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J. Biol. Chem., Vol. 281, Issue 52, 40252-40263, December 29, 2006
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From the Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Received for publication, March 16, 2006 , and in revised form, September 7, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the 
cI repressor, containing an NIapro cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIapro proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The use of inhibitory molecules directly acting at the protein level is thus a complementary approach. This strategy allows the targeting of single functions residing in different domains of multidomain protein complexes or those due to post-translational modifications. An important consideration is also that such proteinaceous inhibitors may be used in the subsequent characterization of the corresponding target protein in vitro or may even serve as first leads in the drug discovery process.
Intracellular proteinases are important regulators of signal transduction, RNA transcription, cell cycle progression, apoptosis, and development (6, 7) and many other processes, and they are also used by viruses in the processing of polyprotein precursors (8). To elucidate their function early in the discovery process, specific small molecule inhibitors will usually not be available, and thus a rapid approach to generate specific inhibitors that function inside the cell would be very valuable.
One way of approaching this challenge would be to use artificial proteinase inhibitors based on proteins. Although a large number of protein families have been used by nature for this purpose (10), the great majority of these inhibitors are secreted proteins and contain disulfide bonds. Thus, they work naturally on secreted proteinases and, consequently, have been re-engineered to target extracellular proteinases (9). Even though there are also natural intracellular proteinase inhibitors controlling many of the processes mentioned above (10), they have not been used as scaffolds for deriving new specificities up to now.
Another approach would be to use scaffolds that are not derived from proteinase inhibitors for this purpose. The generation of novel inhibitors is difficult, because polypeptides are first and foremost substrates of proteinases. The challenge is thus to achieve selective binding without cleavage or by maintaining a stable complex between proteinase and inhibitor even after cleavage of the latter.
An antibody scFv fragment that works in the reducing intracellular milieu (11) has been reported for this purpose. However, because these molecules also rely on disulfide bonds for stability (12-14), they may not provide a general solution for this kind of application. We therefore wished to investigate whether another class of proteins, repeat proteins, can be engineered to act as proteinase inhibitors.
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By structural and sequence consensus analysis of this modular architecture of natural repeat proteins, we constructed highly diverse combinatorial DARPin libraries (15, 21, 22). These libraries consist of an N-terminal capping repeat, a defined number (typically 2 or 3) of engineered randomized internal repeats, and a C-terminal capping repeat (denoted an N2C and an N3C library; Fig. 1) in a single protein chain, and the molecules assume the ankyrin fold. The theoretical diversity exceeds 1014 for the N2C library and 1023 for the N3C library (22).
Unselected members of these libraries show very favorable biophysical properties (16, 22, 23), and selected members interact with their target molecules via their randomized positions in a highly specific manner (16, 24). DARPins do not rely on disulfide bonds for their stability, nor do they contain free cysteines. Furthermore, they do not show structural similarities to known naturally occurring proteinaceous inhibitors.
Here, we investigated whether DARPins can be selected to inhibit the main proteinase responsible for virus maturation of an agriculturally important plant virus, the NIapro proteinase of tobacco etch virus (TEV). NIapro is the main proteinase of potyvirus; it is responsible for two-thirds of all cleavage reactions occurring during the viral infection cycle, and its functionality is vital for successful virus propagation (25-27). Our aim to identify DARPin-based proteinase inhibitors was further encouraged by recent findings that naturally occurring proteinase inhibitors could mediate resistance against potyviruses in transgenic plants (28). In addition, NIapro is structurally highly homologous to the 3C proteinases of the picornavirus family, which are the major cause of numerous human diseases worldwide (29). Furthermore, this proteinase can be expressed in functional form in Escherichia coli without harm to the cell, as its highly specific cleavage reaction does not seem to destroy vital E. coli proteins.
To accomplish our task, we applied a two-step approach of in vitro selection for binding, followed by in vivo activity screening. Although many assays exist to study proteinase activity in vitro (30-32) and in vivo (6, 33, 34), these assays either need purified protein or they lack ease of handling. Therefore, we adapted a known bacterial two-hybrid system (35) to serve as an in vivo proteinase activity screen. We were able to select and characterize in vivo active proteinaceous DARPin NIapro inhibitors. The potential of DARPins as a basis for proteinase inhibition and as a general intracellular target validation tool is discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids—Plasmids used in this study are listed in Table 1, and their construction is described in detail in the Supplemental Material. The sequences of all inserts in plasmids that were generated by PCR were confirmed by DNA sequencing.
