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Originally published In Press as doi:10.1074/jbc.M413623200 on January 7, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11422-11431, March 25, 2005
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Engineering a Selectable Marker for Hyperthermophiles*{boxs}

Stan J. J. Brouns{ddagger}§, Hao Wu{ddagger}, Jasper Akerboom{ddagger}, Andrew P. Turnbull¶, Willem M. de Vos{ddagger}, and John van der Oost{ddagger}

From the {ddagger}Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands and Proteinstrukturfabrik c/o Berliner Elecktronenspeicherring-Gesellschaft für Synchrotronstrahlung GmbH, Albert Einstein Strasse 15, D-12489 Berlin, Germany

Received for publication, December 3, 2004 , and in revised form, December 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Limited thermostability of antibiotic resistance markers has restricted genetic research in the field of extremely thermophilic Archaea and bacteria. In this study, we used directed evolution and selection in the thermophilic bacterium Thermus thermophilus HB27 to find thermostable variants of a bleomycin-binding protein from the mesophilic bacterium Streptoalloteichus hindustanus. In a single selection round, we identified eight clones bearing five types of double mutated genes that provided T. thermophilus transformants with bleomycin resistance at 77 °C, while the wild-type gene could only do so up to 65 °C. Only six different amino acid positions were altered, three of which were glycine residues. All variant proteins were produced in Escherichia coli and analyzed biochemically for thermal stability and functionality at high temperature. A synthetic mutant resistance gene with low GC content was designed that combined four substitutions. The encoded protein showed up to 17 °C increased thermostability and unfolded at 85 °C in the absence of bleomycin, whereas in its presence the protein unfolded at 100 °C. Despite these highly thermophilic properties, this mutant was still able to function normally at mesophilic temperatures in vivo. The mutant protein was co-crystallized with bleomycin, and the structure of the binary complex was determined to a resolution of 1.5 Å. Detailed structural analysis revealed possible molecular mechanisms of thermostabilization and enhanced antibiotic binding, which included the introduction of an intersubunit hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility, and {alpha}-helix stabilization. The potential applicability of the thermostable selection marker is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Despite the vast amount of protein sequences and structures from microorganisms that grow optimally at temperatures above 80 °C, improving the thermal stability of a protein is still a challenging task. This is mainly because the laws governing protein stability are not easily extracted because they are highly variable and complex (1, 2). It seems generally accepted that the extreme stability of certain natural proteins results from the cumulative effect of small adaptations in protein architecture and amino acid composition. Although some of these stabilizing features, such as optimized surface ion pair networks (3), are unlikely to be engineered into a protein of interest, other strategies like {alpha}-helix capping (4) and the introduction of disulfide bonds and prolines in {beta}-turns (5) can be applied very successfully when carefully designed on the basis of a high resolution crystal structure. However, in many cases atomic resolution three-dimensional information of a protein is unavailable. Directed evolution approaches, by contrast, do not require any structural information and commonly rely on random mutagenesis and recombination followed by screening or selection schemes (1, 6). Thermostability screens of mutant libraries are usually carried out by applying a thermal challenge at nonpermissive temperatures after which the remaining functionality of the individual clones is tested (7, 8). To explore sufficient sequence space requires the testing of large numbers of mutant clones, which necessitates high throughput approaches such as the use of robotics. Conversely efficient selection procedures allow the testing of a large set of variants while reducing the effort of finding improved ones to a minimum.

A convenient selection system for finding protein variants in a library with improved thermostability is based on in vivo screening in a thermophilic expression host. Cloning and selection in thermophilic microorganisms such as Geobacillus stearothermophilus (30-60 °C) or Thermus thermophilus (50-80 °C) mimic natural evolution but are only applicable when the gene of interest encodes a protein that is of biological relevance to growth or survival of the host organism (9, 10). The selective pressure can be fine tuned by raising the temperature of growth, enabling only hosts that bear thermoadapted variants to grow on solid media. For instance, a combination of in vitro mutagenesis methods and in vivo selection schemes have led to a highly thermostable kanamycin nucleotidyltransferase gene that is able to function at temperatures up to 79 °C (11). Such mutant selection markers have permitted the development of genetic tools that are very useful in the study of gene-function relationships in thermophilic bacteria (12).

In contrast to thermophiles (optimum temperature for growth, 60-80 °C), antibiotic-based genetic systems for hyperthermophilic bacteria and Archaea (optimum temperature for growth, >80 °C) are still in their infancy. This is primarily due to the absence of thermostable antibiotics and their corresponding resistance factors because most known antibiotic-producing microorganisms are mesophilic bacteria and fungi. Often the common antibiotics cannot be used because many of them are unstable at high temperatures, or hyperthermophiles are simply insensitive to them (13). The glycopeptide bleomycin is an exception since it is a highly thermostable molecule and effective against many aerobic microorganisms and eukaryotic cell lines (14, 15). The bleomycin family of antibiotics, including phleomycin and tallysomycin, are DNA- and RNA-cleaving glycopeptides that are produced by the actinomycetes Streptoalloteichus hindustanus and Streptomyces verticillus. As little as a few hundred bleomycin molecules can effectively kill aerobic cells (16). For this reason, bleomycin is currently clinically used as an antitumor agent against squamous cell carcinomas and malignant lymphomas (17). Resistance against bleomycin-like antibiotics is conferred by N-acetylation, deamidation, and sequestration of the molecule (15). The latter mechanism involves bleomycin-binding proteins (BBPs),1 which have been found only in mesophilic bacteria. Two proteins, Shble and BlmA, provide self-immunity for bleomycin producers S. hindustanus and S. verticillus, respectively (14, 18), and may be involved the transport and excretion of the molecule (19). Two genes, blmT and blmS, are located on the Klebsiella pneumoniae transposon Tn5 (20) and on the Staphylococcus aureus plasmid pUB110 (21), respectively. All four proteins are highly negatively charged cytoplasmic proteins of around 14 kDa that form homodimers that bind two positively charged antibiotic molecules at a hydrophobic subunit interface cleft (19, 22, 23). The small protein size and the wide applicability of the drug have made both shble and blmT popular dominant selection markers in vector systems for lower and higher eukaryotes, bacteria, and halophilic Archaea (15, 24, 25). This prompted us to investigate whether we could thermostabilize Shble and BlmS to allow for their application in aerobic thermophiles and hyperthermophiles.

