Mapping a Disordered Portion of the Brz2001-Binding ...

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Mapping a Disordered Portion of the Brz2001-Binding Site on a Plant Monooxygenase, DWARF4, Using a Quartz-Crystal Microbalance Biosensor-Based T7 Phage Display Yoichi Takakusagi,* Daisuke Manita, Tomoe Kusayanagi, Jesus Izaguirre-Carbonell, Kaori Takakusagi, Kouji Kuramochi, Kazuki Iwabata, Yoshihiro Kanai, Kengo Sakaguchi, Fumio Sugawara

recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

INTRODUCTION

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan. *Present address: National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

ABSTRACT In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor–ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage–displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the aminoacid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule–

I

dentification of small-molecule binding sites on protein is an essential task in medicinal chemistry, chemical biology, and pharmacology. This information serves as a molecular basis for understanding not only the mode of action for the small molecule of interest, but also the biological phenomena associated with the small molecule. While recent proteomics approaches have enabled comprehensive identification of small-molecule/protein interactions in theory, physicochemical properties of experimental samples may restrict their interactions. These are divided mainly into two aspects of problems imposed by those of (1) small molecules and (2) proteins. The former can include low solubility in solvent, low levels of detection on analytical methods due to the small molecular mass of compounds, and inaccessibility of the immobilized small molecular size of compounds to internally located binding sites on larger target proteins. The latter can include the requirement for large amounts of the protein, determination of co-crystallization conditions, molecular size restrictions of the analytical method, and low solubility in solvents that is mainly due to protein misfolding when genetically engineered. Thus, research on undruggable proteome that is a result of these technical limitations should be largely continued.1 A biosensor-based screening of T7 phage-displayed random peptide is a useful strategy for detecting small-molecule/protein interactions, which could circumvent those technical limitations.2–6 This approach employs a high-sensitivity quartz-crystal microbalance (QCM) biosensor that can monitor the decrease of alternating voltage–induced intrinsic crystal vibrations in real time, which indicates a mass increase in the gold electrode deposited on the crystal of the QCM sensor chip (Fig. 1A). The discovery of QCM phenomena in the air phase, which is described as the Sauerbrey’s equation, goes back to the original report in 1959.7 Owing to the reiterated physical and engineering refinements over the past halfcentury, high-resolution QCM biosensors have been developed. They make it possible to weigh the pico- to nanogram scales of bound

ABBREVIATIONS: IR, infrared spectra; QCM, quartz-crystal microbalance; RELIC, receptor–ligand contacts; SAM, self-assembled monolayer; SDS, sodium dodecyl sulfate.

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DOI: 10.1089/adt.2012.478

ONE-CYCLE BIOPANNING COMBINED WITH BIOINFORMATICS ANALYSIS

Fig. 1. A schematic representation of a quartz-crystal microbalance (QCM) biosensor-based T7 phage display and an analysis of the smallmolecule–selected peptide sequence using bioinformatics program in a receptor–ligand contacts (RELIC) suite. (A) One-cycle biopanning of a small-molecule–recognizing peptide sequence on a T7-phage capsid. The T7-phage library is injected into the buffer filled cuvette, and the frequency decrease on binding of the T7 phage to the small molecule on gold is monitored in real time. The 15-mer random peptide (4.5– 5.5 nm in length) on the T7-phage capsid (55 nm) can sterically contact with any small molecules (< 1 nm) immobilized on the gold electrode. The bound T7-phage DNA is directly recovered by host Escherichia coli infection. After plaque isolation, a part of the peptide-encoding region is sequenced according to the general procedure. (B) Illustration of the algorithmic analysis using the MOTIF, MATCH, or HETEROalign program in RELIC suite. The continuous (MOTIF1) or discontinuous (MOTIF2) conserved motif in the small-molecule-selected peptides is detected to evaluate the success of the selection. The subset of short peptides is then used to MATCH or HETEROalign (if PDB data is available) to calculate the weak similarity between the short peptide sequences and a primary protein sequence. The score is calculated by a modified BLOSAM62 amino-acid matrix within each window of five amino acids in length across the entire length of the protein sequence and then cumulatively plotted as a similarity plot. The mapping of the amino-acid sequence is generated at the portion of maximal similarity score.

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molecules even in aqueous solutions, and have been widely applied to studies in life and material sciences.8–10 The use of this platform in a T7 phage display enabled a rapid capture of small-molecule recognizing T7 phages on the gold by sub-minutes of monitoring the frequency decrease, with no need of repeated rounds of biopanning.3,4 This also corresponds to the fact that the QCM-based selection eliminated hitherto inevitable problems associated with phage distribution bias during the amplification process on classical methods. Furthermore, subsequent use of a host Escherichia coli culture realized a rapid and direct recovery of DNA from the trace amount of bound T7 phage, without elute condition exploration. By sequencing the peptide-encoding region of phage DNA, the corresponding amino-acid sequence can be clearly determined. In other words, the T7 phage particle plays a role as a transducer for the highly sensitive biosensor to sequence the trace amount of peptide bound to the small molecule on the gold electrode. Significantly, unlike proteins, the use of random shorter peptides can allow sterical contact with any compounds of small molecular size fixed on the gold electrode (Fig. 1A).3,4 In 2004, Rodi and Makowski and their coworkers launched the receptor–ligand contacts (RELIC) bioinformatics server, and they have since extensively studied the annotation of a small-molecule binding site on protein by a similarity search using affinity-selected random peptides.11–16 According to their earlier studies, phagedisplayed short peptides, most of which are basically disordered, can mimic the disordered loops in naturally occurring proteins that are considered an essential portion in many small-molecule/protein interactions.17–19 Indeed, there are several examples of ligands that occupy sites on proteins which are not the classic ‘‘substrate binding site’’ as well as cases where analogous inhibitors do not bind analogously.20–22 Based on these observations, they have established a suite of bioinformatics programs designed for random peptide phage display. Once the amino-acid sequence of affinity-selected peptides is determined, the use of an appropriate program in RELIC with the sequence allows not only the qualitative and quantitative assessment of the affinity-selected peptide population, but also the annotation of small-molecule binding sites on protein that is de-

pendent on the weak similarity with the limited short peptide sequence data (Fig. 1B).11,13,23,24 In this work, the QCM-based one-cycle biopanning in combination use with RELIC bioinformatics analysis has been extended into plant molecular biology.3,4 Brz2001 (Fig. 2A) is a specific DWARF4 inhibitor in plant hormone brassinosteroid biosynthesis.25,26 Brz2001 belongs to the triazole series of compound that shows low solubility in solvents, as represented by antifungal agents such as fluconazole. In addition, recent studies have revealed that the target protein DWARF4 is a C22 hydrolase which is classified into plant cytochrome P450 (Cyp450) monooxygenase super family and contains heme and iron in an interior location of the enzyme where it catalyzes the hydroxylation reaction.27 This fact implies that the DWARF4 protein requires these cofactors for folding into a functionally correct three-dimensional structure when genetically engineered. Furthermore, it may be also challenging to obtain the amount of purified plant enzyme for interaction analysis by NMR experiment or X-ray crystallography. Using this plant Brz2001/DWARF4 system, which may be time consuming and labor-intensive to refine the direct interaction properties on classical methods, we show that this rapid approach reveals disordered loops within catalytic sites on DWARF4 as an essential portion for Brz2001 recognition. This affinity-based strategy could be applicable for any small-molecule/protein systems under the identical protocol, overcoming the technical difficulties of conventional methods.

