Directed evolution of PDZ variants to generate high-affinity detection reagents

Marc Ferrer1, Jim Maiolo1, Patricia Kratz1, Jessica L. Jackowski2,3, Dennis J. Murphy3, Simon Delagrave2,3,4 and James Inglese1,5

1Department of Automated Biotechnology, Merck and Co., Inc., 502–503 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


    Abstract
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 Abstract
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 Materials and methods
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 References
 
High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific ‘ß1–ß3’ interaction with ligand residue –1, interactions with ligand residues –4 to –7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

Keywords: directed evolution/high-affinity detection reagents/PDZ variants


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 References
 
Proteolytic enzymes are important therapeutic targets for the treatment of viral infections, cancer, Alzheimer's disease and cardiovascular disease, and also conditions linked to inflammation and immune disorders. Measuring protease activity, both in vivo and in vitro, is therefore important for studying the function of these enzymes and developing new therapeutics. Indeed, high-throughput protease assays are currently being applied to the discovery of novel inhibitors of several disease-associated enzymes such as HIV protease (Erickson and Eissenstat, 1999Go), HCV proteases (Whitney et al., 2002Go; Lamarre et al., 2003Go) and {gamma}-secretase (Shearman et al., 2000Go).

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., 2002Go; Neumann et al., 2004Go). Recently, assays have been developed based on epitope unmasking, wherein the enzymatic reaction product—but not the substrate—is recognized by so-called ‘neo-epitope’ antibodies (Zuck et al., 1999Go; Li et al., 2000Go; Shearman et al., 2000Go). 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., 2002Go; Hamilton et al., 2003Go). 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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Fig. 1. PDZ-based homogeneous protease assay. A biotinylated protease substrate (upper left, sequence ... DTVLZ, where Z is an arbitrary sequence of amino acids) is cleaved by a protease at the scissile bond (between P1 and P1') to reveal a peptide product (upper right) with a C-terminus that is recognized by a PDZ domain. Accumulation of the peptide product is revealed by formation of a ternary complex (bottom) comprising the peptide product itself, labeled streptavidin (SA) binding to this peptide's biotinylated N-terminus and a labeled PDZ domain binding to the peptide's C-terminus. The ternary complex is detected by TR-FRET using excitation at 337 nm and emission at 665 nm. While many PDZ domains exist, directed evolution of PDZ domains provides increased flexibility in creating assays for any protease–substrate combination. Amino acid residues of a PDZ ligand are denoted X0 for the C-terminal residue; X–1 for the penultimate residue; and so on. Here, X0 = L, X–1 = V, X–2 = T and X–3 = D, using the single-letter amino acid code. In the present study the Eu3+ label (EuK) is indirectly bound to the PDZ domain via an antibody; see text.

 
The use of PDZ domains as detectors of proteolytic activity is limited by the absence of PDZ domains with sufficient affinity for the many diverse substrate cleavage products. Although a workable compromise can be achieved through the use of substrate peptides whose cleavage products have low but detectable affinity to natural PDZ domains (Hamilton et al., 2003Go), the approach will not work for all enzyme drug targets. An alternative to modifying the substrate sequence is to develop ‘customized’ PDZ domains that bind tightly (Kd in the nanomolar to subnanomolar range) to a desired C-terminal sequence. Here we use in vitro protein evolution to derive PDZ domain variants that recognize a peptide of pre-determined sequence representative of an optimal HIV protease substrate. In vitro evolution, also known as ‘directed evolution’ or ‘applied molecular evolution,’ creates populations of random protein variants through mutagenesis and selects improved variants to achieve significant and useful changes in the function of proteins (Farinas et al., 2001Go; Delagrave and Murphy, 2003Go), including PDZ domains (Schneider et al., 1999Go; Junqueira et al., 2003Go). In the present study, error-prone polymerase chain reaction (PCR) (Leung et al., 1989Go) and phage display (Barbas et al., 2001Go) were used to select three PDZ domain variants that bind the HIV protease cleavage product HRRSARYLDTVL-OH with up to 25-fold higher affinity than the original NHERF PDZ1 domain. By identifying a PDZ variant that binds more tightly to the desired product sequence, we increased assay sensitivity by 5-fold and throughput by at least 3-fold while requiring less PDZ protein to do so. These results represent, to our knowledge, the first reported example of PDZ domain engineering using phage display, yielding new structure–activity observations which we relate to published high-resolution crystallographic data.


