1Department of Automated Biotechnology, Merck and Co., Inc., 502503 Louise Lane, North Wales, PA 19454, 2BioTech Studio LLC, 3701 Market Street, Philadelphia, PA 19104 and 3Fraunhofer USA, Center for Molecular Biotechnology, 9 Innovation Way, Newark, DE 19711, USA
4 To whom correspondence should be addressed, at BioTech Studio LLC
5 To whom correspondence should be addressed. Present address: NIH Chemical Genomics Center, 9800 Medical Center Drive, MSC: 3370, Bethesda, MD 20892-3370, USA. E-mail: sdelagrave{at}biotechstudio.com or jinglese{at}mail.nih.gov
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Abstract |
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Keywords: directed evolution/high-affinity detection reagents/PDZ variants
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Introduction |
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The design of efficient, high-throughput protease assays for drug discovery possesses special challenges requiring new technologies. An ideal protease assay should be sensitive, inexpensive, rapid, applicable to both cell-free and cell-based formats and should utilize a substrate which is structurally as similar as possible to the natural enzyme target. The use of self-quenching substrates, in which a fluorophore and a quencher located on either side of the scissile bond in a peptide substrate are separated by proteolysis, has proven useful but the required substrate modifications can be technically difficult and may render protease digestion so inefficient as to require significant optimization (see, for example, Cummings et al., 2002; Neumann et al., 2004
). Recently, assays have been developed based on epitope unmasking, wherein the enzymatic reaction productbut not the substrateis recognized by so-called neo-epitope antibodies (Zuck et al., 1999
; Li et al., 2000
; Shearman et al., 2000
). This approach is applicable to both cell-free and cell-based assays because it requires minimal modification of the substrate amino acid sequence and less invasive labeling. However, neo-epitope antibodies are particularly problematic because specific recognition of the cleaved product in the presence of the uncleaved substrate is often necessary. Accordingly, the isolation of adequately specific neo-epitope antibodies is technically demanding, costly and time consuming.
The use of PDZ (PSD95/Discs-large/ZO-1) domains as an alternative to neo-epitope antibodies to detect the peptide products of enzymatic reactions was recently successfully demonstrated (Ferrer et al., 2002; Hamilton et al., 2003
). Members of this family of protein domains, which recognize the C-termini of certain receptors and ion channels, have been used in assay systems for hydrolytic enzyme reactions mediated by proteases and phosphatases. Their small size (
100 amino acids) and high expression in Escherichia coli make PDZ domains a particularly attractive reagent in the design of bioassays and analytical methods. In addition to the side chains of the last few residues, the carboxylate group at the C-terminus of a peptide or protein is critical for recognition by PDZ domains, which makes PDZ domains well suited to the task of discriminating between a substrate peptide and its cleavage product. Initial proof-of-principle studies clearly demonstrated that PDZ biosensors detect the newly generated C-terminus resulting from peptidase or phosphatase action if the product peptide has sufficient affinity for the PDZ domain employed (Figure 1).
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Materials and methods |
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Peptides were biotin and fluorescein-labeled at the N-terminus and were purchased from Research Genetics (Huntsville, AL), Princeton Biomolecules (Langhorne, PA) or Synpep (Dublin, CA). Peptides with Tyr in the sequence were dissolved in DMSO to 1 mM solutions (concentration was determined by measuring the UV absorbance at 280 nm, using
Tyr = 1280 l/mol.cm). Fluorescein peptides were dissolved in DMSO to
1 mM solutions (concentration was determined by measuring the UV absorbance at 492 nm, using
FL = 66 000 l/mol.cm). Otherwise, the concentration was estimated from dry weight. Streptavidin-XL665 (molar ratio: 1.9 XL665/streptavidin) was obtained from CIS-US (Boston, MA). [Eu3+]anti-glutathione (GST) chelate mAb was obtained from Perkin-Elmer (Perkin-Elmer, Turku, Finland). HIV protease (HIV-PR) was provided by Dr Lawrence Kuo (Merck Research Laboratories, West Point, PA). Fluorescence emission was measured at 665 nm for the fluorescence energy transfer (FRET) signal and 615 nm (Victor2V, Perkin-Elmer) or 620 nm (Acquest, Molecular Devices) for Eu3+ chelate and results are expressed as the ratio of fluorescence intensities, FI665 nm/FI615 nm.