MATERIALS AND METHODS QCM Apparatus and Reagents The 27-MHz QCM apparatus (AffinixQ) and ceramic sensor chip (SiO2, 0.06 mm thick, 9 mm in diameter, 64 mm2; Au, 0.1 mm thick, 2.5 mm in diameter, 4.9 mm2) were purchased from Initium Inc. T7 select 10-3 OrientExpressTM cDNA Cloning System was from Novagen. The oligonucleotide and primer were purchased from InvitrogenTM Custom Oligonucleotides. Klenow DNA polymerase I and ExoSAP-IT were purchased from USB Corporation. Ex Taq DNA polymerase was obtained from TaKaRa. ABI PRISM! BigDyeTM Terminator Cycle Sequencing Kit was from Applied Biosystems.

Fig. 2. Structure of (A) Brz2001 and (B) a Brz2001 derivative that forms a self-assembled monolayer (SAM) on a gold surface. The molecule comprises four units: (i) sulfide bond for chemisorption on the gold electrode surface of the sensor chip via thiol–Au interactions; (ii) a C11 alkyl chain for accumulation of the Brz2001 on the gold; (iii) diethylene glycol (DEG) for reducing the nonspecific binding (mainly via hydrophobic interaction) with the gold surface or linkers; and (iv) Brz2001 to act as bait during biopanning.

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ONE-CYCLE BIOPANNING COMBINED WITH BIOINFORMATICS ANALYSIS

Construction of the T7 Phage-Displayed Random Peptide Library For the preparation of a duplex DNA library, oligonucleotide GGG GAT CCG AAT TCT (NNK)15 TGA AAG CTT CTC GAG GG (0.056 pM) and CCC TCG AGA AGC TTT CA (0.56 pM) were mixed with Klenow buffer, heated to 95"C for 5 min, and annealed by slowly cooling the mixture to 37"C. The single-stranded regions were converted to duplex DNA by continuing the incubation at 37"C for 2 h in the presence of dNTPs (2.5 mM) and Klenow enzyme (0.5 mU/mL). After the reaction, double-stranded DNA was recovered by EtOH precipitation. The obtained DNA was then digested separately with EcoRI and HindIII

restriction enzyme and inserted into the T7 select 10-3b vector according to the manufacturer’s instructions. The primary titer of this T7 phage library was 1.6 · 107 pfu/mL. For the screening procedure, the phage library was amplified up to 1.7 · 1010 pfu/mL using E. coli (BLT5615) as the host strain.

The QCM Biosensor-Based T7 Phage Display

The protocol is summarized in Table 1. A ceramic sensor chip was attached on the oscillator of a 27-MHz QCM apparatus (AffinixQ), and the intrinsic frequency in the air phase was recorded before compound immobilization. After detaching the chip, a 20 mL aliquot of a Brz2001 derivative solution (1 mM in 70% EtOH) was dropped onto the gold electrode of the ceramic sensor chip and left Table 1. Protocol for the QCM Biosensor-Based T7-Phage Display for 1 h under a humid and shaded atmoand Analysis of RELIC Bioinformatics Server sphere at room temperature. The surface of Step Parameter Value Description the electrode was washed for 10 min in 1 Sensor chip preparation 20 mL 1 mM Brz2001 derivative forming SAM buffer (10 mM Tris-HCl, pH 8.0, and 200 mM NaCl), which was stirred at 2 Setup of QCM apparatus 8 mL 10 mM Tris–HCl, pH 8.0, 200 mM NaCl 1,000 rpm at 25"C (AffinixQ, Initium Inc.). 3 T7-phage library injection 8 mL Injection into the buffer-filled cuvette The sensor chip was set up for the QCM apparatus and recorded the decreased fre4 Monitoring the QCM sensor 10 min Monitoring the frequency decrease for 10 min quency in the air phase to measure the 5 Recovery of bound phage 20 mL Dropping the host E. coli culture on the gold electrode immobilized Brz2001 amount. The Brz2001 derivative was immobilized at *11,000 Hz 6 Phage plaque isolation 3h Incubation at 37"C (330 ng) on average. The QCM sensor was 7 PCR and DNA sequencing 50 mL 20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO4 immersed into the cuvette containing 8 mL of buffer and then allowed to fully stabilize. 8 Analysis of RELIC server Uploading the peptide sequence (.txt) according to the An aliquot of 8 mL of a T7 phage library instruction and executing the program (1.7 · 1010 pfu/mL) was injected into the 9 Sensor chip regeneration 5 mL Swabbing the gold electrode surface by 1% SDS and cuvette. Frequency changes, caused by then dropping the piranha solution binding to the Brz2001 immobilized on the gold electrode surface, were then monitored Step Notes 1. A 20 mL aliquot of a Brz2001 derivative solution (70% EtOH) that forms SAM was dropped onto the gold for 10 min. For the recovery of bound electrode of the ceramic sensor chip and left for 1 h under a humid and shaded atmosphere at room phages, 20 mL of log-phase host E. coli temperature. The immobilized amount on gold was measured as the decreased frequency in the air phase after (BLT5615) solution, which was cultured for immobilization. 30 min at 37"C in the presence of 50 mg/mL 2. The sensor chip was attached on oscillator and immersed; the buffer-filled cuvette was constitutively stirred at of carbenicillin and 1 mM of IPTG before1000 rpm at 25"C (AffinixQ, Initium Inc.). 3. After stabilizing the QCM sensor, the 8 mL of T7 phage library (final > 108 pfu) was injected into the cuvette. hand (OD600 = 1.0), was dropped onto the 4. Frequency decrease by mass increase on binding of T7 phage to Brz2001 on the gold electrode was monitored gold electrode and then incubated at 37"C for 10 min. for 30 min. Another 200 mL of LB medium 5. The sensor chip was detached from the apparatus, and 20 mL of log-phase and T7 phage-infective host was then added to the resulting solution, Escherichia coli (BLT5615) solution was dropped onto the gold electrode to incubate for 30 min at 37"C with which was then subjected to plaque isolashaking. The resulting solution was added to 200 mL of LB medium. 6. The 100 mL of each diluted solution (101 to 106 times) was plated onto LB/carbenicillin plate with 3 mL of tion. After that, the gold electrode surface prewarmed top agarose and 100 mL of host E. coli solution, and incubated for 3 h at 37"C. was swabbed with 1% sodium dodecyl sul7. Each plaque was isolated and suspended into 50 mL phage extraction buffer. Peptide-encoding region on T7 fate, treated with 5 mL of piranha solution phage DNA was amplified by PCR using specific primers and then sequenced. (concentrated H2SO4: 30% H2O2 = 3: 1) for 8. Resulting amino-acid sequence of peptide was listed in .txt file and uploaded to execute an appropriate 5 min, washed with dH2O, and then dried. program in RELIC bioinformatics server according to the instructions. 9. After swabbing the gold electrode surface by 1% SDS, 5 mL of piranha solution (concentrated H2SO4: 30% H2O2 = 3: 1) was dropped and left for 5 min, and then washed by dH2O. QCM, quartz-crystal microbalance; SAM, self-assembled monolayer; RELIC, receptor ligand contacts; SDS, sodium dodecyl sulfate; PCR, polymerase chain reaction.

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Plaque Isolation The recovered phage solution, which contains 5 · 105–5 · 106 pfu/mL of T7 phages on average, was diluted from 101 to

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106 times. A 100 mL aliquot of each solution was mixed with 200 mL of log-phase host E. coli solution and 3 mL of prewarmed top agarose [10 g/L Bacto tryptone, 5 g/L yeast extract, 5 g/L NaCl, 6 g/L agarose], and then seeded onto an LB/carbenicillin plate. The plate was incubated at 37"C for 3 h to form individual phage plaque.