In this study, we performed directed evolution using selection in the thermophilic bacterium T. thermophilus and obtained various mutant proteins that could operate under highly thermophilic growth conditions. Their enhanced performance at high temperature was analyzed biochemically, and possible stabilizing effects were identified.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
All chemicals were of analytical grade and purchased from Sigma. Primers were obtained from MWG Biotech AG (Ebersberg, Germany). Polymerase chain reactions were performed with Pfu TURBO (Stratagene) unless stated otherwise. Bleomycin A2 (Bleocin, Calbiochem) was used for all selections. Escherichia coli HB101 (F- hsdS20 (rB-, mB-) ara-14 galK2 lacY1 leuB6 mcrB mtl-1 proA2 recA13 rpsL20 supE44 thi-1 xyl-5 (StrR)) (26) was used for cloning purposes and routinely transformed by electroporation.

Generation of a Bleomycin-based Shuttle Vector—Bacillus subtilis 168 8G5 carrying pUB110 was kindly provided by Dr. S. Bron (University of Groningen, Groningen, The Netherlands), and the plasmid was isolated by Qiagen Miniprep according to the manufacturer's instructions. The blmS gene was PCR-amplified with primers BG1407 (sense, 5'-GGAGGTGCATATGAGAATGTTACAGTCTATCCC-3') and BG1240 (antisense, 5'-CGCGTCTAGATTAGCTTTTTATTTGTTGAAAAAAG-3') (NdeI and XbaI sites are underlined). Chromosomal DNA of S. hindustanus (ATCC 31158) was prepared according to standard procedures (27) and used for PCR amplification of the shble gene with primers BG1410 (sense, 5'-TGAGGCATATGGCCAAGTTGACCAGTGCCG-3') and BG1411 (antisense, 5'-GATCCTCTAGATTAGTCCTGCTCCTCGGCCACG-3') (NdeI and XbaI sites are underlined). PCR products were digested and ligated into E. coli-T. thermophilus shuttle vector pMK18 (28) (Biotools, Madrid, Spain) thereby replacing the kanamycin nucleotidyltransferase gene. Ligation mixtures were transformed into E. coli HB101, and transformants were plated on 1.5% LB agar plates supplemented with 3 µg/ml bleomycin. Both blmS and shble provided resistance against the antibiotic, giving rise to the 4434-bp plasmid pWUR111 and the 4404-bp plasmid pWUR112, respectively.

Mutant Library Construction—Error-prone PCR was carried out using two different polymerases, namely Taq (Amersham Biosciences) and Mutazyme (Genemorph kit, Stratagene). This approach was chosen to complement the transition and transversion bias of each enzyme to provide a more complete mutational spectrum of the PCR product. For the error-prone amplification, flanking primers BG1412 (sense, 5'-CGACCCTTAAGGAGGTGTGAGGCATATG-3') and BG1408 (antisense, 5'-CGAGCTCGGTACCCGGGGATCCTCTAGATTA-3') (NdeI and XbaI sites are underlined) were designed to allow variation throughout the entire coding sequence between the start and stop codon (indicated in boldface). Taq polymerase-based PCRs were performed as described previously (29). A 50-µl PCR contained 5 ng of pWUR112, 5 pmol of each primer, 0.2 mM dATP and dGTP, 1 mM dCTP and dTTP, 5 units of polymerase, 3 mM MgCl2, and three concentrations of MnCl2 (0.1, 0.3, and 0.5 mM). The mixture was thermocycled as follows: 95 °C (4 min); 30 cycles of 94 °C (30 s), 55 °C (45 s), and 72 °C (25 s); and postdwelled for 4 min at 72 °C. Mutazyme PCRs were prepared according to the manufacturer's instructions and thermocycled as above using an elongation time of 50 s.

Randomly mutated PCR products were cloned into vector pMK18 and transformed into E. coli HB101. A total of ~10,000 Taq- and 10,000 Mutazyme-derived clones were resuspended in 50 ml of LB medium supplemented with antibiotic and grown in 1 liter of medium to early stationary phase. Plasmids were subsequently harvested using a Miniprep plasmid isolation kit (Qiagen).

Selection in T. thermophilus—T. thermophilus HB27 was kindly provided by Dr. J. Berenguer (Autonomous University of Madrid, Madrid, Spain). Cells were routinely cultivated at 70 °C in a Ca2+-(3.9 mM) and Mg2+ (1.9 mM)-rich medium (28) containing 8 g/liter tryptone, 4 g/liter yeast extract, and 3 g/liter NaCl dissolved in Evian mineral water (pH 7.7, after autoclaving) (Evian-les-Bains, France). Transformation of T. thermophilus was essentially performed by the method of Koyama (30). Frozen cell aliquots were resuspended in 25 ml of medium and grown at 150 rpm to an A600 of 0.8. The culture was then diluted 1:1 in preheated medium and incubated for another hour. Next plasmids were added to 0.5 ml of culture, and the mixture was incubated for 2-3 h at 70 °C with occasional shaking before being plated on 3% agar plates (BD Biosciences) supplemented with 30 µg/ml kanamycin or 15 µg/ml bleomycin (Calbiochem) for selection. Colonies appeared within 36 h at 60-70 °C. At temperatures above 70 °C, 1% Gelrite plates (Roth, Karlsruhe, Germany) were used supplemented with 100 µg/ml kanamycin or 20 µg/ml bleomycin for selection. Colonies were grown overnight in liquid medium containing 30 µg/ml kanamycin or 5 µg/ml bleomycin. T. thermophilus plasmid DNA was prepared using a plasmid Miniprep kit (Qiagen) after a preincubation with 2 mg/ml lysozyme for 30 min at 37 °C.