Construction of phage display libraries
Genes encoding the PDZ domains NHERF PDZ1 and PSD95 PDZ3 (NHERF, Na+/H+ exchanger regulatory factor; PSD95, postsynaptic density 95), flanked by BamHI and EcoRI restrictions sites on the 5' and 3' ends, respectively, in the pGEX-2TK vector were obtained as described previously (Ferrer et al., 2002) and amplified by error-prone PCR to yield mutated PDZ domain genes flanked by SfiI and BamHI sites on the 5' end and EcoRI and NotI sites on the 3' end. These PCR products were then digested with SfiI and NotI and cloned into the pCANTAB 5E phagemid (Amersham Biosciences). The library was transformed into electro-competent E.coli XL1-Blue (Stratagene). Each library contained over 106 unique mutants. A mutation rate of
1 amino acid substitution per gene was determined by sequencing randomly picked clones from each library. In parallel, a single-chain Fv (scFv) gene was cloned into the same phagemid, to be used as a negative control in phage display experiments.
Panning
Each library was selected by affinity panning for mutants that have improved affinity for peptide HRRSARYLDTVL-OH, with some modifications on the phagemid manufacturer's instructions (Amersham Biosciences). Phage were prepared by thawing and growing frozen aliquots of the NHERF and PSD95 libraries, superinfecting with helper phage M13K07, precipitating with PEGNaCl and dissolving in Tris-buffered saline (TBS). Each library went through five rounds of panning in polystyrene wells of a microtiter plate. The wells were coated overnight with biotinylated target peptide (HRRSARYLDTVL-OH) and streptavidin, blocked with I-block (Tropix) dissolved in phosphate-buffered saline with Tween 20 (PBST) and an aliquot of the phage libraries was allowed to bind for 12 h at room temperature. The wells were then washed with PBST five (first round) or 10 times (subsequent rounds) and filled with log-phase XL1-Blue E.coli. Infection of the cells by retained phage was allowed to occur for 1 h at room temperature. The cells were then removed from the wells and grown overnight in preparation for a subsequent round of panning. For each round, aliquots of the input and output phage-infected cells were plated onto SOBAG-tet plates, grown at 30°C and colonies were counted the next day. Fifth-round colonies were picked and grown overnight to screen individual phage clones for binding to the target peptide.
Screening of recombinant phage and preliminary characterization
Phage suspensions were prepared for each clone according to standard methods and tested by phage ELISA. Wells of a microtiter plate were coated with streptavidin and the biotinylated target peptide and then blocked with 2% non-fat dry milk or I-block (Tropix). Phage suspensions were added to the wells of the plate and incubated 1 h at room temperature. The wells were then washed with PBST and bound phage were detected with anti-M13 horseradish peroxidase-labeled antibody. Colorimetric substrate was added to each well, 10 N sulfuric acid was added after about 20 min of incubation at room temperature and the plate was read at 490 nm. Three clones (H9, G9, F10) showing strongest binding to the target peptide were grown again on a larger scale to yield more phage suspension, which was tested by phage ELISA using the target peptide as well as other control peptides.
Sequencing of mutants and construction of GST expression vectors
The three mutants F10, G9 and H9 were sequenced according to standard methods. Briefly, the plasmid DNA was prepared by standard purification procedures. The PDZ gene from each mutant plasmid was amplified by PCR, yielding a PCR product which was purified using a Qiagen column and sent to a contractor (SeqWright) for sequencing. The NHERF mutant genes were then digested with BamHI and EcoRI, cloned into pGEX-2TK, transformed into E.coli strain DH10B and grown at 30°C on SOB2% glucose agar with 100 µg/ml ampicillin. Two clones from each transformation were picked and sequenced to verify that the GEX constructs contained the expected mutations. Confirmed clones were then transformed into BL21 (DE3).
Mutant expression and purification
Mutants were expressed in E.coli and purified as GST fusion proteins by standard methods (Ferrer et al., 2002). Briefly, BL21 (DE3) transformants were plated on LB-agarose + ampicillin (AMP) plates. A single colony was seeded overnight in LB with 0.1 mg/ml AMP and the subsequent suspension was used to inoculate 2 l of LB with 0.1 mg/ml AMP. Cells were grown to OD600 = 0.51.0 and induced with 1 mM IPTG for 4 h. Cell pellets were frozen. Thawed cell pellets were lysed in lysis buffer (500 µg/ml lysozyme, 10 mM DTT and protease inhibitor cocktail in PBS) for 30 min at 4°C. The cell suspension was then extruded through a 25 G needle and freezethawed twice. Supernatants were clarified by centrifugation at 27 000 g. GST fusion proteins were then purified using reduced glutathione Sepharose beads (Molecular Probes, Eugene, OR), following protocols provided by the supplier. Protein concentration was determined by the Bradford assay.