PCR and DNA Sequencing Plaques were randomly picked from LB plates, and each was dissolved in 50 mL of phage extraction buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO4). The phages were disrupted by heating the extract to 65"C for 10 min. To amplify the peptide-encoding region, PCR was performed according to the following PCR mixture: 0.3 mL phage solution, 0.05 mL of the 100 mM forward primer 50 -TGC TAA CTT CCA AGC GGA CC-30 and the 100 mM reverse primer 50 AAA AAC CCC TCA AGA CCC GTT TA-30 , 0.25 mM dNTP, 1 mL of 10 · Ex Taq DNA polymerase buffer, and 1.25 U of Ex Taq DNA polymerase. The mixture was diluted up to 10 mL using double-distilled water. PCR condition was 25 cycles each of 94"C for 60 s, 50"C for 30 s, and 72"C for 30 s. The 5 mL of products were treated with 2 mL of ExoSAP-IT (digested at 37"C for 15 min and then inactivated at 80"C for 15 min) and 70% EtOH precipitated. The products were then sequenced using ABI Prism BigDye Terminator Cycle Sequencing Kit and on an ABI Prism3100 Genetic Analyzer (ABI) according to the manufacturer’s protocols.

Analysis Using RELIC Server The stand-alone type RELIC programs were kindly provided by Dr. Lee Makowski (Northeastern University, Boston, MA). AAFREQ, MOTIF1 and 2, and MATCH programs in RELIC were used according to their manuals for analysis of Brz2001-selected peptides and detection of the Brz2001-binding site on DWARF4.13

RESULTS AND DISCUSSION To immobilize Brz2001 (MW: 353.85) on the gold electrode of the QCM sensor chip, we used a derivative that forms a self-assembled monolayer on gold (Fig. 2B, Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/adt).28–30 Apart from its improved high solubility (>10 mM) in 70% EtOH, the larger molecular mass of derivative (MW: 1,490.78) enabled a tight attachment of the compound on the gold and thereby increased the sensitivity of QCM at least thrice greater than that using avidin-biotin immobilization by piggygack or mass enhancer effect.3,4,31–33 Figure 3A shows the representative sensorgram after injecting a T7 phage-displayed random peptide library into a buffer-filled cuvette where the sensor chip was immersed (Fig. 1A). The decreased frequency 10 min after the injection of T7 phage library was 108 – 15 Hz; whereas after the injection of control T7 phage, it was 37 – 15 Hz (Fig. 3B). The sensor chip was dislodged from the device, and the bound T7 phage DNA was directly recovered by host E. coli BLT5615 infection. After the plaque isolation, T7 phage DNA of the peptide-encoding region was sequenced using a specific primer and translated into an amino-acid sequence (Fig. 1A). In total, four sets of one-cycle biopanning identified 34 of the 15-mer peptides that potentially recognize Brz2001 (Table 2).

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Fig. 3. The QCM biosensor-based one-cycle biopanning. (A) A representative QCM sensorgram obtained by an affinity selection of peptide on the QCM apparatus (AffinixQ) using the Brz2001 derivative and a random peptide T7 phage library (8 mL; 1.4 · 108 pfu). The Brz2001-immobilized ceramic sensor chip generated by SAM was attached to the QCM apparatus. After injecting the T7-phage library at the indicated concentration, the frequency decrease was monitored for 10 min. (B) Frequency decrease 10 min after the injection. 1, T7-phage library versus Brz2001; 2, T7-phage library versus gold electrode; 3, Monoclonal T7 phage (displaying no 15-mer peptide) versus Brz2001. Data are means – SD (n = 3). **P < 0.01. In general, small molecules appear to show weak interactions with peptides via surface–surface contacts of continuous or discontinuous motifs of several amino-acid residues. Testing the interaction between individual peptides with small molecules should be experimentally limited to some extent, because there may be a large number of potential small-molecule–recognizing peptides within a library. Furthermore, affinity between immobilized small molecules and short peptides is too weak (KD: 10 - 4–10 - 5 M range) to characterize each affinity status. Nonetheless, algorithmic and heuristic approaches using the RELIC program enabled rapid extraction of sequential information that is potentially associated with the smallmolecule recognition from a subset of affinity-selected peptides, without checking the affinity status of individual peptides.11,13,23,24 Table 3 shows continuous motifs that were detected from 34 Brz2001-selected peptides by the use of the RELIC/MOTIF1 program. In addition to the exact match of short amino-acid stretches, conservative motifs are efficiently extracted from the limited peptide population. Indeed, a pair of six-continuous motifs, 6 kinds of fivecontinuous and 25 of four-continuous motifs, were detected in the subset of peptides (Table 3). Furthermore, the RELIC/MOTIF2 program, which searches for patterns of three amino acids and does not allow conservative amino-acid substitutions, allows identical gap lengths, highlighted VXXFXF (among five individual peptides), CXXVXL, FXVXXG, SXXXVXXXXS, VXLXV (four peptides; Table 4), and others (two or three peptides; data not shown). These motifs were not significantly detected within the 103 peptide sequences (Supplementary Table S1) arbitrarily selected from the unscreened parent library. In addition, frequency of amino acids C F G S V, which are the components of the continuous and discontinuous motifs, also

ONE-CYCLE BIOPANNING COMBINED WITH BIOINFORMATICS ANALYSIS

Table 2. Brz2001-Selected 15-mer Peptide Sequences After Four Sets of Individual One-Cycle Biopanning No.

Sequence

No.

Sequence

No.

Sequence

No.

Sequence

1

VLLAVWSVFHERSWL

10

CLIVCLFIVRCGGCF

19

GCNVWMCPDFCLPAR

28

LGVVGFFPFDPSVLL

2

SLYLPCVLPDLRFRM

11

CGFAFYYVWLMVVMW

20

QFFFSDDGSPPFEVG

29

CSQGLLHSAVFALCF

a

3

MLVCIFFFGMLLIMF

12

FCVCFCGCTIMVLLL

21

SSCNLVLAAHQFCGD

30

4

ASPLIVAAACSSYFL

13

AFVSGADVNVCLHCA

22

LFISIFVLAFCFALV

31

a

a

LSLGSGFVVSYGVSG SCLAVVLNCSGGLWS

SVFCVSGSSSHWLIE

32

FCVNNGTTCSVWRCD

24

YSCDAYVGVRGVLCI

33a

GCVFILWGISSSHDA

SASCFTGGMVRAHVR

25a

LHLIGLAASSGLSDL

34

CVPSFGFVLPNRLAA

17

VRPHPGLARSLPVAR

26

ICNSVSLFVFGPYGA

18

CVYPAFTPFGQYTLL

27

FLPGIGYASVLCCSG

5

FCVSNGTTCVCIGVV

14

TDVDFLFPNIHVSFG

23

6

RVFFVIFLSDLIWPF

15

SNAPCIVDMSSPLVH

7

IFDALSLVCLSVQPS

16a

8

LCCSVNLELSPSMGG

9a

CDFLCVRSAVGTMVA

T7-phage particles were arbitrarily extracted from the resulting solution and then analyzed by PCR amplification of the DNA encoding the fusion peptide. The PCR products were then sequenced. a The peptide directly highlights a disordered portion on DWARF4 using MATCH program (Fig. 5A).

