cys158Ser/ Cys281Ser/Trp326Glu/Glu339Gln) due to its altered properties. Thus, we had to search for novel crystallization conditions using 1248 solutions from commercially available crystallization screens. Well-diffracting crystals were obtained in presence of a PEG3350 precipitant and Table II kcat and KM(tRNA) for Tgt and its mutant variants Tgt varianta Tgt (wild type)a Tgt (Trp326Glu/Glu339Gln) Tgt (Glu339Gln) a

kcat (1023 s21)

KM (mmolL21)

12.0 0.6 2.8

4.8 4.7 1.5

R affinity chromatography. Purified via Strep-tag IIV


a NaI additive at pH 7.8, which allowed structure determination to a resolution of 1.55 A˚. Surprisingly, morphology and unit cell parameters of these crystals were virtually identical to those of customary Tgt crystals indicating a similar arrangement of the protein molecules within the unit cell. We anticipated that the intended direct comparison of this new crystal structure to that of the previously determined structure of Tgt(Lys52Met)21 may be compromised by the different pH conditions applied during crystallization. To assure that the observed structural differences are a genuine effect of the introduced mutations and not simply a consequence of the significantly different pH conditions (pH 8.5 vs. 5.5), we recrystallized Tgt(Lys52Met) at slightly basic conditions (pH 8.5) and refined its structure to a resolution of 1.66 A˚. In fact, the new Tgt(Lys52Met) structure obtained at pH 8.5 differs significantly from the previously determined structure obtained at pH 5.5 (resolution of 2.0 A˚).21 Thus, we will shortly discuss the structural properties of this mutant variant first. In the original structure helix a1 and most of the preceding b1a1 loop (residues 48–62) had remained unassigned due to ill-defined electron density. In the new structure, however, most of this section could be fully traced in the electron density map, except for the incompletely defined residues Ala48 and Ala49. In the new structure, the loop-helix motif adopts two alternative conformations. The less populated conformer (46%) resembles that found in the wild-type structure. In this case, the mutated Met52 residue adopts a geometry closely matching the conformation of the wild-type Lys52 by forming an H-bond to Thr285 instead of a salt bridge to Glu3390 [Fig. 8(B)]. In contrast, in the higher populated conformation (54%) the residues adjacent to the b1a1 loop (residues 46–52) deviate significantly from the wild-type structure. In this case, Met52 adopts a completely different orientation and occupies the position of the adjacent Val51 in the wild-type structure forming hydrophobic contacts to Pro44, Val59, and Ile351. This displacement induces a repositioning of Val51 and alters the course of the b1a1 loop [Fig. 8(B)]. In both conformers found in the new Tgt(Lys52Met) structure, the orientation of helix a1 is identical to the wild-type structure. Although at pH 5.5, the helices aE0 and aF0 are shifted considerably compared to the wild type, at pH 8.5 these helices adopt the same positions commonly observed in Tgt crystal structures. Remarkably, the spatial position of Glu3390 at the apex between helices aE0 and aF0 which is the contact residue of the original Lys52, remains unaffected by the Lys52Met mutation. The crystal structure of Tgt(Cys158Ser/Cys281Ser/ Trp326Glu/Glu339Gln) exhibits the customary crystal packing with a dimeric arrangement in space group C2. At first sight, this seems surprising as this variant is predominantly monomeric in solution. The major part of