Gene Cloning, Overexpression, and Protein Purification—Wild-type and double mutant shble genes were PCR-amplified from their respective pWUR112 plasmids using primers BG1503 (sense, 5'-GATGGCCATGGCCAAGTTGACCAGTGC-3') and BG1504 (antisense, 5'-GCCGCAAGCTTAGTCCTGCTCCTCGGCC-3') (NcoI and HindIII sites are underlined). PCR products were cloned into vector pET26b (Novagen) and fused to an Erwinia carotovora pectate lyase (pelB) signal sequence allowing periplasmic protein overexpression in E. coli BL21(DE3) (Novagen). Periplasmic fractions of 1-liter cultures were prepared by osmotic shock according to the manufacturer's instructions and dialyzed overnight against 20 mM Tris-HCl (pH 7.5). Samples were loaded onto a MonoQ HR 5/50 column connected to a fast protein liquid chromatography system (Amersham Biosciences) and eluted using a 1 M NaCl gradient. Shble-containing fractions were pooled and dialyzed against a 10 mM NaPi buffer (pH 7.0) supplemented with 50 mM NaCl and subsequently purified by size exclusion chromatography using a Superdex 200 HR 10/30 column (Amersham Biosciences).

Synthetic Gene Construction—A synthetic mutant shble gene based on archaeal codon usage was constructed by oligonucleotide assembly PCR (31). This gene contains the point mutations G18E, D32V, L63Q, and G98V and has a GC content of 40.8% compared with 70.2% of wild-type shble. The synthetic gene was designated HTS (high temperature Shble). The sequence has been deposited in GenBankTM under accession number AY780486 [GenBank] .

Assembly PCR mixtures contained 10 oligonucleotides (BG1542-BG1451, Supplemental Table 1) with an overlap of 20 bases. Both flanking primers were 40 bases in length, whereas the eight central primers consisted of 80-90 bases. The assembly PCR mixture contained a 2.5 µM concentration of each primer, 0.2 mM dNTPs, and 0.05 units/µl Pfu polymerase. The mixture was thermocycled at 94 °C (30 s), 55 °C (30 s), and 72 °C (60 s) for 40 cycles. The PCR products were purified over a PCR purification column (Qiagen) and diluted 1:1 in fresh PCR mixture containing only both flanking primers BG1542 and BG1551 at 0.1 µM concentration and were thermocycled according to standard procedures. PCR products of the expected size were isolated from an agarose gel using the Qiaex II gel extraction kit (Qiagen), digested with NdeI and BglII, and cloned into vector pET26b. This allowed for efficient cytoplasmic protein overproduction in E. coli BL21(DE3)-RIL (Novagen). Positive clones were picked from LB agar plates containing 3 µg/ml bleomycin and 50 µg/ml chloramphenicol. A 4-liter culture was grown at 37 °C to an A600 of 0.5, induced with 0.5 mM isopropyl-{beta}-D-thiogalactopyranoside, and incubated for another 5 h. Cells were harvested, resuspended in 20 mM Tris-HCl (pH 7.5), and sonicated. Cell extracts were clarified by centrifugation (30 min at 26,500 x g at 4 °C) and applied to a 70-ml Q-Sepharose Fast Flow (Amersham Biosciences) anion exchange column. Proteins were eluted by a 1 M NaCl gradient, and HTS-containing fractions were pooled and concentrated over a YM10 filter (Amicon) and further purified by size exclusion chromatography as described above.

DNA Sequencing—Inserts of plasmids used in this study were sequenced by Westburg Genomics (Wageningen, The Netherlands).

Protein Quantitation—Protein concentrations were determined by using a Bradford assay (32) (Bio-Rad). Purified proteins were quantified from A280 measurements using a protein extinction coefficient of 29,000 M-1 cm-1 (33).

DNA Protection Assay—Protein functionality assays were essentially performed as described elsewhere (14) using a 10-fold excess molar concentration of protein over bleomycin A2 (Calbiochem). In each assay, 0.2 µg of PstI-linearized plasmid, pUC19, was used. Assays were performed by first incubating DNA and protein shortly at 85 °C after which bleomycin A2, dithiothreitol, and Fe2+ were sequentially added to the reaction mixture.

Circular Dichroism Spectroscopy—CD experiments were performed on a Jasco J-715 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a PTC-348WI Peltier temperature control system. Far-UV CD measurements were conducted with Suprasil quartz cuvettes (Hellma Benelux, Rijswijk, The Netherlands) with a 1-mm cell length. During all experiments, the sample cell chamber was purged by dry N2 gas at a flow rate of 10 liters/min. In temperature-induced unfolding experiments, the cuvette containing a 1.7 µM concentration of protein sample in degassed 10 mM NaPi (pH 7.0) and 50 mM NaCl was heated from 25 to 95 °C at 0.4 °C/min and subsequently cooled to 25 °C at the same rate. The ellipticity at 205 nm was measured every 0.5 °C with a 2-s response time to monitor the loss of {beta}-sheet and {beta}-turn secondary structure elements. The bandwidth of the measurement was set to 1.0 nm, and the sensitivity was set to 100 millidegrees. Data were corrected for the temperature-dependent ellipticity of a blank without protein. Averaged data of two independent scans were fit according to a two-state model of unfolding, and the apparent temperature unfolding midpoint (Tm) was derived from van't Hoff plots.