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pBRcI-T- is a derivative of pBRcI- (35) containing a NIapro cleavage site. pMAKcI-T--pD is a derivative of pQI-pD (39) and constitutively expresses the cI-T- fusion protein under control of the 
-lactamase promoter Pbla.
pMAKcI-T--DL is a derivative of pMAKcI-T--pD in which the pool of DARPins enriched by ribosome display, binding NIapro, replaces phage protein D (gpD). Expression of the DARPin pool is under control of the IPTG-inducible promoter PT5/lac. It was used in all in vivo screening experiments. The open reading frames of the ribosome display-selected DARPins were digested with NcoI and HindIII and ligated into pMAKcI-T--pD, yielding the selection plasmid ready for in vivo screening.
The pMAKcI-T--DL tag is a derivative of pMAKcI-T--DL and was used for the DARPins 9_1s, 13_1b, 20_2b, and E2_5 in the Western blot experiments. In this vector the DARPin genes lack their N-terminal RGS-His6 tag. pAT223-TEV is a derivative of pAT223 and was used for the expression of His-tagged biotinylated and nonbiotinylated gpD-NIapro fusion protein.
Protein Production and Purification—The biotinylated fusion protein pD-NIapro and biotinylated pD alone (plasmids pAT223_TEV and pAT222) were produced by in vivo biotinylation of the N-terminal Avi tag (40) by co-expression of BirA from the plasmid pBirAcm in E. coli XL-1 Blue (Stratagene, La Jolla, CA) according to the protocols of Avidity (Denver) and Qiagen (Hilden, Germany). Efficient biotinylation was confirmed by ELISA and Western blotting using a streptavidinalkaline phosphatase conjugate as detection agent (Roche Applied Science) and by mass spectrometry. Nonbiotinylated pD-NIapro for the ELISA analysis was produced in the same way as the DARPin proteins (22) using pAT223_TEV in E. coli XL-1 Blue. His tag purification of all proteins was carried out as described (22).
In Vitro Selection with Ribosome Display—The DARPin library generation has been described (22). In this study, an N2C and an N3C DARPin library were used, encoding DARPins consisting of a constant N-terminal capping repeat, two or three internal designed ankyrin repeats, respectively, containing randomized residues, and a constant C-terminal capping repeat as a continuous polypeptide chain. The PCR-amplified libraries in the ribosome display format were transcribed in vitro, and four standard ribosome display selection rounds were carried out as described (16, 41).
In Vivo Screening—The pools of NIapro binders selected by ribosome display from selection round three and four, starting from the N2C and the N3C library, were combined and ligated into pMAKcI-T--pD, thereby replacing phage protein D (gpD) and generating pMAKcI-T--DL, co-introduced with pZA55-TEV into E. coli KS1
Z (35) and plated on LB agar plates containing 1% glucose, 50 µg/ml ampicillin, 20 µg/ml tetracycline, 0.2% arabinose, 20 µg/ml X-gal, and 20-25 µM IPTG. Cells were grown at 30 °C overnight and checked for blue color development after various times. pMAKcI-T--DL clones were isolated from different blue colonies and re-introduced together with pZA55-TEV into fresh E. coli KS1Z cells, and the screening step was repeated to confirm the phenotype and to eliminate false positives. The DNA of those clones confirmed twice as positive was sequenced using standard DNA sequencing.
Size-exclusion Chromatography—Immobilized metal ion affinity chromatography-purified DARPins were analyzed on a Superdex 200 HR gel-filtration column (Amersham Biosciences) at room temperature using a SMART chromatography system (Amersham Biosciences) at a flow rate of 60 µl/min. TBS150 (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) was used as running buffer.