    Materials and methods
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General

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 {varepsilon}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 {varepsilon}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., 2002Go) 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 PEG–NaCl 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 1–2 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 SOB–2% 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., 2002Go). 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 = 0.5–1.0 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 freeze–thawed 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., 2003Go). 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., 2002Go) experiments were performed as described previously (Hamilton et al., 2003Go). 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., 2003Go). 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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Directed evolution of PDZ variants

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., 1989Go), 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., 2001Go). 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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Fig. 2. Affinity selection of DTVL-binding PDZ variants from NHERF mutant library. (A) Five rounds of affinity panning were carried out against target peptide HRRSARYLDTVL-OH with two libraries derived from PSD95 PDZ3 (squares) and NHERF PDZ1 (diamonds). On the fifth round of panning, a larger proportion of phage from the NHERF PDZ library than from the PSD95 PDZ library remain bound to the peptide ligand, indicating selection of target peptide-specific PDZ variants. (B) Phage displaying wild-type PSD95 PDZ3, NHERF PDZ1, a negative control (scFv) and three NHERF PDZ variants (F10, H9 and G9) were tested in a phage ELISA against peptides HRRSARYLESSV-OH (PSD95 PDZ3 positive control), HRRSARYLDTVL-OH (target peptide), HRRNEEVQDTRL-OH (wild-type NHERF PDZ positive control) and HRRSARYLDTVLEEMS-OH (substrate peptide).

 
Several individual phage clones were isolated, grown in liquid culture and resulting phage particles were purified by PEG–NaCl precipitation. A phage ELISA screen identified a set of clones from the NHERF library having improved apparent affinity for peptide DTVL. Three of these mutants (F10, G9, H9) and wild-type controls were then tested by phage ELISA (Figure 2B) against peptides HRRSARYLESSV-OH (control peptide bound preferentially by PSD95 PDZ3), HRRNEEVQDTRL-OH [peptide used in Hamilton et al. (2003)Go bound preferentially by NHERF PDZ1], HRRSARYLDTVL-OH (target peptide) and HRRSARYLDTVLEEMS-OH (substrate peptide). Two observations stood out. First, wild-type PSD95 PDZ3 gave a much stronger signal when bound to its preferred ligand (ESSV) than wild-type NHERF PDZ did with its preferred ligand (DTRL). This is inconsistent with expectations based on past affinity measurements and suggests a difference in the efficiency with which NHERF and PSD95 PDZ are displayed on phage. Second, each of the three mutants (H9, G9 and F10) showed dramatically improved ELISA signals for both the HRRNEEVQDTRL-OH and HRRSARYLDTVL-OH peptides compared with wild-type NHERF PDZ; and very low signal with peptides HRRSARYLDTVLEEMS-OH and HRRSARYLESSV-OH. This suggests that these mutants have improved affinity and are specific for HRRSARYLDTVL-OH and HRRNEEVQDTRL-OH.

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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Fig. 3. Nanomolar affinity of selected variants to target peptide. (A) Affinity of wild-type and PDZ variants to target peptide DTVL measured by fluorescence polarization. Serial dilutions of wild-type NHERF GST-PDZ1 (closed squares), PSD95 GST-PDZ3 (inverted closed triangles) and NHERF mutants G9 (closed triangles), H9 (open triangles) and F10 (open squares) were prepared and peptide Fl-HRRSARYLDTVL-OH was added to a final concentration of 10 nM. The samples were incubated for 2 h at room temperature in the dark before reading the FP. Data are the mean of six (wild-type NHERF) or three (all others) independent experiments conducted in triplicate and the error bars represent the standard error in the mean. (B) Affinity of wild-type PDZ domains and PDZ variants to control peptide DTRL. As in (A), but Fl-HRRNEEVQDTRL-OH peptide was used rather than Fl-HRRSARYLDTVL-OH. NHERF WT GST-PDZ1 (closed squares), G9 (closed triangles), H9 (open triangles), F10 (open squares) and PSD95 GST-PDZ3 (closed inverted triangles). Data are the mean of two independent experiments conducted in triplicate.

 

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Table I. Affinity of PDZ domains measured by fluorescence polarizationa

 
Alignment of the wild-type and mutant sequences indicates that minimally two amino acid substitutions are sufficient to improve the affinity of the NHERF PDZ domain for the HRRSARYLDTVL-OH ligand (Figure 4A). In Figure 4B, the observed amino acid substitutions are mapped to a ribbon diagram of the three-dimensional structure of NHERF PDZ1 bound to a polypeptide ligand (Karthikeyan et al., 2001Go). The {alpha}-carbon chain of the last six amino acids of the ligand is shown (purple). The side chains of ligand residues –1, –4, –5 and –6, are shown to highlight residues that differ between peptide DTRL and the target peptide DTVL. All mutants share a common substitution, R87C, which maps to strand ß6 and is on the opposite side of the domain from the peptide binding groove (Figure 4B). All other mutations reside near the ligand binding groove of the PDZ domain. Mutant F10, which shows the smallest increase in affinity for the target peptide, has only one substitution (H29R) in addition to the common R87C change. This amino acid change (H29R) maps near ligand residue –4. Mutant G9 shows the same two substitutions plus a replacement of K32 with a glutamic acid near ligand residues –4 and –5. In addition to the shared R87C, the third mutant, named H9, bears substitutions R40G, located near ligand residue –4, and E43V, proximal to ligand residue –1.