Fluorescence Spectroscopy—Fluorescence experiments were performed on a Varian Cary Eclipse fluorometer (Varian, Middelburg, The Netherlands) equipped with a four-cuvette Peltier multicell holder and PCB-150 waterbath. All measurements were performed in 3-ml Suprasil quartz cuvettes (Hellma Benelux) with a 1-cm path length. A magnetic stirring bar ensured a homogeneous sample temperature. The temperature of the sample was recorded by a temperature probe inside one of the four samples. Spectra and thermal unfolding curves were recorded of 1.7 µM protein solutions in degassed 10 mM NaPi (pH 7.0) buffer supplemented with 50 mM NaCl. Tryptophans were excited at 295 nm, and fluorescence was recorded from 300 to 550 nm with both the excitation and the emission slits set to 5 nm. During temperature-induced unfolding and refolding studies, fluorescence emission intensities were monitored at 315 nm from 27 to 92 °C at a heating and cooling rate of 0.4 °C/min. Data were corrected for the fluorescence emission of corresponding blank solutions. Data of two independent scans were treated and fit as described above.

Differential Scanning Calorimetry (DSC)—DSC measurements were performed on a Microcal III system (Setaram, Caluire, France). Degassed protein samples of 0.28 mg/ml (20 µM) in 10 mM NaPi (pH 7.0) and 50 mM NaCl in the presence and absence of an 8-fold molar excess of bleomycin A2 were heated from 20 to 120 °C at 0.5 °C/min. Midpoint temperatures of unfolding were determined by curved base-line analysis from two independent scans.

Protein Crystallization, Data Collection, and Processing—The HTS protein was extensively dialyzed against 10 mM NaPi, pH 7.0, and was subsequently crystallized using the sitting drop method of vapor diffusion at 20 °C and a protein concentration of 3.3 mg/ml in the presence of a 10-fold molar excess of bleomycin A2 HCl (Calbiochem). Crystals grew optimally using 2.0 M ammonium sulfate as the precipitant in 0.1 M sodium acetate buffer, pH 4.6. Data were collected from a single flash frozen native crystal (100 K) to 1.5-Å resolution using a MAR345 imaging plate at the Protein Structure Factory beamline BL14.2 of the Free University of Berlin at the BESSY synchrotron source (Berlin, Germany). All data were reduced with DENZO and SCALEPACK (34). The crystal used for data collection had unit cell parameters of a = 44.0 Å, b = 66.6 Å, and c = 47.2 Å and {beta} = 117.4° and belonged to space group P21 with a dimer in the asymmetric unit.

The structure of HTS was determined by molecular replacement using the program MOLREP (35) and the S. verticillus BlmA dimer (Protein Data Bank code 1JIE [PDB] ) (23) as the search model. The initial phases were improved using the free atom refinement method together with automatic model tracing in ARP/wARP (36). Translation, libration, and screw rotation (TLS) parameters were determined, and TLS-restrained refinement was performed using REFMAC (37). Several rounds of iterative model building and refinement followed, and water molecules were added using ARP/wARP (36). The final model (comprising 241 amino acids, 288 water molecules, two bleomycin molecules, and three sulfate ions), refined using data between 30- and 1.5-Å resolution, has an R- and free R-factor of 17.4 and 19.4%, respectively, with good geometry. Residues Met-1, Glu-122, Gln-123, and Asp-124 in chain A and Met-1, Gln-123, and Asp-124 in chain B are not visible in the electron density map and therefore have been excluded from the model. Additionally the side chains of two residues in chain A (Asp-36 and Arg-87), four side chains in chain B (Glu-21, Asp-36, Arg-87, and Glu-122), and the {gamma}-aminopropyldimethylsulfonium moiety of bleomycin have been truncated in the final model. The stereochemical quality of the model and the model fit to the diffraction data were analyzed with the programs PROCHECK (38) and SFCHECK (39).

The coordinates and experimental structure factors have been deposited in the Protein Data Bank under accession code 1XRK [PDB] . Figures were prepared with Swiss-PDBviewer version 3.7 SP5 (40) and rendered with POV-Ray version 3.6.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Selection of Stabilized Variants of Shble
Randomly mutated shble genes were introduced in the E. coli-T. thermophilus shuttle vector pMK18 under the control of the promoter of the surface layer protein A (slpA) from T. thermophilus HB8 (41). This promoter is known to drive efficient transcription of the single selection marker in both bacteria. An error-prone library of ~20,000 functional clones was generated in E. coli HB101. Colonies appeared of similar size, and there was no difference between the mutant and wild-type shble phenotype. The plasmid library was harvested and transformed into T. thermophilus HB27, making use of its high natural competence (30). Thermus clones appeared on bleomycin-containing plates up to 65 °C after transformation with the wild-type shble shuttle vector, whereas wild-type blmS was unable to generate a resistant phenotype at either 50 or 65 °C. The transformation efficiency of the shble shuttle vector was approximately 5 times lower at 65 °C than the kanamycin-based vector pMK18 (28). This difference might be due to the lethal effect of bleomycin and the non-catalytic nature of its elimination, which requires at least one protein molecule per bleomycin molecule.