ELISA—The biotinylated antigens (pD or pD-NIapro) were immobilized as follows: neutravidin (66 nM, 100 µl/well; Pierce) in TBS150 was immobilized on a Maxisorp plate (Nunc, Roskilde, Denmark) by overnight incubation at 4 °C. The wells were then blocked with 300 µl of 0.5% bovine serum albumin (Fluka, Buchs, Switzerland) in TBS150 for 1 h at room temperature. Biotinylated antigen (100 µl; 1 µM) in TBS150 with 0.5% bovine serum albumin was allowed to bind for 1 h at 4°C. To test whether the binding of the selected DARPins was specific for NIapro, 100 µl of purified DARPins (1 µM) were applied to wells with or without immobilized antigen for 1 h at room temperature. After extensive washing with TBS150, binding was detected with an anti-RGS-His antibody (Qiagen; detects only the N-terminal RGS-His6 tag of the DARPin but not the internal (pD-NIapro) or C-terminal (pD) His6 tag of the antigen), an anti-mouse-IgG-alkaline phosphatase conjugate (Pierce), and p-nitrophenyl phosphate (Fluka). For competition ELISA, the purified DARPins were incubated with 5 µM of free NIapro prior to and during (4 °C, 100 min) the binding reaction.
Surface Plasmon Resonance—SPR was measured using a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden). The running buffer was 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% Tween 20. A streptavidin SA chip (BIAcore) was used with 2000 response units of biotinylated pD-NIapro immobilized. The interactions were measured at a flow of 50 µl/min with a 5-min buffer flow, a 5-min injection of NIapro binding DARPins in varying concentrations (45 nM to 100 µM), and a dissociation step of 10 min with buffer flow. The signal of an uncoated reference cell was subtracted from the measurements. The equilibrium data of the interaction were evaluated with a global fit using BIAevaluation 3.0 (BIAcore), Scrubber (BioLogic software, Campbell, Australia), and Clamp (42).
Western Blot Analysis—Prior to Western blot analysis, antigenic samples were normalized to cell density (A600), and proteins were separated by standard 15% SDS-PAGE. Western blot analysis was done following the protocol of Ref. 43. An Immobilon-P transfer membrane (Millipore, Billerica, MA) was used for sample transfer using semi-dry electroblotting. Sample detection was achieved using an anti-tetra-His antibody (Qiagen) and an anti-mouse-IgG-alkaline phosphatase conjugate (Pierce) or an anti-cI antibody (Invitrogen) and an anti-rabbit-IgG-horseradish peroxidase conjugate (Sigma). Blots were developed using nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate as substrates or chemiluminescent horseradish peroxidase substrate (Millipore).
In Vitro Inhibition Study—NIapro activity assays were performed essentially according to published procedures (44) with small modifications. Briefly, NIapro (3 µM) and selected DARPins or DARPin E2_5 (300 µM) were preincubated in NIapro reaction buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol) at room temperature for 5 min prior to starting the measurement. The reaction was started by adding 7.5 µl of 200 µM substrate (Ac-TENLYFQ-amc, where amc is 7-amino-4-methyl-coumarin) to the reaction mixture (Vfinal, 100 µl; cfinal substrate, 15 µM), and initial rates of substrate hydrolysis were immediately recorded by fluorometric measurement of the emission intensity. The assay was carried out at room temperature. The excitation wavelength was 360 nm, and the emission wavelength was 465 nm. Initial velocity data of substrate hydrolysis in the presence of the selected DARPins or E2_5 were normalized to initial velocity data obtained from measurements without DARPins; the value obtained here was arbitrarily set to 100%. All experiments were at least done in triplicate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit of E. coli RNA polymerase (RNAP) connected covalently to a DNA-binding protein (bacteriophage repressor cI (35)).
A Genetic Activity Screen for Site-specific Proteolytic Enzymes— The basic strategy of our genetic screen is outlined in Fig. 2. The subunit of the RNA polymerase of E. coli can function as an activator of transcription when connected by a peptide linker to a DNA-binding protein that binds upstream near a promoter sequence (35). We reasoned that insertion of a defined proteinase cleavage site into this linker would allow its cleavage upon co-expression of a corresponding proteinase. This would destroy the activator, thereby abolishing transcription of a reporter gene (-galactosidase) from an appropriately constructed promoter present in the chromosome of E. coli KS1Z (35). Consequently, an inhibitor of that proteolytic enzyme, when additionally co-expressed, would restore the transcription of the reporter gene.
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cI- fusion protein), the gene encoding the cI- fusion protein was placed downstream of an inducible promoter on a plasmid vector, generating plasmid pBRcI- (Table 1). This vector was introduced into an engineered E. coli reporter strain, KS1Z (35). In this strain the chromosomal rpoZ gene, coding for the RNAP subunit, has been deleted. Furthermore, this strain harbors on its chromosome a single copy of a lac promoter derivative (named placOR2-62) bearing a single operator site centered 62 bp upstream of the transcriptional start point (35). In this strain the basal transcription of the -galactosidase reporter gene from this promoter is very weak (in the presence or absence of cI alone) but can be stimulated more than 
70-fold in the presence of the cI- fusion protein (35, 45), which results in blue colonies when grown on plates containing X-gal (Fig. 3A, sector 5).