the protein fold is virtually identical to the wild-type structure and significant changes occur only next to the two mutation sites. The Trp326Glu exchange reduces steric demand; however, the replacement does not induce a local collapse of the folding geometry. Instead, the created vacant space formerly occupied by the indole moiety [Fig. 3(C)] is now filled by six water molecules [Fig. 3(D)], indicating that desolvation of the introduced glutamate residue is energetically unfavorable. Two of the incorporated interstitial water molecules mediate intermolecular H-bonds between Glu3260 and the backbone carbonyl groups of Phe92 and Met93 [Fig. 3(D)]. Obviously, the interstitial water molecules also cushion the repulsive interactions expected for a direct contact between the mutated monomers. Remarkably, the new conformation of the b1a1 loop [residues 46–52; Fig. 8(C)] is unprecedented in any other Tgt structure or any mutant variants thereof. Owing to the altered course of this loop, intermolecular H-bonds are formed between the side chain carboxamide of Gln3390 and the backbone amides of Thr50 and Lys52 as well as between the side chain NH2 group of Gln3390 and the backbone carbonyl group of Ala48. In addition, the backbone carbonyl group of Lys52 forms a strong H-bond (2.8 A˚) to the amide nitrogen of the adjacent Leu3410 . To investigate whether these structural alterations in the loop-helix motif are caused either by the Trp326Glu or the Glu339Gln exchange, we created the single-point mutant variant Tgt(Glu339Gln) and determined its crystal structure to a resolution of 1.80 A˚. In this case, the geometry of the respective region is virtually identical to that observed in the wild-type structure. Compared to the carboxylate of the original Glu3390 , the carboxamide of Gln3390 is slightly tilted concomitant with a marginal shift of an interstitial water molecule located between Gln3390 and Thr50. Nano-ESI-MS experiments with Tgt(Glu339Gln) at a concentration of 10 mmolL21 revealed a significant proportion of monomer (ca. 25%), demonstrating the importance of the Lys52Glu3390 salt bridge for dimer stability (Fig. 10). Consistently, kcat of Tgt(Glu339Gln) was reduced five-fold compared to wildtype Tgt (Table II). Interestingly, crystallization of Tgt(Cys158Ser/ Cys281Ser/Trp326Glu/Glu339Gln) was successful from a buffer containing sodium iodide as additive. The electron density map obtained for the crystal structure of this variant uncovered 10 iodide binding sites, of which two are located on special positions. While eight iodide sites are located at the protein surface, the two partly populated ions are found on special positions close to the loop region comprising residues 46–52. This indicates why iodide was found as important additive, required for successful crystallization. The influence of these ions on the detailed course of this loop region is difficult to assess. In any case, the altered conformation of this section (present with 100% occupancy) creates a new cavity PROTEINS

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in the protein–protein interface, which is partially populated by the iodide ions and two water clusters, each consisting of six water molecules (Supporting Information Fig. S2).

DISCUSSION The Tgt homodimer interface consists of 43 contacting residues that form 18 directional interactions (10 salt bridges, eight H-bonds) between the two dimer mates. More than 60% of the total interface area is hydrophobic. These structural characteristics are frequently found in protein–protein interface architectures and agree with the experience that such interfaces lack, in contrast to solvent-accessible surface regions, significant differentiation in terms of their amino acid composition.53–56 Interestingly, interfaces of homodimers were reported to exhibit a larger number of nonpolar contacts than heterodimers and, on average, larger interfaces seem to have a higher proportion of hydrophobic residues.57 It is difficult to assess whether these observations correlate with an increasing tendency to form permanent instead of transient complexes. In case of Tgt, our statistical analysis of multiple crystal structures shows that only some directional polar contacts are geometrically conserved. These findings are well consistent with the occupancies observed along the MD trajectory of the wildtype protein. However, these analyses provide no definitive evidence for the contributions of these contacts to dimer stability. It is well known that the contributions of individual residues to protein–protein interactions are not equally distributed across the interface but instead localized in some well-defined hot spots.58 This was demonstrated by mutation experiments that systematically replaced interface residues of prototypical protein–protein interactions by alanine.59–62 Applied to our Tgt example, this alanine scanning would have been very laborious. Thus, we first identified potential hot-spot residues in the Tgt interface by computational analyses. For this purpose, we utilized several web services based on heuristic principles, inter alia in combination with computational alanine scanning, and the MD-based MM-GBSA approach.63,64 The latter calculates free binding energies, as a combination of molecular mechanics energies, continuum modelbased solvation free energies, and crude entropy estimates, by averaging over a conformational ensemble obtained from an MD trajectory. However, separate MM-GBSA calculations for a multitude of mutant variants would have been quite computationally demanding. Thus, we used an approximate but faster approach by decomposing the effective energy of binding into contributions of individual residues.43,65–69 All applied prediction tools indicated distinct hot spots in the interface (Fig. 7). The consensus picture suggested,