increased 1.1- to 1.23-fold through the selection, although it decreased or did not change in the other amino acids (Supplementary Tables S2 and S3). Collectively, these results indicate that the information content for Brz2001 recognition significantly increased through the affinity selection for Brz2001. Although the mode of docking of Brz2001 to DWARF4 has still been unsolved by NMR experiment or X-ray crystallography, the subsequent use of the RELIC/MATCH program with the Brz2001selected peptide sequences enabled the prediction of polypeptide portions involved in Brz2001 binding. This program calculates a weak similarity between small-molecule–selected short peptides and a longer target polypeptide, even in the absence of a clearly identifiable consensus sequence motif or a three-dimensional structure of the target (Fig. 1B).3,4,11,13 Figure 4A shows a similarity plot between 34 Brz2001-selected random peptides and the DWARF4 amino-acid sequence (M1-L513). This plot was calculated by a modified BLOSAM62 amino-acid matrix within each window of five amino acids in length across the entire length of the protein sequence.12,14–16 The similarity was cumulatively scored and plotted at each amino-acid residue. Furthermore, scores calculated using 103 unscreened parent random peptides (Supplementary Table S1) were subtracted as a background. As a result, a portion containing S438 in DWARF4 was detected as a maximal similarity score by employing 34 Brz2001-selected peptide sequences. As seen in Figure 5A, this analysis also generated a cluster diagram, indicating that this portion is essential for Brz2001 recognition. A similar trend was noted in the analysis using another 26 Brz2001selected peptides (Figs. 4B and 5B; Supplementary Table S4). In this case, other portions containing N379, P407, and P454, respectively, were also pinpointed, in addition to S438. By contrast, such maximal scores were not observed by analysis using 27 gold electrode–

recognizing control peptides (Fig. 4C; Supplementary Table S5). These results indicate the reproducibility and specificity of this method. Furthermore, a part of the highlighted amino-acid residues in 15-mer peptides overlapped with the potential Brz2001recognizing motifs detected in analysis using RELIC/MOTIF-1 and -2 programs (Tables 3 and 4). Thus, this strategy could be applicable to annotate a small-molecule–recognizing portion on proteins in the case when the three-dimensional structure is unavailable. Significantly, the small-molecule binding site that is internally located on protein can be targeted by this approach. Furthermore, this method is free from both naturally and experimentally occurring protein mutations, deletions, and other modifications that may influence the protein folding and fail to target identification in some cases. According to the information about human cytochrome 2C9, one of a Cyp450 monooxygenase homologous to DWARF4 (E value: 6e-12 on standard blastp) and being available in the three dimensional structure [PDB ID: 1R9O], the primary sequence of the highlighted portion is potentially disordered flexible loops that comprise a substrate-binding site within the vicinity of the catalytic site that is mediated by heme, iron, and a cysteine of the heme axial ligand (Fig. 5A, B; Supplementary Fig. S2).34,35 Indeed, DWARF4 inhibitors having a triazole ring interfere with the redox activity via this portion, which result in the inhibition of brassinosteroid biosynthesis and growth suppression of plants ranging in concentration from 10 - 4 to 10 - 6 M (KD: 10 - 5–10 - 6 M).25–27,36,37 Dynamic mobilization of the flexible portions that have been first detected in this experiment seems to be essential during docking of Brz2001 to the catalytic site. In addition, several other amino-acid residues in DWARF4 were detected by this analysis (Fig. 4A, B). These residues could contribute to the binding of Brz2001to DWARF4, or might just be false positives,

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Table 3. Four to Six Continuous Motifs Extracted from Brz2001-Affinity-Selected Peptides Using the MOTIF1 Program in the RELIC Suite Six continuous motifs

Five continuous motifs

No.

Pos.

Motif

No.

Pos.

Motif

No.

Pos.

Motif

No.

Pos.

Motif

No.

Pos.

3

1

MLVCIF

3

10

MLLIM

4

4

LIVAA

5

11

CIGVV

22

3

ISIFV

10

2

LIVCIF

11

10

LMVVM

21

5

LVLAA

31

2

CLAVV

26

5

VSLFV

12

10

IMVLL

5

5

NGTTC

10

5

CLFIV

33

2

CVFIL

No.

Pos.

No.

Pos.

Motif

12

11

MVLLL

32

a

5

NGTTC

a

Motif

Four continuous motifs No.

Pos.

Motif

No.

Pos.

Motif

No.

Pos.

Motif

Motif

a

1

1

VLLA

3

2

LVCI

5

12

IGVV

8

7

LELS

16

6

TGGM

4

4

LIVA

7

7

LVCL

28

1

LGVV

15

7

VDMS

31

10

SGGL

21

5

LVLA

10

3

IVCL

31

3

LAVV

11

GTMV

17

5

PGLA

1

2

LLAV

24

12

VLCI

6

6

IFLS

27

8

ASVL

27

3

PGIG

25a

3

LIGL

3

8

FGML

22

1

LFIS

10

6

LFIV

22

7

VLAF

1

4

AVWS

31

4

AVVL

6

8

LSDL

22

5

IFVL

28

3

VVGF

31

12

GLWS

3

9

GMLL

25a

12

LSDL

33a

3

VFIL

23a

8

SSSH

2

1

SLYL

31

4

AVVL

7

6

SLVC

12

9

TIMV

33a

10

SSSH

22

4

SIFV

4

2

SPLI

27

9

SVLC

28

12

SVLL

30a

6

GFVV

26

6

SLFV

15

11

SPLV

8

1

LCCS

14

12

VSFG

34

6

GFVL

2

5

PCVL

5

1

FCVS

27

11

LCCS

30a

9

VSYG

3

FCVS

15

4

PCIV

a

23

9a

a

The peptide directly highlights a disordered portion on DWARF4 using MATCH program.

Table 4. Three Discontinuous Motifs Extracted from Brz2001-Affinity-Selected Peptides Using the MOTIF2 Program in the RELIC Suite No. 3

Motif MLVCIFFFGMLLIMF

No. 8

Motif LCCSVNLELSPSMGG

No.

Motif

No.

Motif

No. Motif

5

FCVSNGTTCVCIGVV

4

ASPLIVAAACSSYFL

1

VLLAVWSVFHERSWL IFDALSLVCLSVQPS

22

LFISIFVLAFCFALV

10

CLIVCLFIVRCGGCF

10

CLIVCLFIVRCGGCF

23

SVFCVSGSSSHWLIE

7

26

ICNSVSLFVFGPYGA

26

ICNSVSLFVFGPYGA

30

LSLGSGFVVSYGVSG

30a

LSLGSGFVVSYGVSG

11

CGFAFYYVWLMVVMW

28

LGVVGFFPFDPSVLL

31

SCLAVVLNCSGGLWS

32

FCVNNGTTCSVWRCD

31

SCLAVVLNCSGGLWS

26

ICNSVSLFVFGPYGA

34

CVPSFGFVLPNRLAA Motifs conserving more than 4 peptides are presented in bold. a The peptide directly highlights a disordered portion on DWARF4 using MATCH program.

212 ASSAY and Drug Development Technologies APRIL 2013

ONE-CYCLE BIOPANNING COMBINED WITH BIOINFORMATICS ANALYSIS

Fig. 4. Analysis of the similarity of amino-acid sequence between Brz2001-selected peptides and DWARF4 using the MATCH program in the RELIC suite: similarity plot. Scores for the sequence of DWARF4 (M1-L513) against the sequences of 34 (A) or 26 (B) peptides selected for affinity to Brz2001, or 27 for gold electrode (control) (C) was calculated using RELIC/MATCH program. The similarity scores of random peptides chosen without affinity selection (Supplementary Table S1) have been subtracted from these scores to remove library bias. The portion of maximal similarity score is shown with solid shading in the area under the curve.

Fig. 5. Analysis of the similarity of amino-acid sequence between Brz2001-selected peptides and DWARF4 using the MATCH program in the RELIC suite: cluster diagram. A highlighted mapping of amino-acid sequence between Brz2001-affinity-selected 34 (A) or 26 (B) of peptides and DWARF4 at the portion of maximal similarity. Residues exhibiting identity or similarity to the protein sequence are highlighted in red or orange. The axial ligand of C462 coordinated with iron ion is shown in cyan. 1 Hz = 0.62 ng/cm2.