We tested this screen with NIapro, which displays a high specificity for its 7-amino acid recognition sequence (EXXYXQ
(G/S), where indicates the cleavage point; X indicates any amino acid), at which the TEV polyprotein is cleaved (46). To examine whether the insertion of the recognition sequence would disrupt the transcriptional activation properties of the cI- fusion protein, a 25-amino acid coding region, including the 7-amino acid recognition sequence (ENLYFQS) flanked by a His6 tag and a glycine-serine linker (see vector map in the Supplemental Material), was introduced into the linker region of the cI- fusion protein, thus generating the cI-T- fusion protein. This construct was assayed under the same conditions as the fusion protein without the inserted cleavage sequence, and no difference in transcriptional activation was observed (Fig. 3A, sector 4). Thus, E. coli proteinases do not cleave this cI-T- fusion protein to the extent of abolishing activation.
We next asked whether co-expression of NIapro would affect the function of the cI- or cI-T- fusion protein in vivo. For this purpose, NIapro was expressed in E. coli under the control of an arabinose-inducible promoter (plasmid pZA55-TEV; Table 1) to regulate it independently of the cI- or cI-T- fusion protein and to ensure tight repression in the absence of inducer (37). pZA55-TEV was introduced together with the plasmid vectors encoding cI- or cI-T- fusion protein into E. coli KS1Z. Cells were plated on LB agar plates containing X-gal, IPTG (inducing the activator proteins), and arabinose (inducing the proteinase) in various combinations. Although the cI- fusion protein was still active as transcriptional activator (Fig. 3A, sector 3, blue colonies) in the presence of NIapro, the cI-T- fusion protein was not (Fig. 3A, sector 2, white colonies). Thus, in the presence of NIapro, the cI-T- fusion protein was indeed cleaved and can no longer procure transcriptional activation. In contrast, the cI- fusion protein, which cannot be proteolytically processed, still activated transcription, also indicating that expression of NIapro itself had no influence on the transcriptional activation properties of the cI- fusion protein.
These results show that the transcriptional activation properties of the cI- or cI-T- fusion protein are dependent on the integrity of the fusion proteins and that the cI-T- fusion protein is thus suited to phenotypically monitor proteinase activity in the cytosol of E. coli. The same results were obtained when the cI-T- fusion protein was constitutively expressed under the control of the -lactamase promoter (Fig. 3B, pMAK vector series) instead of under the control of the IPTG-inducible Plac-UV5 promoter (pBR vector series) as used in the experiments described above. The findings presented here are the bacterial counterpart to earlier experiments performed in yeast where the transcriptional activation properties of the GAL4 protein were used to also monitor proteinase inhibition in vivo(34).
In Vitro Enrichment of DARPins for NIapro Binding by Ribosome Display—Ribosome display (RD) selection rounds with both an N2C and an N3C DARPin library (22) on immobilized NIapro were performed as described (16). The DNA libraries used in the selection contained at least 1012 individual members each, as estimated from the initial amount of ligated library DNA (22), and further diversity is introduced by polymerase errors during the PCR cycles intrinsic to each ribosome display selection round. An enrichment of binders was already observed after the second selection round at the level of reverse transcription-PCR (data not shown). The enriched pools of binders (potential inhibitors) from the third and fourth selection rounds from both libraries, N2C and N3C, were combined before screening for intracellularly active NIapro inhibitors.
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, which encodes the transcriptional activator under the control of a constitutive promoter, generating pMAKcI-T--DL (Table 1). Thereby the DARPin genes were placed downstream of an IPTG-inducible T5/lac promoter (47) to ensure controlled and high level expression of the DARPins. The enriched pool was introduced together with pZA55-TEV into E. coli KS1Z, and cells were plated on LB agar plates containing X-gal and the respective inducers. The chosen growth conditions were verified prior to the screening experiment to not influence cell growth, thereby securing both the expression of NIapro and the DARPin library members (data not shown). On average, 105 colony-forming units were plated per plate (24 x 24 cm), of which 0.4-0.6% colony-forming units displayed a "blue" phenotype (Fig. 3D). To test that the restoration of the blue phenotype was indeed due to inhibition of NIapro by a library member, and not because of endogenous mutations, a series of tests was carried out. First, 38 blue colonies were tested for DARPin library member expression and their binding to immobilized NIapro by crude cell extract ELISA. Nearly 80% of the analyzed colonies showed expression of DARPins of the expected size on SDS-polyacrylamide gels. The majority of the selected DARPins was of the N2C type (22), and only two N3C members were found. A positive ELISA for binders to immobilized NIapro was obtained in 75% of the tested cases (data not shown).