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in agreement with general evidence,70 a cluster of four aromatic residues that primarily account for dimer stability. Three of these residues (Trp3260 , Tyr3300 , His3330 ) are contributed by one monomer and reside on a stable helical stretch (aE0 ) whereas the fourth residue (Phe92) is situated in the dimer mate. Due to a crystallographic C2 symmetry, this cluster contributes twice to the interface. Each hot spot is embedded within a ring of hydrophobic residues (O-ring), which supposedly shields the residues important for the dimer contact formation from water access.44 Even though the hot-spot forming residues reside on well-established secondary structural elements, residues from distinct structural elements must come together to complete the hot-spot region. This supports the hypothesis that the tertiary structure of a Tgt monomer is formed before two monomer units assemble to the functional dimer. Hence, no major structural rearrangements are expected to accompany monomer-dimer association. Based on the results of our computational analyses, we focused our intended mutagenesis experiments on the residues of the aromatic cluster. In a former study, a Tyr330Phe exchange had already revealed a slight destabilization of the dimer.21 In the present study, we showed that the Trp326Glu exchange considerably destabilizes the dimer even though its overall architecture remains largely conserved. This observation underlines that minor structural changes may have a huge effect on stability. The transition to a predominantly monomeric state, which was confirmed by native nano-ESI-MS, is accompanied by a significantly decreased enzymatic activity. Most likely, two effects are responsible for the destabilization of the dimer. First of all, the cluster of aromatic interactions formed by Trp3260 , Tyr3300 , His3330 , and Phe92 is disrupted, which certainly exerts a strong influence on the stability of the dimer.71–74 In wild-type Tgt, the arrangement of these aromatic residues is stabilized by a network of interconnected H-bonds in which the donor functionalities of Trp326, Tyr330, and His333 are used to interact with backbone carbonyl groups in the neighboring dimer mate. Second, the polar carboxylate group of the introduced glutamate incorporates several water molecules into the interface as predicted by our MD simulations and subsequently confirmed by crystallography. Obviously, the energetic cost of desolvating this functional group cannot be compensated by any positively charged counter group in the dimer interface. Indeed, the introduced glutamate reduces the hydrophobicity of the interface and thus improves the solvation of the monomer. This explains the altered behavior of this variant during purification and necessitated establishing completely new crystallization conditions. The resulting crystals, however, exhibited unchanged crystal packing suggesting that the Tgt dimer is stabilized by its arrangement present in space group C2. Although it was reported in literature that it is