Upon increasing the temperature of selection, a dramatic decrease in the number of colonies was observed after transformation of 8 µg of mutant library DNA. While 1200 colonies appeared at 67 °C, this number decreased to 800 at 69 °C, 600 at 70 °C, 106 at 75 °C, and eight at 77 °C. No colonies appeared at 78 and 80 °C. Plating efficiencies at these temperatures have been reported to be severely reduced, complicating selection up to 85 °C, the maximum temperature of growth (11). The eight Thermus clones found at 77 °C (termed 77-1 to 77-8) were grown overnight in selective medium at 70 °C; their plasmids were isolated, transformed into E. coli HB101, and subsequently reisolated; and their inserts were sequenced. This revealed that all variants were double mutants bearing, in total, six different amino acid substitutions and three silent mutations (Table I). Five types of double mutants could be distinguished at the protein level, and two sets of double mutants were identical. Remarkably three of six mutations found were glycine substitutions of which glycine 98 was replaced by either a valine or a serine. The fact that only double mutants were found seems to be a clear indication of the high stringency that was used during selection. Interestingly some substitutions, such as L63Q, had occurred in combination with either G18E, D32V, or G98V, which may point to the independent effects of the different mutations. A multiple sequence alignment of BBPs and the position of the mutations are shown in Fig. 1. To assess the reason why these mutants performed better at elevated temperatures in vivo, we produced and purified wild-type Shble and all double mutants and studied their biochemical behavior in vitro. Furthermore a synthetic quadruple mutant gene with low GC content was designed by combining mutations G18E, D32V, L63Q, and G98V. The protein, designated HTS, was produced, purified, and biochemically analyzed. The HTS protein was crystallized in complex with bleomycin A2, and its structure was determined.


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  		TABLE I
Nucleotide and amino acid substitutions of Shble variants

 



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  		FIG. 1.
Structural alignment of bleomycin-binding proteins from different microbial sources. Shble (Protein Data Bank code 1BYL [PDB] ) from S. hindustanus (19), BlmA (Protein Data Bank code 1QTO [PDB] ) from S. verticillus ATCC15003 (45), SvP from S. verticillus ATCC21890, BlmT (Protein Data Bank code 1ECS [PDB] ) from K. pneumoniae transposon Tn5 (22), and BlmS from S. aureus plasmid pUB110 are shown. The alignment was created by backbone superimposition of the three structures and expanded with the SvP and BlmS sequences by realignment using ClustalX version 1.81 (65) while maintaining the original gaps. The HTS structure was used for residue numbering and topology assignment (black arrows, {beta}-strand; checkered boxes, {alpha}-helix). Mutations are indicated by arrows.

 
Thermal Unfolding
Shble variants were subjected to temperature-induced equilibrium unfolding experiments in the presence and absence of bleomycin. The protein was found to unfold largely irreversibly since only 40% of the native folded signal was regained after slow cooling of the thermally unfolded protein. Therefore only apparent midpoint temperatures of unfolding (Tm) could be calculated. The results are summarized in Table II.


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  		TABLE II
Apparent thermal unfolding midpoints (°C) of Shble variants

 
In the absence of the antibiotic, wild-type Shble appears to be a very stable protein. This is remarkable because S. hindustanus grows optimally at 28 °C (42). It is often found, however, that proteins for which low biological turnover is beneficial for a host are prone to little local unfolding and hence are less susceptible to proteolytic attack (43). Structurally Shble, which serves a function of self-immunity, might well be adapted to meet these criteria by its compactness, relatively high secondary structure content, high surface charge, and embedded N and C termini (19). The unfolding data also clearly show the strong stabilizing effect of ligand binding on the thermostability of the BBP since the apparent unfolding midpoint temperature increases 27.3 °C upon bleomycin binding. This effect has also been recognized in other ligand-binding proteins, such as streptavidin and avidin, which become extremely thermostable in the presence of biotin (44). In the absence of bleomycin, the stability of the various mutants is rather different. Of the double mutants, only 77-3 (D32V,L63Q) seems to have a marked increase in Tm as observed with CD spectroscopy and fluorescence spectroscopy, while numbers 77-1 (L63Q,G98V), 77-5 (R31L,G98S), and 77-7 (G18E,L63Q) remain virtually unchanged. Surprisingly mutant 77-4 (R31L,G40A) displays significantly lower Tm values compared with the wild-type. Quadruple mutant HTS, which combines non-redundant mutations found in 77-1, 77-3, and 77-7, displays a profound increase of 13.9, 10.8, and 17.7 °C in stability in the absence of the antibiotic as found by CD, fluorescence spectroscopy, and DSC, respectively. In its presence, the complex becomes hyperthermostable, unfolding at a temperature of just over 100 °C, 5.6 °C higher compared with the wild-type protein.

To our surprise, the double mutants 77-1, 77-5, and 77-7 had almost unchanged apparent melting temperatures compared with the wild-type. This can be understood by realizing that in vivo some amino acid changes may prevent instances of local protein unfolding and therefore may avoid further unfolding and subsequent proteolytic attack. However, this is not necessarily reflected in its in vitro melting temperature, which is a measure of its global stability. Only when the weakest point of a structure was compensated (D32V and L63Q in 77-3), an increase of its melting temperature from 70.8 to 79.5 °C with CD and 67.9 to 69.1 °C with fluorescence spectroscopy was observed. Adding mutation G98V from 77-1 and G18E from 77-7 to 77-3, giving rise to HTS, further increased its melting temperature as one would expect. This observation is analogous to the findings of extensive work that has been conducted with the neutral protease from G. stearothermophilus where interactions close to the N terminus were found to be limiting the global stability (5).

Mutants Improve DNA Protection against Bleomycin at High Temperature
In vitro DNA protection assays were performed with the various Shble mutants to test whether the resistant phenotype of T. thermophilus at 77 °C was due to improved protection against the DNA degrading capability of bleomycin. The result of this is shown in Fig. 2. At 25 °C, no significant differences in band intensities were observed. A 30-min thermal preincubation of the protein at 85 °C, however, revealed a drastic loss of function in mutant 77-4. Differences between the wild-type and mutants became pronounced when bleomycin binding capabilities were tested at 85 °C. At this temperature, the DNA was protected best by 77-1 and HTS followed by 77-4, 77-5, 77-3, 77-7, and the wild type. Surprisingly mutant 77-4, which displayed a low temperature unfolding midpoint and high thermal inactivation at 85 °C, apparently bound bleomycin effectively at high temperature conditions. So although the global stability of this mutant was decreased, it had improved bleomycin binding characteristics, which in itself stabilizes the protein dramatically as observed by DSC measurements for the wild type. These results indicate that some of the double mutants have improved the bleomycin binding properties compared with the wild type, confirming the findings of the in vivo selection procedure in T. thermophilus. Possible structural explanations for the improved functionality at higher temperature are discussed below.