To further verify and confirm the observed phenotype, 10 library members were chosen for a more detailed analysis. For this purpose, freshly retransformed E. coli KS1Z cells harboring pZA55-TEV and one of the 10 selected library members were plated on LB agar plates under screening conditions with or without IPTG for induction of the inhibitory DARPins. As expected, colonies stayed white when IPTG was missing in the growth medium (proteinase cleaves the transcriptional activator) and turned blue when it was present (induction of DARPins which inhibit proteinase; Fig. 3C; cells harboring only pZA55-TEV stayed white in both cases (data not shown)). To ensure that the proteinase recognition site had not been lost during screening, the genes of three selected DARPins (9_1s, 13_1b, and 20_2b) were cut out from their respective plasmid vectors and freshly ligated into the pMAKcI-T--pD backbone (Table 1); the same inhibition phenotype was observed when these clones were analyzed under screening conditions (data not shown). Thus, the phenotype observed in screening was reproducibly confirmed in vivo.
Sequence Analysis of Selected DARPins—We sequenced the 10 selected DARPins (Fig. 4) and found a high content of aromatic amino acids, Trp, Tyr, and Phe at the randomized positions, which are under-represented in the original library design (22). Most interesting in this respect is the high content of Tyr at the variable positions, which can mediate binding via hydrophobic and polar interactions as well as acting as an H donor (48). This is reminiscent of sequences obtained from selections against maltose-binding protein (16) or aminoglycoside phosphotransferase IIIa (17), where similar characteristics of the amino acid composition at the randomized positions, but of course completely different sequences, were found. Nevertheless, binding was entirely specific in each case. This finding also supports the idea that DARPins, similar to antibodies (2), prefer to use a number of aromatic amino acids in their binding site to achieve their binding function (16).
Size-exclusion Chromatography of Selected DARPins—The following experiments were all carried out with immobilized metal ion affinity chromatography-purified protein samples, and the correct molecular mass for all selected DARPins was confirmed by matrix-assisted laser desorption ionization mass spectrometry (Table 2). Size-exclusion chromatography showed that all 10 selected DARPins were predominantly monomeric, and only a single protein species was observed (Fig. 5; Table 2). Only two DARPins (13_1b and 17_2s) showed a small shoulder in the elution profile that could correspond to a dimer (13_1b, see Fig. 4; 17_2s; data not shown). The molecular size values obtained from the gel-filtration studies are given in Table 2. The observed molecular mass is, with the exception of the N3C DARPin 9_1s, always slightly higher than the value calculated from the sequence, which might reflect the elongated shape of ankyrin repeat domains, rather than the formation of higher oligomeric assemblies. These observations are corroborated by size-exclusion studies of full consensus ankyrin repeat proteins with different numbers of repeats in which the correct molecular size was verified by mass spectrometry, and the monomeric nature of the molecules was verified by multiangle light scattering (Table 2).4 For the full consensus ankyrins, the apparent molecular size calculated from the elution volume was also always slightly higher than that expected for a globular protein of the same size (Table 2). The slightly delayed elution behavior of the N3C DARPin 9_1s might be caused by hydrophobic interactions with the column material (this library member has more hydrophobic residues compared with the other selected DARPins).
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protein D (Fig. 6, pD) (49), which was present in the fusion protein used in the selection experiments. All selected DARPins bound their cognate target and did not bind to pD. The binding signal to immobilized NIapro could be suppressed by preincubation with free NIapro, demonstrating that the interaction is specific for the native protein (Fig. 6). The unselected N2C DARPin library member E2_5 (22), used as a control, did not interact with NIapro, indicating that the designed DARPin scaffolds per se do not bind NIapro (Fig. 6). It should be noted that the ELISA signal of some selected DARPins was very weak (2_2b, 7_1b, 12_1b, and 15_3b) and could be improved by reducing the washing time during the ELISA experiment (Fig. 6B). This finding suggests a very fast off-rate of these binders, which was confirmed by SPR experiments (Fig. 7). Thus, under our standard ELISA conditions, these DARPins are washed off before detection with an antibody. To measure the affinity of the interaction of the in vivo selected DARPins specific for NIapro, equilibrium SPR experiments were performed with the DARPins 9_1s, 13_1b, and 20_2b. The KD values of all three NIapro binders were found to be in the low micromolar range (Table 3). The SPR data were fitted with the assumption of a 1:1 interaction with one-site saturation (Fig. 7).