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TAKAKUSAGI ET AL.

as can be seen in the control experiment (Fig. 4C). Conversely, undetected portions or residues involved in the binding of Brz2001 might be present. The introduction of the position of linker on Brz2001, or genetically modified diversity or length of library peptides, might improve these properties. Along with precise docking studies, further experiments make it possible to expand the utility of this approach. Since brassinosteroid has been discovered as a plant growth–promoting hormone, extensive studies have been conducted about its biosynthesis as well as the production of inhibitors as a tool for biosynthesis regulation.25–27,36–41 Brz2001 is one of the synthetic compounds that specifically inhibits DWARF4 activity. These findings in our work will greatly contribute to better understanding the precise mode of docking as well as further designing the DWARF4 inhibitor for studies in brassinosteroid biosynthesis. Moreover, experimental methodology in this work, which could be beyond the technical difficulties of the conventional approach, will be a valuable tool for detecting a small-molecule binding site on a target protein as well as expanding the druggable proteome in a wide range of small-molecule–related studies, including plant molecular biology.

CONCLUSION In this work, the QCM biosensor-based T7 phage display in combination use with bioinformatics analysis has extended into plant molecular biology. A screening of a library of T7 phage-displayed 15mer random peptides was undertaken to identify 34 Brz2001-recognizing peptides. Subsequent qualitative and quantitative assessment of affinity-selected peptides, which is based on an algorithmic and heuristic approach using RELIC programs, predicted the increase in the information content of Brz2001 recognition through the selection, highlighting continuous and discontinuous peptide motifs. Furthermore, a similarity search using the RELIC/MATCH program with Brz2001-selected peptides has identified a potential disordered portion of DWARF4 as a scaffold during Brz2001 docking, even though the three-dimensional structure of this enzyme has been unavailable. A similar trend was also noted by an analysis using another 26 Brzselected peptides and not observed using 27 control peptides, demonstrating the reproducibility and specificity of this method. Beyond the technical limitations associated with physicochemical properties of small-molecule or protein samples, this affinity-based methodology could be applicable to any small-molecule/protein systems under the identical protocol as a high-throughput method for the scanning of small-molecule-recognizing portion. Experimental methodology in this work will greatly contribute to the wide range of small-molecule– related studies, including plant molecular biology.

ACKNOWLEDGMENTS The authors thank Dr. Tadao Asami (University of Tokyo, Tokyo, Japan) for providing Brz2001, and Dr. Lee Makowski (Northeastern University, Boston, MA) for providing the stand-alone type RELIC programs. This work was partially supported by a Grant-in-Aid for Scientific Research (The Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Society for the Promotion of Science).

214 ASSAY and Drug Development Technologies APRIL 2013

DISCLOSURE STATEMENT The authors declare no conflict of interests.

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ONE-CYCLE BIOPANNING COMBINED WITH BIOINFORMATICS ANALYSIS

22. Mattos C, Rasmussen B, Ding X, Petsko GA, Ringe D: Analogous inhibitors of elastase do not always bind analogously. Nat Struct Biol 1994;1:55–58. 23. Makowski L, Soares A: Estimating the diversity of peptide populations from limited sequence data. Bioinformatics 2003;19:483–489. 24. Rodi DJ, Soares AS, Makowski L: Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries. J Mol Biol 2002;322:1039–1052. 25. Sekimata K, Kimura T, Kaneko I, et al.: A specific brassinosteroid biosynthesis inhibitor, Brz2001: evaluation of its effects on Arabidopsis, cress, tobacco, and rice. Planta 2001;213:716–721. 26. Min YK, Asami T, Fujioka S, Murofushi N, Yamaguchi I, Yoshida S: New lead compounds for brassinosteroid biosynthesis inhibitors. Bioorg Med Chem Lett 1999;9:425–430. 27. Asami T, Mizutani M, Fujioka S, et al.: Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta. J Biol Chem 2001;276:25687–25691. 28. Cheng CI, Chang YP, Chu YH: Biomolecular interactions and tools for their recognition: focus on the quartz crystal microbalance and its diverse surface chemistries and applications. Chem Soc Rev 2012;41:1947–1971. 29. Love JC, Estroff LA, Kriebel JK, Nuzzo RG, Whitesides GM: Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chem Rev 2005;105:1103–1169. 30. Wink T, van Zuilen SJ, Bult A, van Bennkom WP: Self-assembled monolayers for biosensors. Analyst 1997;122:43R–50R. 31. Takakusagi Y, Takakusagi K, Ida N, et al.: Binding region and interaction properties of sulfoquinovosylacylglycerol (SQAG) with human vascular endothelial growth factor 165 revealed by biosensor-based assays. MedChemComm 2011;2:1188–1193. 32. Mao X, Yang L, Su XL, Li Y: A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7. Biosens Bioelectron 2006;21:1178–1185. 33. Henne WA, Doorneweerd DD, Lee J, Low PS, Savran C: Detection of folate binding protein with enhanced sensitivity using a functionalized quartz crystal microbalance sensor. Anal Chem 2006;78:4880–4884. 34. Wester MR, Yano JK, Schoch GA, et al.: The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-A resolution. J Biol Chem 2004;279:35630–35637.

35. Williams PA, Cosme J, Ward A, Angove HC, Matak Vinkovic D, Jhoti H: Crystal structure of human cytochrome P450 2C9 with bound warfarin. Nature 2003;424:464–468. 36. Bajguz A, Asami T: Suppression of Wolffia arrhiza growth by brassinazole, an inhibitor of brassinosteroid biosynthesis and its restoration by endogenous 24-epibrassinolide. Phytochemistry 2005;66:1787–1796. 37. Bajguz A, Asami T: Effects of brassinazole, an inhibitor of brassinosteroid biosynthesis, on light- and dark-grown Chlorella vulgaris. Planta 2004;218: 869–877. 38. Yoshimitsu Y, Tanaka K, Fukuda W, et al.: Transcription of DWARF4 plays a crucial role in auxin-regulated root elongation in addition to brassinosteroid homeostasis in Arabidopsis thaliana. PLoS One 2011;6:e23851. 39. Sekimata K, Ohnishi T, Mizutani M, et al.: Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity. Biosci Biotechnol Biochem 2008;72:7–12. 40. Nagata N, Asami T, Yoshida S: Brassinazole, an inhibitor of brassinosteroid biosynthesis, inhibits development of secondary xylem in cress plants (Lepidium sativum). Plant Cell Physiol 2001;42:1006–1011. 41. Nagata N, Min YK, Nakano T, Asami T, Yoshida S: Treatment of darkgrown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants. Planta 2000;211:781–790.

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Address correspondence to: Kengo Sakaguchi, PhD, or Fumio Sugawara, PhD Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science 2641 Yamazaki Noda, Chiba 278-8510 Japan E-mail: [email protected] or [email protected]

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ASSAY and Drug Development Technologies 215

SUPPLEMENTARY DATA General (Chemistry) All nonaqueous reactions were carried out using freshly distilled solvents under an atmosphere of argon. All reactions were monitored by TLC, which was carried out on Silica Gel 60 F254 plates (E. Merck, Darmstadt, Germany). Flash chromatography separations were performed on PSQ 100B (Fuji Silysia Co., Ltd., Japan). The NMR spectra (1H, 13C) were determined on a Bruker 600 MHz or 400 MHz spectrometer (Avance DRX-600, Avance DRX-400) or a JEOL 400 MHz spectrometer ( JNM-LD400), using CDCl3 (with TMS for 1H NMR and chloroform-d for 13C NMR as the internal reference) solution, unless otherwise noted. Chemical shifts were expressed in parts per million (ppm), and coupling constants were expressed in Hertz. Optical rotations were recorded using CHCl3 as a solvent of a JASCO P-1030 digital polarimeter at room temperature, using the sodium D line. Infrared spectra (IR) were recorded on a Jasco FT/IR410 spectrometer using NaCl (neat) or KBr pellets (solid), and were reported as wave numbers (cm - 1). Mass spectra were obtained on an Applied Biosystems mass spectrometer (API QSTAR pulsar i) under conditions of high resolution, using polyethylene glycol as an internal standard.