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  		FIG. 2.
DNA protection assay. Digital photographs of 1% agarose gels showing the degree of DNA protection by Shble variants against the strand scission action of bleomycin A2 are shown. A, assay at 25 °C for 10 min. B, assay at 25 °C for 10 min after protein preincubation at 85 °C for 30 min. C, assay at 85 °C for 10 min. ble, bleomycin A2; WT, wild type.

 
Overall Structure Description
The quadruple mutant HTS was crystallized in the presence of bleomycin A2, and its structure was determined to 1.50-Å resolution (Table III). The crystals grown belong to space group P21 with unit cell parameters of a = 44.0 Å, b = 66.6 Å, and c = 47.2 Å and {beta} = 117.4° and a dimer in the asymmetric unit (Fig. 3, A and B). Representative electron density is shown in Fig. 3C. The structure forms a compact, homodimeric {alpha}/{beta} protein of 121 amino acids (Met-1, Gln-123, and Asp-124 are disordered) in which two bleomycin A2 molecules are accommodated in binding pockets at the dimer interface. These pockets consist of a hydrophilic concavity that runs into a hydrophobic intersubunit crevice. The dimer is maintained by alternate N-terminal {beta}1-strand hydrogen bonding between both monomers and by Van der Waals interactions at the largely hydrophobic subunit contact (19, 45). Three sulfate ions are present at the surface of the dimer of which two form ion pairs with Arg-104 of both chains. The presence of a dimer in the asymmetric unit allowed the identification of certain symmetry deviations between both monomers. A backbone superimposition of both chains (r.m.s.d., 0.38 Å; Table IV) only revealed large differences in a random coil region comprising residues Asp-88, Ala-89, and Ser-90 (Fig. 3, A and B) that is spatially close to the carbamoyl group of the D-mannose moiety of bleomycin (Fig. 4A). Their respective C{alpha} atoms deviate 2.0, 5.1, and 1.6 Å in position while giving rise to almost oppositely pointing amino acid side chains. In contrast to the bleomycin-bound and unbound BlmA structure, backbone B-factors in this region are only marginally higher compared with the average value, suggesting a rigid conformation (23, 45). The difference in orientation of this loop might therefore be the result of sequential binding of two bleomycin molecules. Unlike BlmA, no symmetry-related differences were observed in the region between amino acids 100 and 103. The topology of the HTS protein complex and the mode of bleomycin binding are similar to other BBPs. An overview of available structures is given in Table IV.


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  		TABLE III
Data collection and refinement statistics Values in parentheses refer to the highest resolution shell.

 



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  		FIG. 3.
Structure and electron density of HTS in complex with bleomycin A2. A ribbon diagram showing the dimeric structure of the 4-fold mutant Shble in complex with bleomycin A2 is shown. Mutations are indicated by stick representations. Chain A is in blue, chain B is in red, G18E is in green, D32V is in pink, L63Q is in yellow, and G98V is in orange. A, side view. B, viewed from the N- and C-terminal side (top view). C, stereoview of the electron density around residues Pro-9 and Trp-65 contoured at 2 {sigma}. Residues are colored according to the Corey-Pauling-Koltun color scheme, and water molecules are represented by red spheres.

 


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  		TABLE IV
Crystal structures of bleomycin-binding proteins

 



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  		FIG. 4.
Chemical diagram and electron density of bleomycin A2. A, schematic representation of bleomycin A2. B, electron density around bleomycin A2 contoured at 1.5 {sigma}. The diagram indicates the missing electron density around the {gamma}-aminopropyldimethylsulfonium moiety suggesting a disordered conformation.

 
The structure of the HTS mutant in complex with bleomycin completes the list of structural information of three BBPs with and without their ligands, hereby contributing to our understanding of these proteins in general. Moreover the structure has revealed several molecular features that can account for increased protein stability and improved functionality at higher temperature in vivo and in vitro.

Structural Effects of Mutations
Introduction of an Intersubunit Hydrogen Bond Network—The structure of the dimer shows that each of the two bleomycin A2 molecules is bound by the concerted action of 21 amino acids. Due to its intersubunit location, both binding sites are composed of residues from either subunit. These include Val-32, Phe-33, Glu-35, Phe-38, Ser-51, Ala-52, and Val-53 of one subunit and Pro-59, Asp-60, Asn-61, Thr-62, Gln-63, Trp-65, Phe-86, Ala-89, Trp-102, Ala-107, Arg-109, Gly-113, Cys-115, and His-117 of the other. The crystal structure clearly reveals the central role of mutation L63Q, which was found in three of five different double mutants. Gln-63 is involved in an extensive hydrogen bond network at the bottom of the bleomycin binding concavity (Fig. 5A). It is noteworthy that the carbonyl side chains (O-{epsilon}1) of both Gln-63 residues in the dimer act as terminal hydrogen bond acceptors of a five-molecule water channel present at the dimer interface. A second hydrogen bond is accepted from the side chain hydroxyl group (O-{gamma}) of Ser-51 of the adjacent subunit. The amide side chain (N-{epsilon}2) of Gln-63 forms a hydrogen bond with one of two water molecules trapped between the bleomycin and the surface of the protein. The presence of a leucine at position 63 would most likely not have allowed for a hydrogen bond network of this size. The advantage of an amino acid compatible with hydrogen bonding at position 63 is also evident from the alignment, which indicates that without exception the other four BBPs have a serine at this specific site (Fig. 1). Although the mutant structure without bleomycin is not available, we speculate that an intersubunit hydrogen bond between Gln-63 and Ser-51 can persist even without the antibiotic bound, giving rise to a beneficial interaction that might stabilize the dimer at high temperatures.