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cI-T- fusion protein, as tested by ELISA (data not shown). Protection of the NIapro cleavage site within the cI-T- fusion protein by a DARPin might, in principle, result in the same phenotype. However, because we found no binding of the selected DARPins to the cI-T- fusion protein, we can rule out the latter mechanism for transcriptional activation.
In Situ Inhibition by Selected DARPins—To confirm the direct inhibition of NIapro, we conducted co-expression tests of proteinase and DARPins in the presence of the cI-T- fusion protein in E. coli KS1Z in liquid culture, following the digestion of the transcriptional activator by Western blot analysis in crude cell extracts. For this experiment we used two different antibodies as follows: one was directed against the His6 tag of the cI-T- fusion protein, which is present in its linker sequence (Fig. 8A); and a second one was directed against the cI domain of the cI-T- fusion protein. Upon cleavage of the activator by NIapro, the band corresponding to the full-length activator disappears, whereas the two cleavage products, the His6 tag- domain and the cI domain bands, should appear. However, when performing this experiment, we could not detect the His6 tag- domain alone, as it might get further digested or its band might be too faint to be detected. The band of the full-length activator and the cleavage product could be detected with the anti-cI antibody. This experiment is the most direct means of analyzing the effect of the selected DARPins on NIapro in situ, and in the presence of an inhibitor of NIapro the full-length band of the activator is preserved.
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Z. Cells were grown nearly to the end of the log phase, and DARPin expression was induced by the addition of IPTG. After 30 min an aliquot was withdrawn, and NIapro expression was started by the addition of arabinose. After 2 h another aliquot was withdrawn. The aliquots were normalized to A600, and cells were disrupted by heating at 95 °C for 15 min in SDS-loading buffer, and proteins were subsequently separated by SDS-PAGE. The band of the cI-T- fusion protein was monitored by Western blot analysis with an anti-tetra His-Ab or anti-cI-Ab, respectively. Only the selected DARPins can prevent the digestion of the cI-T- fusion protein by NIapro in vivo (Fig. 8). By contrast, the unselected library DARPin E2_5 (22) was unable to inhibit the digestion of the cI-T- fusion protein by NIapro (Fig. 8, B and C). Using the anti-cI-Ab for detection of the cI-T- fusion protein, we also observed an unspecific degradation of the cI-T- fusion protein by endogenous E. coli proteinases (Fig. 8C), which also occurs in the absence of any DARPins and in the absence of NIapro (Fig. 8D). As expected, this cleavage by E. coli proteinases cannot be inhibited by the expression of our selected DARPins. Nevertheless, enough cI-T- fusion protein is clearly remaining to induce the blue phenotype (Fig. 3). The results of these experiments indicated for the first time the inhibition of a site-specific proteinase by selected DARPins in the intracellular compartment of E. coli.
In Vitro Inhibition by Selected DARPins—To confirm that the in vivo effects observed for the selected DARPins were indeed due to direct inhibition of NIapro, in vitro enzyme assays were performed. Enzyme activity was monitored by the release of the fluorogenic leaving group (7-amino-4-methyl-coumarin (amc)) from a synthetic peptide substrate (Ac-TENLYFQ-amc) as described previously (44) with minor modifications (see under "Materials and Methods") to mimic the in vivo situation. In vivo, the DARPin concentration is much higher than the concentration of NIapro and the substrate, i.e. the cI-T- fusion protein. As estimated from SDS-PAGE analysis (see above) the ratio of DARPin to NIapro to cI-T- fusion protein (i.e. the in vivo substrate) is 800:8:1 µM (see under "Discussion"). Under these conditions, even considering the micromolar KD value, the enzyme should be almost fully complexed with DARPin.