difficult to discriminate ‘real’ protein–protein interface contacts from ‘pure’ crystal packing contacts,75–78 the program PISA29 allows to estimate the contribution of a given protein–protein interface. Yet, in the present case, wild-type Tgt and the mutant variant with the Trp326Glu and Glu339Gln exchange attain nearly identical scoring values by this program. In this respect, however, it should be taken into account that at equilibrium conditions and owing to the law of mass action the proportion of the monomer decreases with increasing protein concentrations. Our native mass spectrometry experiments using a protein concentration of 10 mmolL21 indicate a monomer to dimer ratio of more than 9:1 for the double mutant Tgt(Trp326Glu/ Glu339Gln). Under crystallization conditions, however, the protein concentration is about 10-fold higher clearly supporting dimer formation. Remarkably, in wild-type Tgt, no water molecules are found in the center but only on the brink of the dimer interface. Here, they generate a residual solvation shell around charged functional groups of residues that do not have an oppositely charged interaction partner in the dimer mate. The important role of water in protein–protein interfaces, to ensure complementarity between binding partners, has been discussed in literature, in particular with respect to the tight packing of the molecules.79,80 In our Tgt variant containing the Trp326Glu mutation, we also observe water molecules that mediate contacts between groups that would otherwise repel each other. It is largely unknown whether, and if to what extent, such incorporated water molecules contribute to the destabilization of the interface.81,82 Computational approaches usually do not consider explicit water molecules in interfaces and, thus, reliable energy considerations are not available. All the more, our example underlines the important contribution of water molecules to interface stability or perturbance. Another important aspect regarding protein–protein interface complementarity is the inherent flexibility and plasticity of contacting regions.83–87 In case of the Tgt dimer, enhanced flexibility is observed for the extended loop-helix motif (residues 46–62). In the crystal structure of wild-type Tgt this motif adopts a well-defined geometry. Upon mutation of particular interface residues, however, the inherent flexibility of this stretch becomes obvious. In the structure of Tgt(Lys52Met) crystallized at pH 5.5, no electron density can be attributed to this completely disordered region. In contrast, two conformers are identified in the structure of the same variant crystallized at pH 8.5. Both of them contribute to the interface formation. While one of these conformers is nearly identical to that found in the structure of wildtype Tgt, the second one exhibits a significantly different geometry. In the structure of our double mutant Tgt(Trp326Glu/Glu339Gln), this section follows yet another course. Furthermore, in a recent study of wild-

type Tgt, inhibited by ligands that position extended substituents into this area, an even higher number of conformational states of this region was observed.88 Obviously, this loop-helix motif acts as a kind of shield protecting the hydrophobic interface hot spots from water access. This further underlines the importance of water with respect to the stability or perturbance of protein–protein interactions. Strikingly, the marked adaptability of the loop-helix motif opens the perspective to develop small molecules that stabilize this motif in a conformation detrimental for dimerization. This concept of influencing protein plasticity has already been used successfully for the development of a small-molecule protein–protein interaction modulator of interleukin-2.14 The crystal structure of the Tgt double mutant shows a newly formed cavity located next to the protein–protein interface, which is filled by 2 partly populated iodides and 12 water molecules. This novel cavity may provide a first anchor point for the design of specific inhibitors that stabilize the observed conformation of the loophelix motif and thus interfere with the formation of the interface.

CONCLUSIONS The intrinsic functions of many proteins depend on, or are regulated by, the formation of protein–protein interactions. For this reason, interfering with protein– protein interactions has great therapeutic potential. However, modulating such contacts by small-molecule inhibitors seems notoriously difficult, particularly as the principles of molecular recognition that drive interface formation are only superficially understood. Therefore, we investigated the tRNA modifying enzyme Tgt, whose function depends on a homodimeric quaternary structure. Main goal of this study was to generate a mutant variant that transforms the dimeric wild-type protein into a monomeric state. The computational analysis of the interface, which covers more than 1600 A˚2, shows that a patch of four aromatic hot-spot residues (Phe92, Trp3260 , Tyr3300 , His3330 ) constitutes a stabilizing region in the protein– protein interface. The arrangement of these aromatic amino acid residues is stabilized by H-bonds formed by their donor functionalities. As shown by native nanoESI-MS, the Trp326Glu exchange within this cluster and the concomitant Glu339Gln replacement shift the monomer-dimer equilibrium from a virtually fully dimeric state in the wild type to an almost exclusively monomeric state in the mutant variant. Interestingly enough, the crystal structure of this variant shows unchanged protein packing in the solid state with only minor changes in the interface architecture. Upon mutation, 12 water molecules are introduced into the interface, which inter alia fill the vacant space created by the PROTEINS