Synthesis of the Brz2001 Derivative That Forms the Self-Assembled Monolayer 5-(4-Chlorophenyl)-2,4,5-trideoxy-3-C-phenyl-4-(1H-1,2,4triazol-1-yl)pentonic acid. A solution of KMnO4 (2.71 mg, 0.017 mmol), NaIO4 (159 mg, 0.743 mmol), and K2CO3 (32 mg, 0.232 mmol) in H2O (3 mL) was added to a solution of 1 (52.2 mg, 0.148 mmol) in acetone (2 mL). The reaction was stirred at room temperature for 3.5 h before the addition of more KMnO4 (2.0 mg, 0.013 mmol) to the mixture. The layers were separated, and the aqueous layer was extracted with EtOAc ( · 3). The combined organic layer was washed with brine, dried (Na2SO4), and evaporated. The residue was purified by silica gel chromatography (CHCl3: MeOH = 20:1 with 1% AcOH) to yield 2 (48.9 mg, 89.3%) as a colorless powder. 1H NMR (600 MHz, CDCl3, major diastereomer) d = 7.83 (1H, s), 7.57 (1H, s), 7.36 (2H, d, J = 7.6 Hz), 7.31 (2H, d, J = 7.6 Hz), 7.26 (1H, m), 7.10 (2H, d, J = 8.4 Hz), 6.76 (2H, d, J = 8.4 Hz), 6.76 (2H, d, J = 8.4 Hz), 4.70 (1H, dd, J = 11.8 Hz, 2.5 Hz), 3.33 (1H, dd, J = 14.2 Hz, 11.8 Hz), 3.26 (1H, d, J = 16.3 Hz), 3.16 (1H, dd, J = 14.2 Hz, 2.5 Hz), 3.11 (1H, d, J = 16.3 Hz); 13 C NMR (100 MHz, DMSO-d6) d = 173.0, 150.1, 144.8, 143.4, 137.2, 131.1, 130.7 ( · 2), 128.2 ( · 2), 127.7 ( · 2), 127.0, 125.6 ( · 2), 76.1, 69.0, 42.7, 33.9; IR (neat) 3407, 2930, 1713, 1496, 1444, 1408, 1279, 1209, 1137, 1093, 1024, 886, 817, 757, 705, 663 cm - 1; HRMS (ESI) calculated for C19H17N3O3NaCl [M + Na] + : 370.0963, found to be 370.0969. 4-Nitrophenyl 5-(4-chlorophenyl)-2,4,5-trideoxy-3-C-phenyl-4-(1H-1, 2,4-triazol-1-yl) pentonate. EDCI (17.0 mg, 0.089 mmol) was added to a solution of 2 (15.5 mg, 0.042 mmol), p-nitrophenol (17.0 mg, 0.12 mmol), and DMAP (1.23 mg, 0.010 mmol) in CH2Cl2 (2 mL), and the mixture was stirred at room temperature for 1.5 h. The mixture was then

concentrated, and the residue was purified by silica gel chromatography (hexane: EtOAc = 2:1) to yield 3 (17.6 mg, 85.5%) as a yellow powder. 1H NMR (600 MHz, CDCl3, major diastereomer) d = 8.21 (2H, d, J = 8.5 Hz), 7.85 (1H, s), 7.55 (1H, s), 7.44 (2H, d, J = 7.4 Hz), 7.36 (2H, d, J = 7.4 Hz), 7.31 (1H, m), 7.12 (2H, d, J = 8.3 Hz), 6.99 (2H, d, J = 8.5 Hz), 6.80 (2H, d, J = 8.3 Hz), 4.89 (1H, dd, J = 11.7 Hz, 2.8 Hz), 3.53 (1H, d, J = 15.7 Hz), 3.41 (1H, dd, J = 14.0 Hz, 11.7 Hz), 3.34 (1H, d, J = 15.7 Hz), 3.28 (1H, dd, J = 14.0 Hz, 2.8 Hz); 13C NMR (100 MHz, CD3OD-CDCl3) d = 169.9, 156.2, 151.2, 146.9, 146.4, 143.4, 137.4, 133.6, 131.5 ( · 2), 129.5 ( · 2), 129.3 ( · 2), 129.1 ( · 2), 126.9, 126.0 ( · 2), 123.8 ( · 2), 78.0, 71.2, 44.2, 35.2; IR (neat) 3,120, 3,026, 2,927, 2,855, 1,755, 1,592, 1,523, 1,496, 1,446, 1,341, 1,290, 1,206, 1,138, 1,014, 930, 860, 812, 755, 704, and 672 cm - 1; HRMS (ESI) calculated for C25H21N4O5NaCl [M + Na] + : 515.1092, found to be 515.1117.

Brz2001 Derivative DMAP (1.1 mg, 0.009 mmol) was added to a solution of 3 (17.6 mg, 0.035 mmol) and 4 (18.4 mg, 0.018 mmol)S1 in pyridine (2 mL), and the mixture was stirred at room temperature for 18 h. Then, the mixture was concentrated, and the residue was purified by silica gel chromatography (CHCl3: MeOH = 20:1 to 4:1) to yield Brz2001 derivative (13.7 mg, 51%) as a colorless oil. 1H NMR (600 MHz, CDCl3, major isomer) d = 7.77 (1H · 2, s), 7.54 (1H · 2, s), 7.32 (2H · 2, d, J = 7.4 Hz), 7.26 (2H · 2, d, J = 7.4 Hz), 7.21 (1H · 2, m), 7.10 (2H · 2, d, J = 8.4 Hz), 6.85 (2H · 2, d, J = 8.4 Hz), 6.22 (1H · 2, m), 6.09 (1H · 2, s), 4.83 (1H · 2, dd, J = 11.8 Hz, 2.8 Hz), 3.65–3.60 (4H · 2, m), 3.59–3.55 (3H · 2, m), 3.51 (2H · 2, t, J = 4.5 Hz), 3.47–3.43 (4H · 2, m), 3.39– 3.30 (4H · 2, m), 3.21 (1H · 2, dd, J = 14.2 Hz, 11.8 Hz), 3.02 (1H · 2, d, J = 14.9 Hz), 2.87 (1H · 2, d, J = 14.9 Hz), 2.67 (2H · 2, t, J = 7.4 Hz), 2.15 (2H · 2, t, J = 7.4 Hz), 1.65 (2H · 2, m), 1.61 (2H · 2, m), 1.36 (2H · 2, m), 1.25 (10H · 2, brm). 13C NMR (100 MHz, CDCl3) d = 173.4 ( · 2), 170.7 ( · 2), 151.1 ( · 2), 144.5 ( · 2), 142.0 ( · 2), 135.7 ( · 2), 132.6 ( · 2), 129.9 ( · 4), 128.7 ( · 4), 128.2 ( · 4), 127.7 ( · 2), 125.4 ( · 4), 70.39 ( · 2), 70.36 ( · 2), 70.2 ( · 2), 70.1 ( · 2), 70.0 ( · 2), 69.8 ( · 2), 69.6 ( · 2), 43.0 ( · 2), 39.2 ( · 2), 39.1 ( · 4), 39.0 ( · 2), 36.7 ( · 2), 34.1 ( · 2), 29.5 ( · 2), 29.43 ( · 2), 29.37 ( · 2), 29.3 ( · 2), 29.21 ( · 2), 29.18 ( · 2), 28.5 ( · 2), 25.7 ( · 2). IR (neat) 3,296, 3,087, 2,926, 2,856, 1,648, 1,551, 1,495, 1,447, 1,350, 1,278, 1,137, 1,096, 1,016, 934, 812, 755, 703, 665 cm - 1; HRMS (ESI) calculated for C76H110N10O12Na2S2Cl2 [M + 2Na]2 + , 767.3453, found to be 767.3489.