The enzymatic hydrolysis of our in vitro substrate (i.e. Ac-TENLYFQ-amc peptide) was found to be very slow (Km = 60 µM; kcat = 1.54 x 10-5 s-1), but clearly distinguishable from samples containing no proteinase (background). For our in vitro experiments we used a ratio of DARPin to NIapro to peptide of 100:1:5 (300:3:15 µM), which should mimic the in vivo situation quite closely.
For all DARPins, which were analyzed in more detail (9_1s, 13_1b, and 20_2b) and which show NIapro inhibition in vivo, enzyme inhibition was detected, whereas the control protein (E2_5) showed no significant influence on NIapro activity (Fig. 9). Under the assay conditions chosen, the inhibition was not complete for this synthetic substrate; some residual activity, ranging from 5 to 32%, depending on the individual inhibitor, was observed (Fig. 9). The different inhibition efficiencies of the selected DARPins do not correlate with differences in KD values under the assay conditions.
There are several reasons why the in vitro inhibition assay may be only partial. First, the substrate and the inhibitor may compete at least partially for the same site. As we are forced to use higher substrate concentrations (to detect the reaction) in vitro, compared with the in vivo situation, the inhibitor may be partially displaced by the substrate. Conversely, the DARPins may not completely block the binding of the peptide substrate, but only decrease its affinity, as the DARPin-binding site may be adjacent and thus have a more dramatic effect only on (longer) protein substrates. Because of the extremely slow turnover of the peptide substrate with NIapro, it would be very difficult to conduct a full kinetic analysis of the inhibition mode (17). Nevertheless, the fact that KD values and the extent of inhibition do not seem to correlate argues for a mixed inhibition as found previously (17).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite the great variety of natural proteinaceous inhibitors of proteolytic enzymes, they show some convergence of design and/or mechanism. The key challenge is how a protein can avoid being a substrate and instead become an inhibitor. A tight binding Michaelis complex, a tight binding product complex, or even a stable acyl-enzyme intermediate may be formed as a result of an inhibitor loop entering the active site of the proteinase (55). This tight binding efficiently prevents turnover. Alternatively, a loop binding nonproductively in the opposite orientation or a steric blockage by the whole inhibitor protein, without a peptide necessarily occupying the active site, are additional modes of inhibition (55).
Our DARPins are not proteinase inhibitors per se, and the input DARPin library of our experiments is completely unbiased for function. Nevertheless, we were able to selected specific NIapro-inhibiting DARPins. DARPins also lack pronounced extended flexible loops, which could reach into the active site of the proteinase, and thus it is more likely that inhibition is realized by steric blockage of the active site access or, alternatively, by an allosteric effect that arrests the proteinase in a nonproductive conformation, comparable with the mechanisms found for the inhibition of a prokaryotic kinase by a DARPin (24). The exact mechanism of action can only be elucidated from the crystal structure of the complex.
It should be mentioned that the identification of inhibiting DARPins within the obtained pools of NIapro binders was a rare event, because only 0.5% of all (by ribosome display) preselected binders screened gave rise to a blue phenotype. Nevertheless, the results presented here show that the fundamental structural requirements of proteinase inhibitors can be fulfilled by repeat proteins, whose versatility is thus further underlined. Although, to the best of our knowledge, no repeat protein has yet been ascertained as a natural proteinase inhibitor, the binding partners of most of the many members of the repeat protein families have not yet been identified. Thus, our results would make it not surprising if a natural repeat protein was found to act as a proteinase inhibitor. Their large, rigid interaction surface could clearly block an active site by avoiding all direct contacts with the catalytic center or even by an allosteric effect.
At first sight it may be surprising that the relatively low affinities of the DARPins identified as inhibitors could be sufficient for mediating such a profound in vivo effect. But these relatively low affinities of the inhibitors found in the present experiment can be explained, even though DARPin-based binders with KD values in the low nanomolar range could be routinely obtained against a wide variety of targets from our combinatorial DARPin libraries (16, 17). The estimation of the intracellular DARPin concentrations during the screening experiment, calculated based on the densitometric analysis of protein bands from SDS-polyacrylamide gels of crude cell extracts, revealed an intracellular concentration between 800 and 900 µM for the selected DARPins (assuming an average E. coli cell volume of 10-15 liters and 1 A600 corresponding to 5 x 108 cells/ml (56)). This is far above the determined KD values. Thus, these findings might demonstrate that in intracellular selections determinants other than affinity, such as activity, solubility, and expression (see below), govern the selection process of specific protein-protein interactions. Furthermore, only a very small subset of binders is expected to also be inhibitors. Therefore, these results have no bearing on the general high affinity of selected DARPins, which are in the nanomolar to picomolar range (16).5
Our findings presented here are corroborated by recent findings with the protein complementation assay selection system (58). In these studies, antibody scFv fragments were selected against various targets directly from a diverse library (59). In those cases where specific intracellular activity was obtained without prior in vitro selection, micromolar affinities were also observed (59, 60). Stability, expression level, solubility, and the monomeric state of the scFv fragments may be the main determinants for successful selection of these specific binders in this intracellular selection system (59). Thus, in vivo selections may not by themselves lead to high affinity binders without additional in vitro affinity maturation steps.