Fluorescence polarization ligand binding assay
Fluorescence polarization experiments were performed as described previously (Hamilton et al., 2003). Briefly, serial dilutions of GST-fusion PDZ domains were prepared in PSB with Tween 20 and bovine serum albumin (PBSTB) buffer (155 mM NaCl, 1 mM KH2PO4, 3 mM Na2HPO4, 0.05% Tween-20, 0.1% BSA, pH 7.4). Serial dilutions (19 µl) were transferred in triplicate to a Greiner 384-well black, medium binding, small volume plate. One microliter of fluoresceinated peptide (Fl-HRRSARYLDTVL-OH or Fl-HRRNEEVQDTRL-OH) was added as a 200 nM stock. The plates were covered to prevent evaporation and incubated for 2 h at room temperature in the dark. Fluorescence polarization (FP) was measured on the Acquest (G = 0.8) or the Victor2V (G = 1.07) and data were analyzed using GraphPad Prizm. Data shown are the mean of six (Fl-HRRSARYLDTVL wild-type NHERF), three (Fl-HRRSARYLDTVL-OH all others) or two (Fl-HRRNEEVQDTRL-OH) independent experiments performed in triplicate.
Time-resolved (TR) FRET binding assay
TR-FRET (Ferrer et al., 2002) experiments were performed as described previously (Hamilton et al., 2003
). Briefly, a dilution series in the concentration of biotinylated HIV product peptide (biotin-HRRSARYLDTVL-OH) was prepared in dynamic range buffer (1:20 enzyme assay buffer in PBSTB) and biotinylated substrate peptide (biotin-HRRSARYLDTVLEEMS-OH) was added to maintain the concentration of peptide at 200 nM. GST-fusion PDZ domains were diluted to 40 nM and 5 µl of GST-fusion PDZ domain was incubated with 5 µl of product substrate mixture for 10 min at room temperature in medium binding Greiner 384-well small volume black plates. Subsequently, 10 µl of detection mixture (10 nM anti-GST [Eu3+], 25 nM streptavidin-XL665) were added and the plates were covered and incubated for 1 h at room temperature in the dark. Plates were read using the Victor2V: samples were excited at 340 nm and emission was measured at 615 and 665 nm. Data are the mean of two independent experiments conducted in triplicate.
HIV protease activity FRET assay
HIV protease kinetics assay was performed as described previously (Hamilton et al., 2003). Serial 2-fold dilutions of HIV protease were prepared in enzyme assay buffer (0.1 M NaOAc, 1.0 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/ml BSA, pH 4.7). HIV protease was mixed with an equal volume of 2 mM substrate peptide (biotin-HRRSARYLDTVLEEMS-OH) and the reaction was allowed to proceed at 37°C. Reactions were quenched by removing 10 µl into 40 µl of PBSTB. Five microliters of quenched reaction were removed into 5 µl of 40 nM GST-fusion protein in triplicate and the mixture was incubated for 10 min at room temperature. Subsequently, 10 µl of detection solution (10 nM anti-GST [Eu3+], 25 nM streptavidin-XL665) were added to each well and the plate was covered and incubated for 1 h at room temperature in the dark. Plates were read using the Acquest: samples were excited at 340 nm and emission was measured at 620 and 665 nm. Data shown are the mean of two (wild-type and H9) or three (G9 and F10) independent experiments conducted in triplicate and error bars represent the standard error of the mean.
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Results |
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The phage display libraries of PDZ domains were designed based on two scaffolds: PDZ1 of NHERF and PDZ3 of PSD95. The NHERF and PSD95 wild-type PDZ domains were mutated by error-prone PCR (Leung et al., 1989), yielding two mutant libraries each containing over 106 unique mutants. The observed mutation rate corresponded to
1 amino acid substitution per gene as determined by DNA sequencing of randomly picked clones. Each library was selected for mutants that have improved affinity for peptide HRRSARYLDTVL-OH (the target peptide). Phage were prepared from frozen stocks of the NHERF and PSD95 libraries according to standard methods (Barbas et al., 2001
). Each library was carried through five rounds of panning. The input phage titer (number of phage added to the immobilized target peptide) and output phage titer (phage remaining bound to the target after washing off non-specific phage) from each round were determined. The ratio of output phage to input phage for the NHERF library panning showed a clear trend of phage amplification after round 3, suggesting selection of mutants specific for target peptide HRRSARYLDTVL-OH (Figure 2A).