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  		FIG. 5.
Structural effects of the individual mutations. A, L63Q. A ribbon diagram showing the hydrogen bond network at the dimer interface is shown. Thr-62, Gln-63, Ser-51, and bleomycin A2 are shown together with the intersubunit water channel. B, D32V. A ribbon diagram showing the hydrophobic intersubunit bleomycin tail binding crevice is shown. Val-32 may be involved in improved hydrophobic packing of this surface indentation among amino acids Phe-33, Phe-38, Val-42, Thr-47, and Phe-49. C, G98V. A ribbon representation showing a loop between Pro-92 and Pro-111 that is involved in bleomycin binding is shown. Val-98 is located at a former hinge region that enables Trp-102 to stack the bithiazole tail against Phe-33 and Phe-38. The electron density revealed two alternative side chain rotamers for Val-98 in chain A (not shown) and a single side chain conformation in chain B. D, G18E. A side-by-side comparison of {alpha}-helix 1 formed between Asp-15 and Leu-27 in the wild-type and mutant crystal structures of Shble. The existence of a surface ion pair between Glu-21 and Arg-26 is visible in the electron density (contoured at 1.5 {sigma}). Interatomic distances are indicated in Å. Corey-Pauling-Koltun color coding was used for amino acids and bleomycin A2. Chain A is indicated in blue, and chain B is indicated in red. Water molecules are represented by red spheres. Hydrogen bonds are depicted by green dotted lines.

 
Hydrophobic Packing of Surface Indentations—In wild-type Shble and BlmA, Asp-32 is located on the edge of the intersubunit binding groove for the bithiazole moiety and tail region of bleomycin (Fig. 4A) (19, 23, 45). In bleomycin A2 and B2 and in phleomycin D1 the tail is positively charged, suggesting involvement of Asp-32 in electrostatic stabilization or ligand recognition. From NMR studies it has become clear that in the bound state no strong interactions occur between the protein and the positively charged tail of bleomycin (46). These data are supported by the absence of electron density for the {gamma}-aminopropyldimethylsulfonium moiety of bleomycin A2 in the binary complex structure of BlmA and HTS, suggesting a disordered conformation of the tail end (Fig. 4B) (23). This might have allowed for an amino acid substitution to valine, which extends the hydrophobic bithiazole binding cleft at the dimer interface fitting nicely within a highly hydrophobic environment consisting of Phe-33, Phe-38, Val-42, Thr-47, and Phe-49 (Fig. 5B). In addition, both BlmT and BlmS sequences also contain a valine at the corresponding position (Fig. 1). From a thermodynamic point of view, a mutation introducing surface hydrophobicity is generally believed to be unfavorable and has therefore rarely been investigated in directed mutagenesis studies. Nevertheless some studies have reported significant improvements in protein stability by placing bulky hydrophobic amino acids at the surface of a neutral protease from G. stearothermophilus (47). Recent findings using Bacillus licheniformis {alpha}-amylase have clearly indicated that hydrophobic surface residues can indeed be extremely stabilizing by improving hydrophobic packing of surface indentations, thereby reinforcing subsurface secondary structure elements (48). This might explain the enhanced secondary structure preservation at high temperatures as inferred from CD of mutant 77-3. Strikingly modeling of G40A into the wild-type structure (not shown) revealed close spatial proximity to Asp-32 (5 Å between C{alpha} and 3.5 Å between C{beta} atoms) that may also underline a similar need for hydrophobicity in this part of the protein. Mutation Arg-31 to Leu, which occurred in two types of double mutants, came as a surprise since it is involved in a surface ion pair with Asp-25 in wild-type Shble. Apparently this electrostatic interaction does not counterweight the beneficial effects of improved hydrophobic packing among residues Val-20, Thr-24, Val-34, and Val-41.

Reduction of Surface Loop Flexibility—From previous crystallographic and NMR studies of BBPs, it has become clear that the loop following Gly-98 in Shble will change its conformation upon binding of the antibiotic (22, 23, 45, 46). This conformational change enables the tryptophan at position 102 to stack optimally with the hydrophobic bithiazole moiety of bleomycin (Fig. 4A), packing both thiazole rings tightly against Phe-33 and Phe-38 of the adjacent subunit. In both BlmA and Shble, Gly-98, located on the edge of a small {beta}-strand leading toward the binding loop, seems to have a hinge function (Fig. 5C). The bending motion of the backbone is also clearly reflected in large {varphi} and {Psi} torsion angle changes of more than 20° upon the binding of bleomycin. At high temperatures, however, this flexibility might have caused problems leading to local unfolding or a decreased bleomycin binding ability. A substitution for either a valine or serine as observed would increase the rigidity of the loop and could therefore restore the binding capacity at high temperature.

{alpha}-Helix Stabilization—Mutation G18E introduces a glutamate at position N-3 in the first turn of the largest {alpha}-helix of the protein. Statistical analysis and experimental studies have shown that glutamates are energetically highly favored over glycines at the third position in an {alpha}-helix (49, 50). This effect is most likely caused by the stabilizing effect of the negatively charged side chain on the helix macrodipole. To our surprise, chain A of the crystal structure revealed the formation of a genuine i, i + 5 {alpha}-helix surface ion pair between Glu-21 and Arg-26 that was absent in the wild-type structure (Fig. 5D). This new ion pair may have been the result of repulsion of anionic glutamate side chains of positions 18 and 21, directing the latter toward the C-terminal arginine. Although i, i + 5 {alpha}-helical surface ion pairs do not give rise to strong ionic interactions at ambient temperatures (51), they might be more favorable at higher temperatures. Theoretical models have indicated that the energetic cost of desolvating charged groups is much less at 100 °C due to a drop in the dielectric constant of water (52). This is currently the best explanation for the fact that proteins from extreme thermophiles have large ion pair networks at their surfaces that are thought to be involved in maintaining structural integrity (3). Additionally a minor beneficial effect of this mutation could be the introduction of additional negative surface charge, which enhances electrostatic attraction of the cationic antibiotic under physiological conditions.