A Genetic Screen to Isolate Inhibitors of Site-specific Proteolytic Enzymes—Although the in vitro selection of binding molecules against almost any target from large combinatorial libraries using techniques such as ribosome or phage display (41, 61) is rapid and usually straightforward, the subsequently necessary assay to identify any additional desired functionality must be tailored to the specific requirement. Usually, only a small subset of the still large pool of binding molecules after the selection process will mediate the desired function (e.g. inhibition of an enzyme).
We modified a previously reported bacterial two-hybrid system (35), in which the transcriptional activation of a reporter gene is now coupled to the inhibition of a site-specific proteolytic enzyme. Our genetic screen allows the convenient screening of a large number of clones, and it tests them directly in the intracellular environment.
Nevertheless, as pointed out above, high affinity binders do not necessarily have an advantage in this screen. If high affinity binding is a requirement for later applications, either the amount of substrate has to be increased, the amount of inhibitor has to be decreased, or an affinity maturation step has to be added to the initial in vitro selection procedure. Only the latter step would be feasible for NIapro because of its relatively low turnover, which in consequence makes high cellular concentrations of this enzyme necessary to enable the assay to work.
This screen can in principle be adapted to any proteinase (as the cleavage site within the fusion protein can easily be exchanged), provided that such a proteinase is not toxic for E. coli. Also, the system is not limited to DARPins or even proteins, because cell-permeable compounds can be tested as well.
Conclusions and Perspectives—We report here for the first time DARPin-based proteinase inhibitors and chose the example of an agriculturally important plant virus fully active in the cell. The favorable biophysical properties of DARPins, in particular their high rigidity and stability, might have enabled us to generate inhibitors structurally distant from known naturally occurring inhibitors (10). Selected DARPins showing not only specific binding, but also providing a desired functionality for a given substrate, can be used as valuable tools in target validation experiments, extra- and intracellular (17). By arresting the protein in an inactive conformation and co-crystallization of such protein-protein complexes (24), the process of drug discovery even for small molecules may be accelerated.
Apart from the general applicability of DARPins as target validation tools, in plants DARPins might open a new general means to engineer resistance against those plant viruses or parasites possessing proteinase activity as part of their natural processing or insecticidal transmission mechanism (62). The selected DARPins presented here might potentially serve as leads to engineer resistance.
In human or animal health, the general applicability of DARPins as specific intracellular enzyme inhibitors in an organism is currently still hampered by the unsolved problem of delivering DARPins to the cytosol of the target cells, which has to await future progress in DNA/RNA or protein delivery strategies. Nevertheless, because of the ease of transfection of cell lines, protein knock-outs may become an important part of the drug discovery process.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Materials and Methods and Fig. S1.
1 Present address: Molecular Partners AG, % Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich.
2 To whom correspondence should be addressed. Tel.: 41-44-635-55-70; Fax: 41-44-635-57-12; E-mail: plueckthun{at}bioc.unizh.ch.
3 The abbreviations used are: DARPin, designed ankyrin repeat protein; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl--D-thiogalactopyranoside; NIapro, potyvirus nuclear inclusion-a proteinase (25 kDa, not containing the viral genome-linked protein domain VPg); RNAP, RNA polymerase; SPR, surface plasmon resonance; TEV, tobacco etch virus; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside.
4 S. Wetzel, unpublished results.
5 C. Zahnd, E. Wyler, J. M. Schwenk, D. Steiner, C. Ward, F. Pecorari, T. O. Joos, and A. Plückthun, manuscript in preparation.
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-tetra His-Ab following 15% SDS-PAGE and Western blotting. pMAKcI-T-