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replacement of the bulky tryptophan residue. In addition, these water molecules generate a solvation shell for the charged glutamate carboxylate, which is not able to form a salt bridge to an oppositely charged functional group in the interface. Ultimately, these water molecules assemble at interstitial sites to mediate and moderate interactions with the mismatching functionality introduced into the interface. Overall, we conclude that the incorporated water molecules are, together with the misplaced charge and disrupted arrangement of aromatic residues, responsible for the predominantly monomeric state of the mutated Tgt. Remarkably, a loop-helix motif, which is involved in many polar contacts across the interface and known to exhibit enhanced flexibility, adopts an alternative conformation in the Tgt variant containing the Trp326Glu exchange. In wild-type Tgt, this motif adopts a geometry that effectively shields the aromatic hot-spot region from water access. Furthermore, the crystal structure of the Tgt(Lys52Met) mutant variant, which is only marginally monomeric in solution, yet shows another conformation of this motif. This suggests that the actually adopted loophelix conformation has an important effect on the properties of the formed interface, even though its stabilization is mainly determined by the patch of aromatic residues. Influencing the geometry of this loop-helix motif by a small-molecule may provide a promising perspective to develop a novel modulator of the enzymatic function of Tgt, which constitutes a putative target for the design of drugs against Shigellosis. Further mutational studies of the aromatic amino acids residues comprising the hot-spot region will enhance our understanding of the structural aspects of protein–protein interface stability of Tgt. ACKNOWLEDGMENTS The authors gratefully acknowledge the beamline staff at BESSY II (Helmholtz-Zentrum Berlin) in Berlin, Germany, for providing us outstanding support. We thank Andreas Heine for his help during crystallography and Christian Sohn for his support during "in house" X-ray data collection. We thank the GIS IBiSA for financial support of a Synapt G2 HDMS mass spectrometer and we are grateful for the financial support by Deutsche Forschungsgemeinschaft. REFERENCES 1. Zinzalla G, Thurston DE. Targeting protein-protein interactions for therapeutic intervention: a challenge for the future. Future Med Chem 2009;1:65–93. 2. Fischer PM. Protein-protein interactions in drug discovery. Drug Des Rev 2005;2:179–207. 3. Xenarios I, Eisenberg D. Protein interaction databases. Curr Opin Biotechnol 2001;12:334–339. 4. Archakov AI, Govorun VM, Dubanov AV, Ivanov YD, Veselovsky AV, Lewi P, Janssen P. Protein-protein interactions as a target for drugs in proteomics. Proteomics 2003;3:380–391.

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Comparative analysis of the spatial analysis methods for hotspot identification.

Cellular enzyme activity at the polymer-tissue interface: a review.

Structural analysis of BRCA1 reveals modification hotspot.

Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.

Researchers Dissect the Cost of Targeted Agents.

Suicides on the Austrian railway network: hotspot analysis and effect of proximity to psychiatric institutions.

An integrated bioinformatics analysis to dissect kinase dependency in triple negative breast cancer.

Monoclonal antibodies - tools to dissect the nervous system.

A visual interface to computer programs for linkage analysis.

Cooperativity induced by a single mutation at the subunit interface of a dimeric enzyme: glutathione reductase.

Free-Energy Density Functional of Ions at a Dielectric Interface.

[Importance of morphological and functional diagnostics of the vitreoretinal interface].

Combining Quantitative Genetics Approaches with Regulatory Network Analysis to Dissect the Complex Metabolism of the Maize Kernel.

Integrating Image-Based Phenomics and Association Analysis to Dissect the Genetic Architecture of Temporal Salinity Responses in Rice.

Control of RecBCD enzyme activity by DNA binding- and Chi hotspot-dependent conformational changes.

2-Aminopurine as a fluorescent probe of DNA conformation and the DNA-enzyme interface.

Fish functional traits correlated with environmental variables in a temperate biodiversity hotspot.

Mapping paths: new approaches to dissect eukaryotic signaling circuitry.

Analysis of a theoretical model for anisotropic enzyme membranes application to enzyme electrodes.

Analysis of Different Classification Techniques for Two-Class Functional Near-Infrared Spectroscopy-Based Brain-Computer Interface.

The Use of Advanced Mass Spectrometry to Dissect the Life-Cycle of Photosystem II.

Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

Recent advances in high-throughput approaches to dissect enhancer function.

Mutagenesis and TILLING to Dissect Gene Function in Plants.