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We used FP on purified PDZ variants to obtain quantitative affinity measurements on isolated protein rather than protein displayed on phage. PDZ domain variants and wild-type PDZ domains were converted to GST-fusion proteins, purified and their affinities determined (Figure 3 and Table I). Using these GST-fusion proteins, FP-based measurements confirmed the improved binding of NHERF PDZ variants to target peptide DTVL relative to wild-type NHERF PDZ (Figure 3A). FP measurements also showed significantly decreased affinity for peptide DTRL (Figure 3B). Table I highlights the affinity changes resulting from the directed evolution process. Mutant H9 has a 25-fold increase in affinity for target peptide DTVL, binding it with a dissociation constant (Kd) of 620 nM. Mutant G9 is the most specific, having a nearly 1.5-fold higher affinity for the target peptide than peptide DTRL and confirming that ligand specificity was successfully switched.
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The NHERF PDZ domain variants were compared with wild-type for their ability to detect HIV protease cleavage product. The dynamic range and lower detection limits were determined by varying the productsubstrate peptide mixture against a constant concentration of each of the GST-PDZ mutants using TR-FRET detection (Figure 5A and B). The signal-to-background ratio obtained using 10 nM of each PDZ for a range of product:substrate peptide ratios clearly demonstrates the greater sensitivity of the mutant PDZ domains in detecting HRRSARYLDTVL-OH product peptide (Figure 5A and B). In this assay, the G9 and H9 variants provide signal-to-background ratios >10 at a 10:90 product:substrate peptide ratio (i.e. simulating 10% turnover) and the lower limit of detection is 2 nM. The F10 variant gives a signal-to-background ratio above 10 at
15:85 product:substrate peptide ratio (i.e. simulating 15% turnover) and the lower limit of detection is
5 nM. These numbers compare favorably with the wild-type NHERF PDZ1, which requires 30% substrate turnover to obtain a 10-fold signal-to-background ratio and whose lower limit of detection is
10 nM.
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We chose to use the HIV protease as a test system because it is a prototypical antiviral target and a PDZ-based detection assay has already been engineered to serve as the model system (Ferrer et al., 2002; Hamilton et al., 2003
). HIV protease cleaves a polyprotein sequence at different positions to yield several functional viral proteins. The recognition sequence includes a large hydrophobic or aromatic residue at the P1 position. The peptide HRRSARYLDTVLEEMS-OH was predicted to be a substrate for HIV protease, yielding the product peptide HRRSARYLDTVL-OH, and this was verified experimentally (Hamilton et al., 2003
). While wild-type NHERF PDZ1 binds tightly to a similar peptide (HRRNEEVQDTRL-OH), this specificity is not useful to study HIV protease because peptide HRRNEEVQDTRLEEMS is not a good substrate. Hence our task was to create a PDZ variant recognizing a peptide (DTVL) that differs substantially from peptide DTRL (compare residues 1, and 4 to 8 of peptides DTVL and DTRL; see bottom of Figure 1 for PDZ ligand numbering scheme).
Using directed evolution to enhance binding affinity of a PDZ domain for the desired product peptide is an appealing strategy to develop a sensitive detection reagent for a variety of protease assays. Although Skelton et al. (2003) first described the use of phage display to study PDZligand interactions, they did not increase the affinity or alter the specificity of the Erbin PDZ domain under investigation. Computer-based design of PDZ domains has also been employed (Reina et al., 2002
) to yield PDZ variants with substantial specificity changes and affinities in the range 1100 µM. The in vitro protein evolution techniques used here yielded a PDZ binding domain with high affinity (620 nM) to the desired peptide sequence HRRSARYLDTVL-OH. Affinity measurements of purified PDZ variants clearly indicate that substrate specificity of mutants G9 and H9 was switched from peptide DTRL to peptide DTVL. It is noteworthy that although no negative selection against substrate peptide was used in the panning process, all the mutants retain selectivity for the product sequence over the substrate.
As few as two mutations were needed to achieve the observed specificity change, which is generally consistent with a previous report indicating that a similarly small number of substitutions is needed to achieve substantial specificity changes in PDZ domains (Schneider et al., 1999). The roles of the various amino acid substitutions observed can be rationalized using the data presented here and grouped into three categories.