SUPPLEMENTARY REFERENCES S1. Takakusagi Y, Kuramochi K, Takagi M, et al.: Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool. Bioorg Med Chem 2008;16:9837–9846. S2. Williams PA, Cosme J, Ward A, Angove HC, Matak Vinkovic D, Jhoti H: Crystal structure of human cytochrome P450 2C9 with bound warfarin. Nature 2003;424:464–468. S3. Wester MR, Yano JK, Schoch GA, et al.: The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-A resolution. J Biol Chem 2004;279:35630–35637.

Supplementary Fig. S1. Synthesis of Brz2001 derivative.

Supplementary Fig. S2. Three-dimensional structure of human cyochrome 2C9, a representative enzyme of Cyp450 super family. (A) Part of the homology between DWARF4 and human CYP2C9 (E value: 6e-12). The disordered portion of catalytic site identified by Brz2001-selected peptides in DWARF4, along with the cognate region in CYP2C9, is shown in gray. The heme axial ligand of Cys is shown in cyan. (B) Threedimensional structure of human CYP2C9 complexed with flurbiprofen (PDB ID: 1R9O).S2,S3 The peptide backbone of CYP2C9 is shown in cartoon form. Heme is shown in CPK. Oxygen in heme is shown in red. The Fe ion is shown in yellow. For convenience, flurbiprofen is not shown. (C, D) Close-up view of a part of the CYP2C9 molecule. The homologous region with potential Brz2001-binding site in DWARF4, which was identified using Brz2001-selected peptides, is shown in cartoon form in gray. The heme and axial ligand of C435 are shown in stick form.

Supplementary Table S1. 15-mer Peptide Sequences Randomly Extracted from the Parent Library of T7 Phage-Displayed Peptides No.

Sequence

No.

Sequence

No.

Sequence

No.

Sequence

1

VFCGSIFNGRAVWWT

27

CSFFSSLLLETCGLC

53

FNCRCLLLFSNLAFL

79

ICCLLLYLIRGCAPL

2

SVRVRLDLGSLYYGR

28

FLGAYYSHMHQSGCL

54

HRGVLRCACIGETGL

80

LFVGGPDFVANHNCL

3

RVPFPLHLRWLCIAR

29

RCKALTLDLQSPLCD

55

IFVFYSVGSGVLSSA

81

PLYTLNFGFHVVYVV

4

GVLHVKSLLVLSSAD

30

IFSASVLGIWGFWSE

56

SVHYLNLAYCSCLLV

82

ACYGYMPSLDCSASQ

5

RLVLLWWSNAGRHFR

31

CPHNFYFAGVDDINA

57

ELLVLVGGGSCRNRL

83

VCYLILLFSLPCLPH

6

DYNCIPSNAGHVFCA

32

YFALGLLVNNGLPCW

58

WFVFFSVSFVLSFWT

84

ICFGLHMPFPHGAWR

7

AFQLTQGSASSWVLG

33

SCIFRVVSCIHHHLP

59

LGDSGGRGAWYQYRL

85

PHCAYTVASSFCLCL

8

ISCFCVNDDSSDIIA

34

CIPILLSTIALRYVH

60

DFFCCASALSILVVP

86

CSSMFFMFFGIMVYC

9

LLFTFSSSRVQMISC

35

CHCLSLVEISSSYWL

61

FLSLYFSPSLSPPQW

87

ALVCVRILFLDLCGC

10

VVCVRHNDMCCAYAT

36

YLFLGTTGLCRGNEL

62

YVFFLWPLFVNSPVS

88

RSSNCCIYVLDFVVC

11

CTGVRWPLHLRIALS

37

GVVSLSGRRVPMYAV

63

FLYIRCNPGGSSVLA

89

CVFILLFVVLFILFD

12

CRRGWRGVRVLRGGN

38

CIALGISQLKFVRVC

64

GRGLLFAVERAPCRW

90

YFGAASCRSCGVTAI

13

VLGPISDFLSPFRPS

39

SGVIFNDLVFYGAVT

65

FAFILYKSYWGPLAA

91

SFVYRGNGWVSHMSI

14

LYAPGFVFSSNSIWT

40

GSLLLGHVLAISSVR

66

IVYCILLCLLYMPFF

92

CRLFLVTLMLVFFLA

15

IPLCVSCGCDSLRVV

41

RCVCASPLIGKACPW

67

FVSVSLASSPFNPSP

93

FLYVVAVGFGLNGWR

16

LFLACDRTILGLDVD

42

LYVPVCLGSYLHVPM

68

AFVCVGPLTLAVMDY

94

IFGVFISSCLCITTL

17

LCAFISYPDARLYVT

43

CFNALVLPLVSARGV

69

FSLNSLKHCVLVVRG

95

RLPSTGVFIHVPSFD

18

LFDVCALALPCSPWR

44

LCHFCRAVPYFTSYS

70

VGLACSCVGSSIFRS

96

SANFTGFGILPRLRS

19

SAFLCCMIFPHPPVS

45

PLVPSFIFSLVPSIA

71

FRCFRVRYAVKSDEC

97

IEFVVIYCCVFIFGW

20

MYGLAGYSDSFPIWF

46

MCSVAFIGQRLDHAP

72

DFLCWPAYVHFSRLR

98

FVYGLPGVYAVRVVS

21

VHFFGHIDILSCVNA

47

FGCYIMAHVNDFFIC

73

LGGLLQLASFLPRAR

99

ANLLSPDRLGPALTC

22

GRESDVTLLFTLDAV

48

SFFCLVLFYFWIFKI

74

LRFGVLTLSCYTGVP

100

ICNPEFCGALSSMAF

23

FCPLMFVLPCRASRV

49

VFFWVYVVVHCSSES

75

IVCYCSVAHHAGDLA

101

AGVVVILHWAVGVGG

24

SGYLVTSLCYVFFNA

50

AGCHRVWPVGLTSGP

76

LSHASCEPGDAPLEY

102

GRQFDCVRDQTCIRA

25

CVLTSCPPIAHPFRV

51

GLSMGQSVVYFSSTS

77

TLDVYCAPELASHAC

103

26

SFGLRGLLHAYFRSS

52

RMARVLLGCDASLLR

78

FPLIYPCNIAACDRF

These peptide sequences were subjected to AAFREQ, and MATCH programs in the RELIC bioinformatics server as a background. RELIC, receptor ligand contacts.

LRLFHLLSCASPTDL

Supplementary Table S2. Frequency of Each of the 20 Amino Acids in the Parent Library of T7 Phage-Displayed Peptide Calculated Using AAFREQ Program in RELIC Suite a.a.