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Fig. 4. Alignment and structural model of wild-type NHERF PDZ 1 from rabbit, NHERF variants H9, F10, G9 and wild-type human PSD95 PDZ3. (A) Amino acid substitutions found in the variants are highlighted (black letters on white background). Numbering is according to Karthikeyan et al. (2001)Go and PDB file 1I92. (B) The crystal structure of NHERF (orange) in complex with a ligand (purple), as described by Karthikeyan et al. (2001)Go, PDB file 1I92. Side chains of ligand residues which differ between peptides HRRSARYLDTVL-OH and HRRNEEVQDTRL-OH are shown in stick representation and labeled (–1 and –4 to –6). All amino acid substitutions found in the three NHERF mutants isolated in this study are shown in stick form and colored red. Only substitution R87C is found in all three mutants. Figure generated using DeepView3.7 and POV-Ray3.5.

 
Comparison of wild-type and variant PDZ domains in protease assay

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 product–substrate 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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Fig. 5. Comparison of wild-type and variant PDZ domains in simulated protease assay. (A) A dose–response titration of biotinylated product peptide (biotin-HRRSARYLDTVL-OH) was prepared with enough biotinylated substrate peptide (biotin-HRRSARYLDTVLEEMS-OH) to maintain 200 nM total peptide concentration. A 5 µl volume of these samples was incubated in triplicate with 5 µl of GST-conjugated PDZ domain [40 nM; NHERF WT GST-PDZ1 (closed squares), G9 (closed triangles), H9 (pen triangles), F10 (open squares) and PSD95 GST-PDZ3 (closed inverted triangles) for 10 min at room temperature]. Then 10 µl of detection reagent (25 nM streptavidin-XL665, 10 nM anti-GST[Eu3+]) were added and the samples were covered and incubated for 1 h at room temperature in the dark. Using the Victor2V, samples were excited at 340 nm and emission was measured at 615 and 665 nm. The 665:615 nm emission ratio was calculated and the data were fitted to a sigmoidal dose–response binding model with a variable Hill coefficient. Data are the mean of two independent experiments conducted in triplicate and the error bars represent the standard error in the mean. (B) Data were collected as in (A), but the 665:615 nm emission ratio is plotted against the percentage turnover, calculated as the ratio of product peptide to total peptide. Data are the mean of two independent experiments conducted in triplicate and the error bars represent the standard error in the mean.

 
In the actual enzymatic reaction, the mutants G9, H9 and F10 were tested for their ability to detect substrate turnover at varying enzyme concentrations. As shown in Figure 6, all three mutant GST–PDZ fusion proteins detect product formation in the time course experiment more effectively than the wild-type NHERF PDZ1 domain. A 10-fold assay window was obtained for H9 and G9 in 30 min, using ~0.3 nM of HIV protease, whereas F10 required 0.6 nM and wild-type required 1.25 nM. As pointed out in the previous paragraph, a 10-fold assay window for mutants H9 and G9 corresponds to 10% turnover, whereas for wild-type ~30% substrate conversion is required.



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Fig. 6. Comparison of wild-type and variant PDZ domains in HIV protease assay. HIV protease enzyme was allowed to react with 1 mM biotinylated substrate peptide (biotin-HRRSARYLDTVLEEMS-OH) at 37°C in assay buffer at the indicated concentrations (closed squares, 10 nM; closed triangles, 5 nM; closed inverted triangles, 2.5 nM; closed diamonds, 1.25 nM; closed circles, 0.625 nM; open squares, no enzyme). Reactions were quenched at the indicated time intervals by diluting them 1:5 into PBSTB, pH 7.4. For detection, 5 ml of quenched reaction were incubated for 10 min at room temperature with 5 ml (40 nM) of the indicated GST fusion PDZ domain [(A) NHERF; (B) G9; (C) H9; or (D) F10), then 10 ml of detection solution (25 nM streptavidin-XL665, 10 nM anti-GST[Eu3+]) were added and the samples were covered and incubated for 1ne h at room temperature in the dark. Using the Acquest, samples were excited at 340 nm and emission at 620 and 665 nm was measured. Data are the mean of two (wild-type and H9) or three (G9 and F10) independent experiments conducted in triplicate and the error bars represent the standard error of the mean.

 
Discussion

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., 2002Go; Hamilton et al., 2003Go). 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., 2003Go). 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)Go first described the use of phage display to study PDZ–ligand 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., 2002Go) to yield PDZ variants with substantial specificity changes and affinities in the range 1–100 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., 1999Go). 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 ‘ß1–ß3’ interactions (Fuh et al., 2000Go). 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 X–S/T–X–{phi} recognition sequence (where {phi} is hydrophobic). Moreover, Karthiekeyan et al. (2001)Go 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 620–1700 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 streptavidin–peptide 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 {alpha}-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.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 References
 
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Received October 3, 2004; revised January 17, 2005; accepted January 18, 2005.

Edited by William DeGrado