First, our data support the notion that PDZ domains interact with side chains at ligand position 1 via ß1ß3 interactions (Fuh et al., 2000). Substitution E43V in mutant H9 is of particular interest in this regard because it maps near position 1 in the target peptide and substitution of a hydrophobic residue for an acidic one (E43V) is consistent with the presence of a valine rather than an arginine in the target peptide. Position 1 is the only one which differentiates peptides DTVL and DTRL in the consensus XS/TX
recognition sequence (where
is hydrophobic). Moreover, Karthiekeyan et al. (2001)
have previously identified E43 as a determinant of NHERF PDZ specificity with respect to position 1 of the peptide ligand, which agrees with our observation of increased affinity and specificity in mutant H9.
Second, our data also indicate that the NHERF PDZ domain can be engineered to distinguish ligand residues in position 4 and beyond. This is suggested by the observation that several mutations are near the ligand-binding groove but away from the carboxyl-binding pocket. Substitutions R40G found in mutant H9 and K32E found in mutant G9 each cause a net increase in the acidic character of the ligand-binding groove, consistent with the net increase in positive charge of residues in positions 6 and 7 of peptide DTVL compared with peptide DTRL. Replacement of His at position 29 with Arg in mutant F10, which has the smallest increase in affinity for peptide DTVL of the three mutants, is likely to be directly responsible for this affinity increase because the only other substitution (R87C) in this variant is assigned to a different role.
Third, substitution R87C can be assigned to the apparent improvement in the efficiency with which NHERF PDZ is displayed on phage. Given that wild-type NHERF PDZ, which has an affinity of 311 nM for peptide DTRL, gives a low phage ELISA signal, it would be reasonable to expect that NHERF PDZ mutants having an affinity for peptide DTRL of 6201700 nM would produce a similarly low signal. However, the phage ELISA signal of these mutants is actually much greater, indicating that mutations have improved independently both phage ELISA signal and affinity. At the genotype level, all three mutants share only substitution R87C, providing circumstantial evidence linking the mutation with improved phage ELISA signal. Furthermore, R87 is on the opposite face of the protein from its binding pocket, whereas the other substituted residues all seem likely to interact directly with the peptide ligand. The substitution of Cys for Arg could improve phage display efficiency by improving NHERF PDZ domain expression or by facilitating the formation of dimeric PDZ domains, thereby affording a selective advantage via avidity enhancement. Soluble PDZ domain variants produced by incomplete suppression of the opal stop codon between the PDZ gene and phage gene 3 could conceivably form disulfide-linked dimers with monovalent phage-displayed PDZ domains.
It is possible that our affinity measurements are affected by avidity effects caused by the likely dimeric state of GST-PDZ fusions and the probable multivalent state of streptavidinpeptide complexes. However, most of our conclusions are based on affinity changes and should therefore not be affected by such effects. Moreover, for the purposes of creating highly sensitive assays, such avidity effects, if they occur, would be beneficial. On the other hand, FP, used here to measure affinity, is less likely to be affected by avidity effects than methods relying on immobilized ligands such as surface plasmon resonance.
The pervasiveness of antibodies in diagnostics and drug discovery is well known and great potential for their use in proteomic analysis exists. The generation of antibodies on a routine basis for novel peptide epitopes in useful quantities is not always possible. This is particularly true for the raising of antibodies to short peptide neo-epitopes, where the exposure of a new carboxyl terminal amino acid defines the only difference between the epitope and its encrypted form. PDZ domains have evolved to recognize the terminal four to five amino acids of cognate protein partners with the -carboxylate contributing critical hydrogen-bonding interactions to the specificity and stability of peptide-domain complex. From an assay development perspective, these modular proteins are ideally suited as probes or sensors of the many processing enzymes that transform proteins and peptides. Such enzymes include viral and host proteases, convertases, phosphatases, amidases and esterases, among others. Identification of a PDZ domain with marginal affinity to the product peptide of an important therapeutic target, HIV protease and subsequent in vitro protein evolution allowed the development of a high sensitivity (<10% substrate conversion yields a 10-fold signal-to-background ratio, a 3-fold increase over the previous assay), using 5-fold less enzyme, an important factor in efficient, cost-effective, large-scale screening.
We envision that by coupling in vitro evolution and affinity maturation, it should be possible to identify PDZ domains that bind a wide range of protease product sequences with nanomolar affinity, allowing the development of antibody-independent analysis of both in situ and cellular peptide and protein processing.
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Received October 3, 2004; revised January 17, 2005; accepted January 18, 2005.
Edited by William DeGrado