#1

#2

#3

#4

#5

#6

#7

#8

#9

#10

#11

#12

A

7

3

5

9

4

3

6

8

5

11

8

5

C

12

12

11

9

10

8

6

2

9

6

6

D

3

0

3

0

2

1

5

4

4

4

E

1

1

1

0

1

0

1

1

2

F

13

19

14

15

6

8

5

8

G

7

8

10

7

8

8

5

H

1

3

4

2

1

3

I

11

2

1

6

6

K

0

0

1

0

L

13

15

13

M

2

1

N

0

P

#13

#14

#15

Total

Frequency

6

11

12

103

0.0667

9

3

6

10

119

0.077

4

3

5

2

5

45

0.0291

1

0

1

0

4

1

15

0.0097

9

4

9

7

9

5

4

135

0.0874

14

7

8

8

5

5

8

3

111

0.0718

2

4

3

6

5

4

4

0

2

44

0.0285

5

5

1

11

2

3

6

7

3

3

72

0.0466

0

1

2

0

0

1

2

0

0

1

0

8

0.0052

18

20

15

17

18

14

16

12

9

10

10

12

212

0.1372

0

2

1

2

3

0

3

0

0

4

3

0

1

22

0.0142

2

4

3

0

3

4

3

2

2

4

2

3

3

1

36

0.0233

3

3

4

5

1

6

6

9

2

4

5

12

7

5

6

78

0.0505

Q

0

0

2

0

0

3

0

1

1

2

2

1

0

1

1

14

0.0091

R

7

9

2

2

9

4

3

4

4

4

4

6

7

10

10

85

0.055

S

10

7

7

4

10

12

11

11

11

12

14

17

10

7

11

154

0.0997

T

1

1

0

3

3

4

4

2

1

0

3

3

4

3

6

38

0.0246

V

7

13

13

13

12

10

12

10

9

12

8

7

10

14

8

158

0.1023

W

1

0

0

1

2

3

2

0

2

4

1

1

2

8

5

32

0.0207

Y

4

4

8

4

7

4

4

3

4

4

5

1

8

2

2

64

0.0414

1545 Occurrence of each of the 20 amino acids at each recombinant insert position and the overall position-independent frequency of each amino acid are shown.

Supplementary Table S3. Frequency of Each of the 20 Amino Acids in the Brz2001-Selected Peptide Calculated Using AAFREQ Program in RELIC Suite a.a.

#1

#2

#3

#4

#5

#6

#7

#8

#9

#10

#11

#12

#13

#14

#15

Total

Frequency

A

2

1

1

4

2

1

2

5

5

0

0

2

1

3

5

34

0.0667

C

6

8

3

4

3

2

1

1

4

1

5

1

2

5

0

46

0.0902

D

0

2

1

2

0

1

2

1

1

3

0

0

0

2

2

17

0.0333

E

0

0

0

0

0

0

0

1

0

0

1

0

1

0

1

4

0.0078

F

4

4

5

3

5

4

7

2

3

3

1

3

1

2

4

51

0.1

G

2

2

0

3

3

6

3

4

1

1

5

3

2

3

5

43

0.0843

H

0

1

0

1

0

0

1

0

0

2

2

0

3

0

1

11

0.0216

I

2

0

2

1

5

2

0

1

1

2

0

2

1

1

1

21

0.0412

K

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

L

5

5

4

3

3

5

6

3

2

2

5

4

7

4

6

64

0.1255

M

1

0

0

0

0

1

0

0

2

1

2

0

2

2

1

12

0.0235

N

0

1

2

2

2

1

0

1

2

0

1

0

0

0

0

12

0.0235

P

0

0

4

2

2

0

0

4

1

2

3

3

1

2

0

24

0.0471

Q

1

0

1

0

0

0

0

0

0

0

2

0

1

0

0

5

0.0098

R

1

1

0

0

0

0

1

0

1

2

1

3

1

1

3

15

0.0294

S

6

5

1

6

2

3

1

3

5

9

4

4

3

2

2

56

0.1098

T

1

0

0

0

0

1

3

2

1

0

0

1

1

0

0

10

0.0196

V

2

4

8

3

6

4

4

6

4

6

1

5

4

5

2

64

0.1255

W

0

0

0

0

1

1

1

0

1

0

0

2

1

2

1

10

0.0196

Y

1

0

2

0

0

2

2

0

0

0

1

1

2

0

0

11

0.0216

0

510 Occurrence of each of the 20 amino acids at each recombinant insert position and the overall position-independent frequency of each amino acid are shown.

Supplementary Table S4. Brz2001-Selected 15-Mer Peptide Sequences After Three Sets of Individual One-Cycle Biopanning No. 0a

Sequence

No. Sequence 0a

No. Sequence

No. Sequence

1

AGPFDSVSGLWSTFG

8

FVLFGSPRLCTQSCG

150

LHLLWSSVCSGIYKC

220

VLSAFCCGFCSDNAF

20

CDVFCFVASDSHGAN

90

FVRVVVYAWHLMSFA

160 a

MLLVDWRRLLGTSTS

230

VQVSRCLSTVAGHSV

30 a

CLVISAFCDHCVSTV

100

FYLLMSDIIEPSPPM

170

PCLVVSHLHVLASSC

240

VSSPWMSLRYLVPPV

40

CMQTLLAAATVCDMG

110

HFADYRLSVVEDYCW

180 a

SFVVRFVDSVRALTL

250

VYDCLADDFSAYRGV

50

CNCDMNVINSDRYGA

120

IVLPHPISYTSGLLP

190

SLLSDSPAAHTLPWV

260

WIASPFLGVGVRDPP

60 a

ELLVVPSSFSAWNQV

130

LCLYWFDAWLSEFMG

200

TCDSHIGIMAEYALR

70 a

FCGGSAVRVSDLGWK

140

LCPSFLLSSETSLIG

210

VIVDHLLQEIAPGAY

T7 phage particles were arbitrarily extracted from the resulting solution and then analyzed by PCR amplification of the DNA encoding the fusion peptide. The PCR products were then sequenced. a The peptide directly highlights a disordered portion on DWARF4 using the MATCH program (Fig. 5B). PCR, polymerase chain reaction.

Supplementary Table S5. Gold Electrode-Recognizing 15-Mer Peptide Sequences (Control) After Three Sets of Individual One-Cycle Biopanning No.

Sequence

No. Sequence

No. Sequence

No. Sequence

1†

AFFLHLFVCVLLSLS

8†

FGLRNVEVFCLPRLM

15†

LYVSSCMLAVHQSHL

22†

VGFMWTPRLTSLRHV

2†

AGLVSFVPTLLRSSM

9†

FLASVHRRLVTSSFL

16†

PSGLPRSGLEVAKTG

23†

VLLPCRVWMCTLLFC

3†

CFEMLQRFFPCTSLC

10†

GALVFPAMPAHIDVR

17†

PVYFFLDTAFFASLY

24†

VYSHVCAANSDFWDC

4†

CLPWLSLNFVNMLGP

11†

GCSSDSCGAVRIFCQ

18†

RCYRITFSANDFTWS

25†

YLCGGFAVMCGGDVV

5†

CYCFQNNQVSCLCVV

12†

HLCDEFRSIFYLRPV

19†

RSYIFGHVPGSLSTR

26†

YVCFAVFVAWPLQRA

6†

CYFCFVYVSDRFSSA

13†

LCWRDPLMDVRAVTA

20†

SIFVAFCFVGDFLGP

27†

YYGMCLPPFHLFSWC

7†

FACSLGVFGICSHYI

14†

LGFDLIRVLGVRNRG

21†

VCVFGLSGRVVRAAA

T7 phage particles were arbitrarily extracted from the resulting solution and then analyzed by PCR amplification of the DNA encoding the fusion peptide. The PCR products were then sequenced.