Laboratory Versus Natural Evolution of Thermostability—In this study, several possible mechanisms of adaptation to high temperature were identified, such as the introduction of a hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility, and {alpha}-helix stabilization. Remarkably half of all mutations found were glycine replacements, which could point to protein stabilization by decreasing the entropy of the unfolded state (53). Although this could be a general strategy of stabilization, proteins from hyperthermophiles do not have a lower glycine content than their mesophilic counterparts but rather a slightly increased one (54). Their predicted proteomes do have an increased propensity for charged (Arg, Lys, and Glu) and bulky aliphatic (Ile and Val) amino acids that has mostly come at the cost of polar residues (Asn, Gln, Ser, and Thr) (54). This is fully in agreement with the requirements for the elevated numbers of surface salt bridges and improved hydrophobic core packing that has generally been recognized in these types of proteins. These are just two of a multitude of mechanisms that proteins from hyperthermophilic microorganisms have used to deal with extreme temperatures (2, 55, 56).

Recently several other random mutagenesis studies have also reported large improvements in thermostability by applying directed evolution approaches. A mesophilic xylanase of family 11 was stabilized by over 35 °C by combining nine mutations found separately after extensive screening. The activity of this mutant was optimized by saturation mutagenesis of all mutated positions, yielding an enzyme variant with highly enhanced properties for high temperature applications (8). In another study, a highly thermostable esterase containing seven mutations was evolved in six rounds of random mutagenesis, recombination, and screening (7). The resulting enzyme was crystallized, and its structure was determined (57). The structure revealed that improved stability was due to altered core packing, {alpha}-helix stabilization, the introduction of surface salt bridges, and reduction of flexibility in surface loops. From these and many other directed evolution and site-directed mutagenesis studies, it has become apparent that (i) proteins can be stabilized substantially by small numbers of mutations, (ii) these mutations are often located at the protein surface, and (iii) their effects are usually additive. As few as two of 12 amino acid differences between a mesophilic and thermophilic cold shock protein turned out to be responsible for the difference in thermostability (58). The remaining variation in sequence might just have occurred as a result of neutral sequence drift or specific properties required by the host, such as solubility, turnover, and molecular interactions (59).

Despite the fact that only a small number of mutations are required to render a protein thermostable, finding those mutations remains a difficult task. Apart from screening vast numbers of random mutants in microtiter plates, in vitro and in vivo selection schemes offer great advantages to reduce the effort to encounter improved variants. Currently two main strategies are available.

One of these thermostabilizing selection methods is called Proside (protein stability increased by directed evolution) which is based on the empirically derived inverse correlation between protein thermostability and proteolytic susceptibility (60). In a phage display-like procedure, a library of mutants is fused between two domains of E. coli filamentous phage Fd gene-3-protein, which is then subjected to proteases while inducing local unfolding of the target protein by means of temperature or chemical denaturants. The resulting unstable fusion protein variants are cleaved, causing only the surviving, more stable phages to be found after infection. It was found that in an ionic denaturant, non-polar surface interactions were optimized, whereas at elevated temperature variants with improved surface electrostatics were selected (59).

Cloning and selection in a thermophile, as conducted in this study, is a second directed evolution strategy that can lead to rapid improvements in thermostability. Despite its simplicity, very few studies using this technique have been reported. This is most likely due to its limited applicability because the gene of interest rarely confers any biologically relevant function for growth or survival of the thermophile. Nonetheless functionally stabilized mutants have been reported up to 79 °C for a kanamycin nucleotidyltransferase (9, 11) and 58 °C for a chloramphenicol acetyltransferase (61). Auxotrophic knock-out strains for leucine biosynthesis were complemented with thermolabile counterpart genes from B. subtilis and Saccharomyces cerevisiae and adapted to higher temperature by serial accumulation of beneficial mutations in the in trans introduced 3-isopropylmalate dehydrogenase genes (62, 63). A hybrid {alpha}-galactosidase consisting of Bacillus stearothermophilus and T. thermophilus peptide regions was adapted to function at 67 °C by selection for growth on melibiose as the sole carbon and energy source (64).

Concluding Remarks
In this study, several double mutants of a bleomycin-binding protein were isolated with enhanced performance at high temperature both in vivo and in vitro. Structural analysis showed that the mutations gave rise to different means of stabilization in four parts of the protein. A combined mutant gene with a low GC content was created that can serve as an antibiotic resistance marker for aerobic and microaerophilic mesophiles, thermophiles, and hyperthermophiles. This may allow the development of efficient shuttle vectors and knock-out strategies for hyperthermophilic Archaea and bacteria based on positive selection schemes. Moreover the high GC content double mutant genes will now permit multigene knock-out strategies in thermophiles such as T. thermophilus, allowing further exploration and exploitation of thermophilic microbial sources.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY780486 [GenBank] .

The atomic coordinates and structure factors (code 1XRK) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by a grant from the European Union in the framework of the SCREEN project (Contract QLK3-CT-2000-00649). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Table 1. Back

§ To whom correspondence should be addressed. Tel.: 31-317-483110; Fax: 31-317-483829; E-mail: stan.brouns{at}wur.nl.

1 The abbreviations used are: BBP, bleomycin-binding protein; Shble, BBP from S. hindustanus; BlmA, BBP from S. verticillus; BlmT, BBP from transposon Tn5; BlmS, BBP from plasmid pUB110; HTS, high temperature Shble; r.m.s.d., root mean square deviation; DSC, differential scanning calorimetry. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. Berenguer for helpful suggestions and Anton Korteweg for technical assistance with DSC.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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